纳米级微泡增强乳腺肿瘤显像效果及高强度聚焦超声消融效果的实验研究
|
摘要
分子生物学（molecular biology）及其分支学科分子影像学(molecular imaging)技术发展迅速，并扩展到超声分子成像（ultrasound molecular imaging）领域。
分子影像技术的关键在于分子探针（molecular probe）的选择，超声造影剂（Ultrasound contrast agent，UCA）作为新型的超声分子影像探针，是目前研究的热点，UCA是一种稳定的含气泡的物质，临床常用于发现微小病变以及区分良、恶性肿瘤等方面，其原理是改变声速（sonic speed）、声衰减系数（attenuationcoefficient）、增强后散射（backscattering）等，利用组织与气泡之间产生的声阻抗（acoustic impedance）差，增加组织与血流之间的回声信号，提高组织血流灌注的超声显像分辨率，从而提高超声诊断的特异性、灵敏度。
传统的的UCA属血池显像剂（blood pool imaging agent），一定程度上影响了超声分子成像及治疗的发展，近年纳米级造影剂（nanoscale contrast agent）不断涌现，纳米级UCA具有很强的穿透力，能穿过血管壁，直达靶区细胞，其开发和应用有利于发展靶向性、高效性、小型化且具有辅助治疗作用的新型UCA。
超声造影（contrast enhanced ultrasound，CEUS）技术为乳腺癌等的诊断、鉴别等提供了有力的帮助，研究表明，常用的CEUS属于非特异性显影，UCA对组织没有特异性结合力，仅能观察感兴趣区血管短暂显影（动脉相），不能在特定组织内长时间驻留，影响了诊断率；近年特异性CEUS成为研究的热点，其核心内容是在微泡表面连接特异性抗体，利用其与靶区相应受体特异性结合的特点，使微泡选择性浓聚于靶区而产生特异性增强显影，导向分子和UCA的小型化是该技术的关键；近来兴起的纳米抗体(nanobody)及小分子抗体模拟肽（peptide mimics），具有与抗原特异性结合的特性，可以克服传统单克隆抗体分子量过大、穿透力较弱等缺点，易于大量生产；靶向性的纳米级UCA聚集于特定区域时产生显影，未聚集则不产生显影，有利于提高诊断的准确性。
高强度聚焦超声(High intensity focused ultrasound，HIFU）在治疗恶性肿瘤方面取得了很大进展，临床HIFU术中，存在辐照剂量较大、辐照时间较长等问题，易引起并发症，学者们提出改变组织结构、密度、血供、功能等组织声环境（AET）,以加速声能沉积,提高HIFU疗效,减少并发症；UCA可以提高HIFU疗效，其机理可能是：①增强空化声孔(sonoporation)效应，②升高靶区温度，③影响细胞增殖，④毛细血管破坏；常用的UCA粒径较大，不能透过血管内皮间隙直达肿瘤细胞，并且，声通道组织中的微泡过大过多，可导致聚焦超声不能聚焦或焦点偏移等问题，纳米级微泡（nanobubble，NB）有望弥补常规微泡的弱点，实现肿瘤的血管外显影和治疗。
我们在成功研制微米级微泡的基础上，优化制备工艺研制NB，对分析其基本性质，在兔VX2乳腺肿瘤模型上，研究其增强实质显像的效果，以及作为增效剂在增强HIFU消融效果中的作用。
另一方面，常规超声技术对HIFU实时疗效评价主要依赖于灰阶（grey scale）值变化，这种方法存在灵敏度较差、依赖操作医生主观判断等问题，灰阶值变化不明显时，易发生误判，导致辐照剂量增大、治疗时间增多，引起不必要的并发症，目前急需寻找一种更为敏感、准确、可靠的评价方法；以前，超声散斑（speckle）被认为是一种干扰信号而在超声成像时被计算机自动过滤清除，近来，人们开始认识到散斑也是一种信息载体，从而促进了散斑计量术的诞生，有研究表明，声像图散斑图像的相关函数（correlation）、基于小波变换（wavelet transform）的Tamura斑纹分析（texture analysis）、多分辨分析（multi-resolutionanalysis）等可用于判断HIFU辐照的靶区是否发生凝固性坏死（Coagulative Necrosis，CN），并优于灰阶值评价，有望成为新的HIFU实时疗效评价方式。
综上，本课题研究主要包括以下四个部分：
目的：在成功研制微米级微泡的基础上，优化制备工艺研制NB，对其基本性质进行初步的研究，以便于下一步应用。方法：应用优化的工艺制备纳米微泡，二棕榈酰磷脂酰胆碱（DPPC）、二棕榈酰磷脂酸（DPPA）等及辅助成分，按比例混合（脂质微泡获国家专利，可广泛应用于动物实验中，增强肝脏，心脏、肾脏等脏器的成像），超纯水将上述成分充分混悬均匀，经一定工艺制成混悬液，分装于锥形瓶中，使用优化的工艺冻干混悬液，以提高脂质膜的密封性，加入水合液（自制）重新水合，注入八氟丙烷（C3F8）气体，水平往复式机械振荡，频率≧4500次/分，振幅:(15士1) mm，振荡约90s左右，低速离心法对已制备的原始微泡分级分离，300RPM离心3分钟后，微泡悬浮液分为上下两部分，上层粒度较大的微泡被丢弃，留取下层粒度较小的微泡进行下一步实验。生理盐水稀释50倍后，进行肉眼观察、光镜观察、电镜观察、荧光显微镜观察，Zeta Sizier3000测定微泡的粒径、表面电位。结果：自制的微泡，肉眼观察呈白色凝乳状，封装于西林瓶中，振荡前为清亮的半透明液体，振荡后呈白色凝乳状的混悬液，表面未见泡沫，黏稠，不透明；光镜下观察，微泡呈类圆形，数量较多，分布均，无聚集，偶见较大的脂质体小颗粒；电镜下，微泡粒径不足1μm；粒径测试结果均值623.4nm，分布于278.8nm～697.0nm之间，表面电位均值1.3mV，分
布于-3.2mV～+9.5mV之间。结论：各种成膜材料中，脂质（lipids）的弹性较好、对温度要求不高、毒副作用少、无免疫原性，在本实验中，我们以脂质为成膜成分，在人体安全剂量条件下，改进各类脂质及辅助成分配方比例，加入C3F8（惰性气体，可安全地经肺排除）；考虑到机械振荡法较常用的声振法产生的热量小、对脂质体等成分活性破坏少、产泡量高、污染小等特点，我们采用合适的振荡频率和时间机械振荡制备微泡；采用优化后的冻干工艺（合适的预冻温度、冻干时间、冻干速度、真空压力等）及灭菌方式，最后与低速离心法相结合，得到NB，肉眼及镜下观察微泡呈类圆形，数量较多，分布较均匀，无聚集，稳定性较好，理论上可以通过肿瘤组织的血管内皮间隙。
目的：在制备NB的基础上，选择小分子多肽AHNP(序列为：FCDGFYACYMDV，荧光标记)与NB结合，研究其体外寻靶作用，在兔VX2乳腺肿瘤模型上，研究NB增强实质显像效果。方法：①活性剂与NB混匀，加入激活剂碳化二亚胺（EDC）与AHNP混合，室温反应30min，机械振荡后静置片刻，去下清液，缓冲液（PBS）冲洗，得到靶向NB。花环形成实验、花环形成阻断实验、免疫荧光染色观察靶向NB体外寻靶效果。②健康纯种新西兰白兔（famale）30只，麻醉后将提前制备好的VX2肿瘤组织悬液注入双侧乳腺组织各1ml，两周后经logiq9彩色多普勒超声诊断仪检查，选取乳
腺区肿瘤组织直径大小约15mm左右的兔24只进行实验，实验兔麻醉后，随机分组为靶向NB组10只，声诺维（Sonovue）组10只，PBS组4只，耳缘静脉分别注射靶向NB、Sonovue、PBS，Logiq9超声诊断仪记录并观察体内增强显像效果。结果：标记荧光的携AHNP的NB，呈浅黄色，光镜下，微泡均匀细密分布，检测微泡的粒径与非靶向微泡相比稍大，荧光显微镜下观察，靶向NB见绿色荧光，非靶向微泡偶见零星的微弱荧光，花环形成率达80%以上，特异性结合靶向微泡的细胞阳性率约60%；实验兔超声显示乳腺肿瘤边界清楚，截面呈椭圆形，内部低回声，彩色多普勒显示肿瘤内部血供丰富，频谱多普勒显示为动脉频谱，实验兔注射靶向NB后，平均8s（5s～12s）出现血管增强显像，注射后平均15s（9s～20s），实质回声增强，增强效果最长持续时间15min左右，Sonovue组注射后平均11s（7s～19s）出现增强显像，最长持续时间20min左右，PBS组无增强效应。结论：AHNP为抗p185her2/neu的特异性单克隆抗体CDR-H3环的模拟多肽，与HER2具有抗体样的特异性结合能力，AHNP与NB结合，有较强的体外寻靶效果，在兔VX2乳腺肿瘤模型中，靶向NB能达到增强肿瘤实质显像的效果，实验中及实验后，各组实验兔均无不良反应，亦证实了自制NB的安全性。
目的：在HIFU消融兔VX2乳腺肿瘤的过程中，联合使用自制的NB，观察其在不同辐照声功率、不同辐照时间条件下，对HIFU消融的增效作用，探讨NB在超声治疗中的应用价值。方法：携VX2乳腺肿瘤兔52只，随机分组为HIFU+NB组26只，HIFU+PBS组26只，脱毛，0.15mg/Kg剂量的“速眠新”麻醉，经耳缘静脉通道推注2.5%戊巴比妥钠（25mg/kg）持续麻醉，固定，启动聚焦超声肿瘤治疗系统，定位，辐照前10s，经耳缘静脉通道注射PBS（0.2ml/kg）或NB（0.2ml/kg），继而生理盐水1ml推注，调整焦点使其位于肿瘤内部，调整辐照声功率（150W，120W，100W）及时间（3s，5s）行HIFU辐照，系统自动分析辐照前后靶区超声灰阶变化值。HIFU辐照后1天，做病理检查，统计数据并分析。结果：HIFU治疗前超声显示肿瘤边界清楚，截面呈椭圆形，内部低回声，彩色多普勒显示肿瘤内部血供丰富，频谱多普勒显示为动脉频谱，HIFU治疗后靶区血供基本消失，偶见靶区周边点状血供；HIFU治疗前肿瘤造影显示为增强显像，HIFU治疗后肿瘤造影显示为靶区无明显增强显像，靶区周边增强显像，形成所谓“负显影”效果；HIFU治疗中及治疗后，实验兔无明显不良反应，1例实验兔HIFU+NB治疗两周后，乳腺肿块完全消失（考虑是否与HIFU治疗后促进了肿瘤细胞凋亡等有关）；HIFU辐照后靶区灰度出现增强，5s时间条件下，不同辐照声功率（120W、150W），HIFU+NB组靶区辐照后平均灰阶变化明显高于HIFU+PBS组，统计学分析有明显差异（P<0.01），但在100W、5s条件下，HIFU+NB组靶区辐照后平均灰阶差变化与HIFU+PBS组相比较，无明显差别，HIFU+NB组内分析，发现在150W和120W条件下，5s辐照组的平均灰阶差均明显高于
3s辐照组（P<0.05）；150W、5s同等条件下HIFU辐照后，肉眼观察辐照区呈灰白色灶,坏死灶分界清，HIFU+NB组坏死范围明显大于HIFU+PBS组；HE染色结果显示，HIFU+NB组与HIFU+PBS组的靶区组织内均见明确的凝固性坏死，HIFU+NB组发生凝固性坏死范围明显大于HIFU+PBS组（P<0.001），HIFU+NB组内对比分析，在同等辐照时间下，150W辐照声功率较120W辐照声功率损伤范围增加，在同等辐照声功率下，5s辐照时间较3s辐照时间损伤范围增加，120W、5s辐照条件与150W、3s辐照条件造成的的损伤效果基本一致。结论：在HIFU消融过程中，NB可以利用“负显影”效果，判断HIFU有效治疗区域；在同等辐照声功率、辐照时间条件下，HIFU+NB组较HIFU+PBS组，能有效提高辐照后灰阶变化，明显增大辐照后凝固性坏死的范围，提示NB能减少辐照强度，缩短辐照时间,达到增强HIFU损伤效果的目的。NB可同时应用诊断和治疗领域，其具体作用机制（如空化效应、声孔效应）以及辐照后远期致细胞凋亡效应等还有待进一步深入研究。
目的：采用纹理分析、相关函数评价HIFU+NB增强乳腺肿瘤消融的效果，寻求比灰度变化更有效的实时判断HIFU疗效的方法。方法：携VX2乳腺肿瘤兔60只，随机分组为HIFU+NB组30只，HIFU+PBS组30只，麻醉固定后，JC-200聚焦超声肿瘤治疗系统消融，辐照前10s，经耳缘静脉通道注射PBS（0.2ml/kg）或NB（0.2ml/kg），继而生理盐水1ml推注，采集辐照前后图像；病理检查；Matlab截取感兴趣去（ROI），进行两次小波变换，提取纹理分析1和2等参数；Matla软件截取辐照前后靶区声像图，像素大小30*30，计算辐照后即刻与辐照前靶区声像图的相关函数最大值；分析得出的结果录入WEKA3.7，采用支撑适量机（SVM）建立决策平面，分析样本；统计数据。结果：HIFU+PBS组，纹理分析1和2判断CN特异度与敏感度与灰阶判断有统计学差异，各组P＜0.05。HIFU+NB组，纹理分析1和2判断CN特异度与敏感度与灰阶判断有统计学差异，各组P＜0.05。HIFU+NB组与HIFU+PBS组辐照前后声像图应用相关函数对CN的判断灵敏度高于灰阶判断（P<0.05）。应用相关函数总判对率高于灰阶(P<0.05)。结论:初步证实纹理分析与灰阶评价相比，在判断CN的灵敏度及特异度上，更有优越性。而相同辐照条件下，相关函数分析对CN的总判对率高于灰阶。
With the development of molecular biology and molecularimaging,ultrasound molecular imaging has been advanced rapidly.Thekey is the molecular probe.Ultrasound contrast agent(UCA) as a newmolecular imaging ultrasound probe,is the hot spot.UCA have beenused to increase the sensitivity and specificity of the detection ofdisease due to the increase of signal reflection from blood flow,it canenhance the backscattered signal and improve the resolution.The majorprinciple behind all ultrasound contrast agent is an impedancemismatch between the surrounding medium (blood and soft tissue) andthe agent(gas).
As blood pool imaging agents,conventional contrast agents containmicrobubbles that can not permeate through blood vessel wall,whichlimits the research on ultrasound molecular imaging.Over recentyears,many nanoscale UCA including liposome,fluorocarbonemulsions have been developed.These nanoscale UCA represent the trend of developing highly efficient,miniaturized with sound targetingperformance in molecular imaging and therapy.
It was shown that only particles with a diameter＜700nm canpermeate through the spaces between vascular endothelial cells in suchdiseases as tumor.Nanoscale UCA can permeate through vascularendothelium into tissue spaces,thus allowing for imaging ofextravascular target tissues.Oeffinger et al.demonstrated the feasibilityof using nanoscale microbubbles to enhance the performance ofultrasound contrast.Meanwhile,as the microbubble size is reduced tonanoscale,the molecular properties of UCA change substantially,withemergence of some unique properties,such as long half-lives,highsurface reactivity,strong absorbability,and resistance to enzymaticdegradation.All these properties make possible new applications ofnanoscale UCA in medicine.
In the present study,after having successfully developed aconventional micrometer-grade UCA,we further optimized thepreparation process and method to develop the nanoscale lipid-coatedUCA through low-speed centrifugation.We prepared and preliminarilycharacterized a nanobubble.We researched it’s effect of the echointensity,and the ablation effect of high intensity focused ultrasound,inbreast VX2tumor model of rabbit.
On the other hand,the conventional ultrasonic imaging technology to real-time HIFU effect assessment mainly dependent on thegrey-scale,but this approach has many problems,such as lowsensitivity,subjective judgment.When the grey-scale is not evident,themisjudgment would be maked in HIFU ablation process,and lead toirradiation increased,treatment time increase,and unnecessarycomplications.There is an urgent need to seek a more sensitive,accurateand reliable evaluation method.Speckle refers to the irregular smallobjects formed in ultrasonic scattering.Researchers think speckle isalso a kind of information carrier,the speckle measurement techniquehas been born.Recently study shows that,the ultrasonic specklecorrelation function,the amura markings analysis based on wavelettransform,multi-resolution analysis index can be used to judge whetherthe target area of HIFU irradiation have coagulative necrosis,and betterthan the grey-scale values evaluation,is expected to become the newHIFU real-time effect assessment method.
This subject was studied mainly from the following four parts.
Objective:The purpose of the study is to prepare a nanobubble and toinvestigate it’s characterization.Methods:Lipids(DSPC,DPPC,DPPA)and other constituents were mixed at appropriate ratios and prepared into a suspension.A1-ml suspension was sub-packaged into penicillinampules(actual volume3.5ml) and lyophilized using an optimizedprocess to enhance the tightness of lipid coating.The lipid bubbles wereself made,which was awarded by National Invention Patent of China in2005and widely applied in experimental studies in contrast imaging inliver,heart and kidney in animal study.1ml of the hydration solutioncontaining hyperosmotic sugar and surfactants was added into thelyophilized preparation,and then C3F8was slowly infused into thepenicillin ampule to replace air in the ampule top,followed by90s ofhorizontal mechanical vibration[working frequency≥4500vibrationsper minute,vibrationamplitude:(15±1)mm].The prepared microbubbleswere separated by low-speed centrifugation(300rpm,3min) into twofractions.The microbubbles in the upper fraction were larger than thosein the lower fraction.The upper fraction was discarded,and themicrobubbles in the lower fraction were further analyzed.Thedistribution and appearance of the microbubble were observed under alight microscope and a electron microscope.The diameter and the zetapotential of the nanobubble were measured using a Zeta SIZIER3000electric potential analyzer.HER2antibody had been mixed withmicrobubble.Results:Macroscopic observation,the suspension of themicrobubble was white,milky,viscose,and opaque,with no obviousfoams on the surface.Microscopic observation,the microbubble was found to be round,distribute evenly,and did not aggregate in normalsaline.The diameter of the microbubble was623.4nm on average(278.8nm-697.0nm),and the zeta potential was1.3mV on average(3.2mV-+9.5mV).Microbubble can be binding with antibody by staticadsorption method.Conclusions:Because perfluorocarbon-filled lipidmicrobubbles were sufficiently stable during circulating in thevasculature as blood pool agents,phospholipids were used as thematerials of forming membrane in the study.UCA injected parenterallymust be less than8μm in diameter in order to traverse the capillariesin the pulmonary bed,these UCA remain in the vasculature until theyare eliminated from the body by a variety of mechanisms.Targeting ofcells outside the capillaries requires UCA diameters＜700nm,andpreferably＜700nm to enable escape through the larger-than-usualpores that have been noted in the leaky vasculature of a tumor.UCAhave the potential to be used to directly target cells if the agent can bemade small enough to pass through the vessels and be modified toattach to specific sites in the targeted cells.We have demonstrated thatthe stable nanobubble can be prepared by machine vibration and lowspeed centrifugation.This study provides an important platform forminiaturizing and improving the targeting performance of ultrasoundcontrast agents.
Objective:AHNP mix with nanobubble.We Research the targetfunction of the AHNP-Nanobubble compound in vitro and invivo.Methods:Preparation the nanobubble,add sugar solution andsurfactant shaken,add EDC-AHNP compound that marked fluorescentin normal atmospheric temperature for30min.After the oscillation byusing the mechanical vibration tester,choose appropriate power andtime,we can get initial targeting nanobubble.Rosette formationtest,rosette forming block experiment and immunofluorescence areused for observation targeting nanobubble in vitro.Rabbits with VX2tumors were provided by Chongqing Medical University.Tumor tissueswere resected from rabbit liver under aseptic conditions and washed inphysiological saline,and the tumor tissue was excised from the tumormargin and cut into small pieces with scissors.Thirty healthy femaleNew Zealand rabbits weighing2.5–3.0kg were used in the study.Underanesthesia,the bilateral breasts tissue was injected with tumor tissuesuspension (1ml per side).The tumors developed in rabbits breasts2weeks after the injection and they measured15mm in diameter using a5-10MHz linear array transducer(LOGIQ9),and randomized intogroups.The hair over the breast was shaved,and ultrasound couplingmedium was applied.Color ultrasonograph(LOGIQ9) was used for contrast-enhanced imaging with the probe frequency of7.0MHz,themechanical index of0.13,the dynamic range of51,and the soundoutput power of5%.The parameters of time gain compensation andfocus range were adjusted to the optimum.Bolus injections of thetargeting nanobubble,or sonovue,or PBS were given through a20-gauge catheter in an ear vein at the0.2ml/kg.The tubing wasflushed with1ml saline after injection.The dynamic and staticimageswere stored for further analysis.Results:The targeting nanobubble arelight yellow color in visual observation.In light microscopy,thetargeting nanobubble are uniform distribution.There are no obviousdifference between the targeting nanobubble and nomal microbubble inZETA SIZIER3000determination.In fluorescence microscope,thetargeting nanobubble are light yellow color,nomal microbubble can notbe detected,or an sporadic weak fluorescent.Rosette formation test rateof more than80%,conjunct ratio achieved60%.The targetingnanobubble can enhance the echo intensity of rabbit VX2breasttumor,8s after injection,the echo intensity of vessels was enhanced,and15s after injection,the echo intensity of parenchyma wasenhanced.Significant enhancement was still observed after15min.11safter injection of Sonovue,the echo intensity of vessels wasenhanced,and significant enhancement was still observed after20min.PBS group have not enhance effect.Conclusions:The penetrating potential of nanoscale UCA is stronger than that of micrometer-grade.The targeting nanobubble are prepared successfully,which havestrongly targeted and stability in vitro,and have better targeted contrastimaging performance in vivo.We should further enhance the imagingperformance, stability,and antibody binding of this nanobubble.
Objective:Breast tumor is one of the most common malignancies inwomen.Surgical treatments including radical mastectomy,modifiedradical mastectomy and breast conservation are invasive.HIFU is aminimally invasive treatment tool for tumor.HIFU can penetrate theskin and focus low energy ultrasonic waves onto the target tumorarea,causing coagulation necrosis of the tumor tissue by means oftransient high temperature (above65℃),cavitation and mechanicaleffects,without obvious injury to the adjacent normal tissues.Thepotential advantage of therapeutic HIFU is not only to provide aminimally invasive treatment for malignant tumors,but also to sparebreast tissue.Ultrasonic wave energy attenuates with increasingtransmission distance in tissues.Ultrasound energy is unlikely toaccumulate in the target tissue due to uniform tissue texture andinsignificant differences in acoustic resistance between adjacent tissue interfaces.For achieve tumor ablation,large doses and long time periodsare usually employed in HIFU,increasing the risk of complicationssuch as burns.In order to increase the efficiency of HIFU and reducethe complications,researchers have attempted to change the intrinsicacoustic properties for increasing ultrasound energy accumulation inthe target tissues by changing their structure,density,bloodperfusion,and functional status(remodeling the acoustic environment oftissue,RAET).One way to mitigate the disadvantage of HIFU might beto use UCA.UCA have been applied in conventional ultrasoundimaging not only for differentiating malignant tumors from benignones,but also for improving the detection of small ill-defined tumors onconventional grayscale imaging.UCA are important potentiators forincreasing the tissue coagulation range following HIFU.They may alsoserve as ultrasound cavitation cores,which may increase tissueabsorption of ultrasonic wave energy and rise tissue temperature.UCAmay enhance the thermal effect and cavitation of HIFU,while reducingthe tissue injury threshold to HIFU.To further investigate the propertiesof nanobubble,we evaluated the effectiveness of combiningnanobubble with HIFU on the treatment of breast cancer VX2tumorsin rabbits with ablation.Methods:Eighty female rabbits with10mmVX2breast tumor(per side),weighing2.5–3.0kg,were used in thestudy,randomized into groups.The JC-200focused ultrasound system(Haifu Technology) was used for tumor treatment.Underanesthesia,the breast tumor rabbit was immobilized on the treatmentcouch.Tumors were located,PBS(1ml) or nanobubble(0.2ml/kg) wereinjected into the ear rim vein10s prior to HIFU irradiation.The focusof the treatment system was within the tumor,and the ultrasoundimages were acquired immediately following HIFU irradiations withdifferent settings (150W for3s,150W for5s,120W for3s,120W for5s and100W for5s).Images demonstrating changes of the grayscale inthe target area before and after HIFU irradiation were stored in thecomputer for further analysis.Pathologic examination after2days.Statistical analysis data of grayscale value and area ratio ofnecrosis.Results:The increase of grayscale value of ultrasoundimaging is the result of HIFU exposure induced cavitation and bubbleformation,which causes a rapid temperature rise in tissues andstructural changes in coagulative necrotic tissues.The grayscale changein the target area was significantly higher in the HIFU+NB group thanthe HIFU+PBS group after5s irradiation (120W or150W)(p<0.01),butthere was no significant difference between both groups after5s at100W irradiation.Macroscopic observation,the necrotic area wassignificantly larger in the HIFU+NB group than in the HIFU+PBSgroup(5s,150W).HE staining showed the area of coagulation necrosiswas significantly larger in the HIFU+NB group than the HIFU+PBS group(p<0.001).These findings demonstrated that nanobubbleincreased the HIFU effectiveness.In the HIFU+NB group,the area ofdamage after irradiation at150W was larger than after irradiation at120W,given the same irradiation time.The area of damage after HIFUfor5s was larger than that after HIFU for3s,given the same irradiationpower.These findings suggested that the area of coagulation necrosiswas positively correlated with the dose and duration of HIFUirradiation.Conclusions:Poliachik et al. found that microbubbles serveas cavitation cores in blood and reduce the cavitation threshold, thuspromoting cavitation.Bailey et al.found that microbubbles affected themorphology of the HIFU treated area in vivo experiments involving ahydrostatic pressure chamber.Yu et al.conducted HIFU treatment inrabbit kidneys and found that its efficacy was significantly enhanced inthe microbubble group when compared to the control group; the tissuenecrosis rate increased by a factor of3.1–3.4and no viable tissues wereidentified pathologically in the focused area in either group.Sokka etal.performed an in vitro study to investigate the thermal effect offocused ultrasound plus microbubbles in rabbits,and found thatmicrobubbles significantly accelerated tissue heating and expanded thearea of tissue damage by a factor of2–3.In the present study,it wasdemonstrated that HIFU-induced lesions were larger in tumors thatcontained nanobubble than in those that contained PBS.The area of tissue damage in VX2breast tumors in rabbits was increased,and thestrength and time of HIFU irradiation were reduced significantly in theHIFU+NB group.Therefore,the nanobubble could function as anenhancer of HIFU ablation.During HIFU treatment,real-timeevaluation of the grayscale value determines whether coagulativenecrosis has occurred.When the grayscale value reaches a certainvalue,it represents coagulative necrosis.We analyzed and calculated thedifference in the grayscale values between pre-HIFU and post-HIFUexposure.As a result, the difference in grayscale value in theHIFU+NB group was higher than in the HIFU+PBS group.Thissuggested that the effectiveness of treatment by means of the size anddegree of coagulative necrosis was significantly different between theHIFU+NB group and the HIFU+PBS group.Therefore,the utilization ofreal-time ultrasound to guide,locate, monitor and evaluate therapeuticefficacy of HIFU with nanobubble should be encouraged.Themechanism of the heating effect produced by microbubbles andnanobubbles presenting in the ultrasound field remains unclear.Twopossible factors are: Heating by oscillation and/or explosion ofmicrobubble contrast agents exposed to HIFU,and cavitation bubblesgenerated by HIFU exposure.However,in terms of generatingheat,which one plays more important role than the other betweencavitation and microbubble oscillation is still unknown.To understand the interactions between ultrasound and contrast agents,it is importantto compare the effectiveness of microbubbles with nanobubbles inHIFU treatment.Consequently,our study suggested that a nanoscaleultrasound contrast agents appears to be a useful enhancer not only forincreasing the detection of tumors,but also for improving theeffectiveness of treatment of tumors with HIFU.
Objective:Texture analysis and correlation analysis are used to judgecoagulative necrosis after HIFU irradiation with nanobubble,andcompare with grey-scale values.Methods:One hundred and twentyhealthy female VX2breast tumor rabbits,weighing2.5–3.0kg,wereused in the study,randomized into groups.The JC-200focusedultrasound system was used for tumor treatment.Images demonstratingchanges of the grayscale in the target area before and after HIFU withnanobubble irradiation were stored in the computer for furtheranalysis.In Matlab software,extract four texture feature parametersfrom the wavelet ultrasonographic(kurtosis,skewness,mean,variance)as a Texture1.Another Tamura texture feature parameters extraction ofcoarseness,contrast and directionality,as a Texture2,and the correlation of the target area was computed.Establish decision surfaces by meansof support vector machine(SVM),and analyzing samples.Histology andmicroscopic examination were harvested2days after HIFU.Statisticalanalysis,compare with the gray scale.Results:Texture analysis ofcoagulative necrosis judgment,accuracy and sensitivity are better thangray evaluation(P<0.05).Accuracy rate of Correlation analysisis higherthan the accuracy rate of gray scale(P<0.05).Conclusions:Theaccuracy and sensitivity judgment of coagulation necrosis by means ofwavelet transformation texture analysis is higher than grey scaleevaluation.Correlation analysis data can be used to predict coagulationnecrosis of target tissues after HIFU irradiation with nanobubble,it ismore exact and sensitive than traditional gray-scale evaluation method.The next step,more samples should be use to confirmed these results.
分子影像技术的关键在于分子探针（molecular probe）的选择，超声造影剂（Ultrasound contrast agent，UCA）作为新型的超声分子影像探针，是目前研究的热点，UCA是一种稳定的含气泡的物质，临床常用于发现微小病变以及区分良、恶性肿瘤等方面，其原理是改变声速（sonic speed）、声衰减系数（attenuationcoefficient）、增强后散射（backscattering）等，利用组织与气泡之间产生的声阻抗（acoustic impedance）差，增加组织与血流之间的回声信号，提高组织血流灌注的超声显像分辨率，从而提高超声诊断的特异性、灵敏度。
传统的的UCA属血池显像剂（blood pool imaging agent），一定程度上影响了超声分子成像及治疗的发展，近年纳米级造影剂（nanoscale contrast agent）不断涌现，纳米级UCA具有很强的穿透力，能穿过血管壁，直达靶区细胞，其开发和应用有利于发展靶向性、高效性、小型化且具有辅助治疗作用的新型UCA。
超声造影（contrast enhanced ultrasound，CEUS）技术为乳腺癌等的诊断、鉴别等提供了有力的帮助，研究表明，常用的CEUS属于非特异性显影，UCA对组织没有特异性结合力，仅能观察感兴趣区血管短暂显影（动脉相），不能在特定组织内长时间驻留，影响了诊断率；近年特异性CEUS成为研究的热点，其核心内容是在微泡表面连接特异性抗体，利用其与靶区相应受体特异性结合的特点，使微泡选择性浓聚于靶区而产生特异性增强显影，导向分子和UCA的小型化是该技术的关键；近来兴起的纳米抗体(nanobody)及小分子抗体模拟肽（peptide mimics），具有与抗原特异性结合的特性，可以克服传统单克隆抗体分子量过大、穿透力较弱等缺点，易于大量生产；靶向性的纳米级UCA聚集于特定区域时产生显影，未聚集则不产生显影，有利于提高诊断的准确性。
高强度聚焦超声(High intensity focused ultrasound，HIFU）在治疗恶性肿瘤方面取得了很大进展，临床HIFU术中，存在辐照剂量较大、辐照时间较长等问题，易引起并发症，学者们提出改变组织结构、密度、血供、功能等组织声环境（AET）,以加速声能沉积,提高HIFU疗效,减少并发症；UCA可以提高HIFU疗效，其机理可能是：①增强空化声孔(sonoporation)效应，②升高靶区温度，③影响细胞增殖，④毛细血管破坏；常用的UCA粒径较大，不能透过血管内皮间隙直达肿瘤细胞，并且，声通道组织中的微泡过大过多，可导致聚焦超声不能聚焦或焦点偏移等问题，纳米级微泡（nanobubble，NB）有望弥补常规微泡的弱点，实现肿瘤的血管外显影和治疗。
我们在成功研制微米级微泡的基础上，优化制备工艺研制NB，对分析其基本性质，在兔VX2乳腺肿瘤模型上，研究其增强实质显像的效果，以及作为增效剂在增强HIFU消融效果中的作用。
另一方面，常规超声技术对HIFU实时疗效评价主要依赖于灰阶（grey scale）值变化，这种方法存在灵敏度较差、依赖操作医生主观判断等问题，灰阶值变化不明显时，易发生误判，导致辐照剂量增大、治疗时间增多，引起不必要的并发症，目前急需寻找一种更为敏感、准确、可靠的评价方法；以前，超声散斑（speckle）被认为是一种干扰信号而在超声成像时被计算机自动过滤清除，近来，人们开始认识到散斑也是一种信息载体，从而促进了散斑计量术的诞生，有研究表明，声像图散斑图像的相关函数（correlation）、基于小波变换（wavelet transform）的Tamura斑纹分析（texture analysis）、多分辨分析（multi-resolutionanalysis）等可用于判断HIFU辐照的靶区是否发生凝固性坏死（Coagulative Necrosis，CN），并优于灰阶值评价，有望成为新的HIFU实时疗效评价方式。
综上，本课题研究主要包括以下四个部分：
目的：在成功研制微米级微泡的基础上，优化制备工艺研制NB，对其基本性质进行初步的研究，以便于下一步应用。方法：应用优化的工艺制备纳米微泡，二棕榈酰磷脂酰胆碱（DPPC）、二棕榈酰磷脂酸（DPPA）等及辅助成分，按比例混合（脂质微泡获国家专利，可广泛应用于动物实验中，增强肝脏，心脏、肾脏等脏器的成像），超纯水将上述成分充分混悬均匀，经一定工艺制成混悬液，分装于锥形瓶中，使用优化的工艺冻干混悬液，以提高脂质膜的密封性，加入水合液（自制）重新水合，注入八氟丙烷（C3F8）气体，水平往复式机械振荡，频率≧4500次/分，振幅:(15士1) mm，振荡约90s左右，低速离心法对已制备的原始微泡分级分离，300RPM离心3分钟后，微泡悬浮液分为上下两部分，上层粒度较大的微泡被丢弃，留取下层粒度较小的微泡进行下一步实验。生理盐水稀释50倍后，进行肉眼观察、光镜观察、电镜观察、荧光显微镜观察，Zeta Sizier3000测定微泡的粒径、表面电位。结果：自制的微泡，肉眼观察呈白色凝乳状，封装于西林瓶中，振荡前为清亮的半透明液体，振荡后呈白色凝乳状的混悬液，表面未见泡沫，黏稠，不透明；光镜下观察，微泡呈类圆形，数量较多，分布均，无聚集，偶见较大的脂质体小颗粒；电镜下，微泡粒径不足1μm；粒径测试结果均值623.4nm，分布于278.8nm～697.0nm之间，表面电位均值1.3mV，分
布于-3.2mV～+9.5mV之间。结论：各种成膜材料中，脂质（lipids）的弹性较好、对温度要求不高、毒副作用少、无免疫原性，在本实验中，我们以脂质为成膜成分，在人体安全剂量条件下，改进各类脂质及辅助成分配方比例，加入C3F8（惰性气体，可安全地经肺排除）；考虑到机械振荡法较常用的声振法产生的热量小、对脂质体等成分活性破坏少、产泡量高、污染小等特点，我们采用合适的振荡频率和时间机械振荡制备微泡；采用优化后的冻干工艺（合适的预冻温度、冻干时间、冻干速度、真空压力等）及灭菌方式，最后与低速离心法相结合，得到NB，肉眼及镜下观察微泡呈类圆形，数量较多，分布较均匀，无聚集，稳定性较好，理论上可以通过肿瘤组织的血管内皮间隙。
目的：在制备NB的基础上，选择小分子多肽AHNP(序列为：FCDGFYACYMDV，荧光标记)与NB结合，研究其体外寻靶作用，在兔VX2乳腺肿瘤模型上，研究NB增强实质显像效果。方法：①活性剂与NB混匀，加入激活剂碳化二亚胺（EDC）与AHNP混合，室温反应30min，机械振荡后静置片刻，去下清液，缓冲液（PBS）冲洗，得到靶向NB。花环形成实验、花环形成阻断实验、免疫荧光染色观察靶向NB体外寻靶效果。②健康纯种新西兰白兔（famale）30只，麻醉后将提前制备好的VX2肿瘤组织悬液注入双侧乳腺组织各1ml，两周后经logiq9彩色多普勒超声诊断仪检查，选取乳
腺区肿瘤组织直径大小约15mm左右的兔24只进行实验，实验兔麻醉后，随机分组为靶向NB组10只，声诺维（Sonovue）组10只，PBS组4只，耳缘静脉分别注射靶向NB、Sonovue、PBS，Logiq9超声诊断仪记录并观察体内增强显像效果。结果：标记荧光的携AHNP的NB，呈浅黄色，光镜下，微泡均匀细密分布，检测微泡的粒径与非靶向微泡相比稍大，荧光显微镜下观察，靶向NB见绿色荧光，非靶向微泡偶见零星的微弱荧光，花环形成率达80%以上，特异性结合靶向微泡的细胞阳性率约60%；实验兔超声显示乳腺肿瘤边界清楚，截面呈椭圆形，内部低回声，彩色多普勒显示肿瘤内部血供丰富，频谱多普勒显示为动脉频谱，实验兔注射靶向NB后，平均8s（5s～12s）出现血管增强显像，注射后平均15s（9s～20s），实质回声增强，增强效果最长持续时间15min左右，Sonovue组注射后平均11s（7s～19s）出现增强显像，最长持续时间20min左右，PBS组无增强效应。结论：AHNP为抗p185her2/neu的特异性单克隆抗体CDR-H3环的模拟多肽，与HER2具有抗体样的特异性结合能力，AHNP与NB结合，有较强的体外寻靶效果，在兔VX2乳腺肿瘤模型中，靶向NB能达到增强肿瘤实质显像的效果，实验中及实验后，各组实验兔均无不良反应，亦证实了自制NB的安全性。
目的：在HIFU消融兔VX2乳腺肿瘤的过程中，联合使用自制的NB，观察其在不同辐照声功率、不同辐照时间条件下，对HIFU消融的增效作用，探讨NB在超声治疗中的应用价值。方法：携VX2乳腺肿瘤兔52只，随机分组为HIFU+NB组26只，HIFU+PBS组26只，脱毛，0.15mg/Kg剂量的“速眠新”麻醉，经耳缘静脉通道推注2.5%戊巴比妥钠（25mg/kg）持续麻醉，固定，启动聚焦超声肿瘤治疗系统，定位，辐照前10s，经耳缘静脉通道注射PBS（0.2ml/kg）或NB（0.2ml/kg），继而生理盐水1ml推注，调整焦点使其位于肿瘤内部，调整辐照声功率（150W，120W，100W）及时间（3s，5s）行HIFU辐照，系统自动分析辐照前后靶区超声灰阶变化值。HIFU辐照后1天，做病理检查，统计数据并分析。结果：HIFU治疗前超声显示肿瘤边界清楚，截面呈椭圆形，内部低回声，彩色多普勒显示肿瘤内部血供丰富，频谱多普勒显示为动脉频谱，HIFU治疗后靶区血供基本消失，偶见靶区周边点状血供；HIFU治疗前肿瘤造影显示为增强显像，HIFU治疗后肿瘤造影显示为靶区无明显增强显像，靶区周边增强显像，形成所谓“负显影”效果；HIFU治疗中及治疗后，实验兔无明显不良反应，1例实验兔HIFU+NB治疗两周后，乳腺肿块完全消失（考虑是否与HIFU治疗后促进了肿瘤细胞凋亡等有关）；HIFU辐照后靶区灰度出现增强，5s时间条件下，不同辐照声功率（120W、150W），HIFU+NB组靶区辐照后平均灰阶变化明显高于HIFU+PBS组，统计学分析有明显差异（P<0.01），但在100W、5s条件下，HIFU+NB组靶区辐照后平均灰阶差变化与HIFU+PBS组相比较，无明显差别，HIFU+NB组内分析，发现在150W和120W条件下，5s辐照组的平均灰阶差均明显高于
3s辐照组（P<0.05）；150W、5s同等条件下HIFU辐照后，肉眼观察辐照区呈灰白色灶,坏死灶分界清，HIFU+NB组坏死范围明显大于HIFU+PBS组；HE染色结果显示，HIFU+NB组与HIFU+PBS组的靶区组织内均见明确的凝固性坏死，HIFU+NB组发生凝固性坏死范围明显大于HIFU+PBS组（P<0.001），HIFU+NB组内对比分析，在同等辐照时间下，150W辐照声功率较120W辐照声功率损伤范围增加，在同等辐照声功率下，5s辐照时间较3s辐照时间损伤范围增加，120W、5s辐照条件与150W、3s辐照条件造成的的损伤效果基本一致。结论：在HIFU消融过程中，NB可以利用“负显影”效果，判断HIFU有效治疗区域；在同等辐照声功率、辐照时间条件下，HIFU+NB组较HIFU+PBS组，能有效提高辐照后灰阶变化，明显增大辐照后凝固性坏死的范围，提示NB能减少辐照强度，缩短辐照时间,达到增强HIFU损伤效果的目的。NB可同时应用诊断和治疗领域，其具体作用机制（如空化效应、声孔效应）以及辐照后远期致细胞凋亡效应等还有待进一步深入研究。
目的：采用纹理分析、相关函数评价HIFU+NB增强乳腺肿瘤消融的效果，寻求比灰度变化更有效的实时判断HIFU疗效的方法。方法：携VX2乳腺肿瘤兔60只，随机分组为HIFU+NB组30只，HIFU+PBS组30只，麻醉固定后，JC-200聚焦超声肿瘤治疗系统消融，辐照前10s，经耳缘静脉通道注射PBS（0.2ml/kg）或NB（0.2ml/kg），继而生理盐水1ml推注，采集辐照前后图像；病理检查；Matlab截取感兴趣去（ROI），进行两次小波变换，提取纹理分析1和2等参数；Matla软件截取辐照前后靶区声像图，像素大小30*30，计算辐照后即刻与辐照前靶区声像图的相关函数最大值；分析得出的结果录入WEKA3.7，采用支撑适量机（SVM）建立决策平面，分析样本；统计数据。结果：HIFU+PBS组，纹理分析1和2判断CN特异度与敏感度与灰阶判断有统计学差异，各组P＜0.05。HIFU+NB组，纹理分析1和2判断CN特异度与敏感度与灰阶判断有统计学差异，各组P＜0.05。HIFU+NB组与HIFU+PBS组辐照前后声像图应用相关函数对CN的判断灵敏度高于灰阶判断（P<0.05）。应用相关函数总判对率高于灰阶(P<0.05)。结论:初步证实纹理分析与灰阶评价相比，在判断CN的灵敏度及特异度上，更有优越性。而相同辐照条件下，相关函数分析对CN的总判对率高于灰阶。
With the development of molecular biology and molecularimaging,ultrasound molecular imaging has been advanced rapidly.Thekey is the molecular probe.Ultrasound contrast agent(UCA) as a newmolecular imaging ultrasound probe,is the hot spot.UCA have beenused to increase the sensitivity and specificity of the detection ofdisease due to the increase of signal reflection from blood flow,it canenhance the backscattered signal and improve the resolution.The majorprinciple behind all ultrasound contrast agent is an impedancemismatch between the surrounding medium (blood and soft tissue) andthe agent(gas).
As blood pool imaging agents,conventional contrast agents containmicrobubbles that can not permeate through blood vessel wall,whichlimits the research on ultrasound molecular imaging.Over recentyears,many nanoscale UCA including liposome,fluorocarbonemulsions have been developed.These nanoscale UCA represent the trend of developing highly efficient,miniaturized with sound targetingperformance in molecular imaging and therapy.
It was shown that only particles with a diameter＜700nm canpermeate through the spaces between vascular endothelial cells in suchdiseases as tumor.Nanoscale UCA can permeate through vascularendothelium into tissue spaces,thus allowing for imaging ofextravascular target tissues.Oeffinger et al.demonstrated the feasibilityof using nanoscale microbubbles to enhance the performance ofultrasound contrast.Meanwhile,as the microbubble size is reduced tonanoscale,the molecular properties of UCA change substantially,withemergence of some unique properties,such as long half-lives,highsurface reactivity,strong absorbability,and resistance to enzymaticdegradation.All these properties make possible new applications ofnanoscale UCA in medicine.
In the present study,after having successfully developed aconventional micrometer-grade UCA,we further optimized thepreparation process and method to develop the nanoscale lipid-coatedUCA through low-speed centrifugation.We prepared and preliminarilycharacterized a nanobubble.We researched it’s effect of the echointensity,and the ablation effect of high intensity focused ultrasound,inbreast VX2tumor model of rabbit.
On the other hand,the conventional ultrasonic imaging technology to real-time HIFU effect assessment mainly dependent on thegrey-scale,but this approach has many problems,such as lowsensitivity,subjective judgment.When the grey-scale is not evident,themisjudgment would be maked in HIFU ablation process,and lead toirradiation increased,treatment time increase,and unnecessarycomplications.There is an urgent need to seek a more sensitive,accurateand reliable evaluation method.Speckle refers to the irregular smallobjects formed in ultrasonic scattering.Researchers think speckle isalso a kind of information carrier,the speckle measurement techniquehas been born.Recently study shows that,the ultrasonic specklecorrelation function,the amura markings analysis based on wavelettransform,multi-resolution analysis index can be used to judge whetherthe target area of HIFU irradiation have coagulative necrosis,and betterthan the grey-scale values evaluation,is expected to become the newHIFU real-time effect assessment method.
This subject was studied mainly from the following four parts.
Objective:The purpose of the study is to prepare a nanobubble and toinvestigate it’s characterization.Methods:Lipids(DSPC,DPPC,DPPA)and other constituents were mixed at appropriate ratios and prepared into a suspension.A1-ml suspension was sub-packaged into penicillinampules(actual volume3.5ml) and lyophilized using an optimizedprocess to enhance the tightness of lipid coating.The lipid bubbles wereself made,which was awarded by National Invention Patent of China in2005and widely applied in experimental studies in contrast imaging inliver,heart and kidney in animal study.1ml of the hydration solutioncontaining hyperosmotic sugar and surfactants was added into thelyophilized preparation,and then C3F8was slowly infused into thepenicillin ampule to replace air in the ampule top,followed by90s ofhorizontal mechanical vibration[working frequency≥4500vibrationsper minute,vibrationamplitude:(15±1)mm].The prepared microbubbleswere separated by low-speed centrifugation(300rpm,3min) into twofractions.The microbubbles in the upper fraction were larger than thosein the lower fraction.The upper fraction was discarded,and themicrobubbles in the lower fraction were further analyzed.Thedistribution and appearance of the microbubble were observed under alight microscope and a electron microscope.The diameter and the zetapotential of the nanobubble were measured using a Zeta SIZIER3000electric potential analyzer.HER2antibody had been mixed withmicrobubble.Results:Macroscopic observation,the suspension of themicrobubble was white,milky,viscose,and opaque,with no obviousfoams on the surface.Microscopic observation,the microbubble was found to be round,distribute evenly,and did not aggregate in normalsaline.The diameter of the microbubble was623.4nm on average(278.8nm-697.0nm),and the zeta potential was1.3mV on average(3.2mV-+9.5mV).Microbubble can be binding with antibody by staticadsorption method.Conclusions:Because perfluorocarbon-filled lipidmicrobubbles were sufficiently stable during circulating in thevasculature as blood pool agents,phospholipids were used as thematerials of forming membrane in the study.UCA injected parenterallymust be less than8μm in diameter in order to traverse the capillariesin the pulmonary bed,these UCA remain in the vasculature until theyare eliminated from the body by a variety of mechanisms.Targeting ofcells outside the capillaries requires UCA diameters＜700nm,andpreferably＜700nm to enable escape through the larger-than-usualpores that have been noted in the leaky vasculature of a tumor.UCAhave the potential to be used to directly target cells if the agent can bemade small enough to pass through the vessels and be modified toattach to specific sites in the targeted cells.We have demonstrated thatthe stable nanobubble can be prepared by machine vibration and lowspeed centrifugation.This study provides an important platform forminiaturizing and improving the targeting performance of ultrasoundcontrast agents.
Objective:AHNP mix with nanobubble.We Research the targetfunction of the AHNP-Nanobubble compound in vitro and invivo.Methods:Preparation the nanobubble,add sugar solution andsurfactant shaken,add EDC-AHNP compound that marked fluorescentin normal atmospheric temperature for30min.After the oscillation byusing the mechanical vibration tester,choose appropriate power andtime,we can get initial targeting nanobubble.Rosette formationtest,rosette forming block experiment and immunofluorescence areused for observation targeting nanobubble in vitro.Rabbits with VX2tumors were provided by Chongqing Medical University.Tumor tissueswere resected from rabbit liver under aseptic conditions and washed inphysiological saline,and the tumor tissue was excised from the tumormargin and cut into small pieces with scissors.Thirty healthy femaleNew Zealand rabbits weighing2.5–3.0kg were used in the study.Underanesthesia,the bilateral breasts tissue was injected with tumor tissuesuspension (1ml per side).The tumors developed in rabbits breasts2weeks after the injection and they measured15mm in diameter using a5-10MHz linear array transducer(LOGIQ9),and randomized intogroups.The hair over the breast was shaved,and ultrasound couplingmedium was applied.Color ultrasonograph(LOGIQ9) was used for contrast-enhanced imaging with the probe frequency of7.0MHz,themechanical index of0.13,the dynamic range of51,and the soundoutput power of5%.The parameters of time gain compensation andfocus range were adjusted to the optimum.Bolus injections of thetargeting nanobubble,or sonovue,or PBS were given through a20-gauge catheter in an ear vein at the0.2ml/kg.The tubing wasflushed with1ml saline after injection.The dynamic and staticimageswere stored for further analysis.Results:The targeting nanobubble arelight yellow color in visual observation.In light microscopy,thetargeting nanobubble are uniform distribution.There are no obviousdifference between the targeting nanobubble and nomal microbubble inZETA SIZIER3000determination.In fluorescence microscope,thetargeting nanobubble are light yellow color,nomal microbubble can notbe detected,or an sporadic weak fluorescent.Rosette formation test rateof more than80%,conjunct ratio achieved60%.The targetingnanobubble can enhance the echo intensity of rabbit VX2breasttumor,8s after injection,the echo intensity of vessels was enhanced,and15s after injection,the echo intensity of parenchyma wasenhanced.Significant enhancement was still observed after15min.11safter injection of Sonovue,the echo intensity of vessels wasenhanced,and significant enhancement was still observed after20min.PBS group have not enhance effect.Conclusions:The penetrating potential of nanoscale UCA is stronger than that of micrometer-grade.The targeting nanobubble are prepared successfully,which havestrongly targeted and stability in vitro,and have better targeted contrastimaging performance in vivo.We should further enhance the imagingperformance, stability,and antibody binding of this nanobubble.
Objective:Breast tumor is one of the most common malignancies inwomen.Surgical treatments including radical mastectomy,modifiedradical mastectomy and breast conservation are invasive.HIFU is aminimally invasive treatment tool for tumor.HIFU can penetrate theskin and focus low energy ultrasonic waves onto the target tumorarea,causing coagulation necrosis of the tumor tissue by means oftransient high temperature (above65℃),cavitation and mechanicaleffects,without obvious injury to the adjacent normal tissues.Thepotential advantage of therapeutic HIFU is not only to provide aminimally invasive treatment for malignant tumors,but also to sparebreast tissue.Ultrasonic wave energy attenuates with increasingtransmission distance in tissues.Ultrasound energy is unlikely toaccumulate in the target tissue due to uniform tissue texture andinsignificant differences in acoustic resistance between adjacent tissue interfaces.For achieve tumor ablation,large doses and long time periodsare usually employed in HIFU,increasing the risk of complicationssuch as burns.In order to increase the efficiency of HIFU and reducethe complications,researchers have attempted to change the intrinsicacoustic properties for increasing ultrasound energy accumulation inthe target tissues by changing their structure,density,bloodperfusion,and functional status(remodeling the acoustic environment oftissue,RAET).One way to mitigate the disadvantage of HIFU might beto use UCA.UCA have been applied in conventional ultrasoundimaging not only for differentiating malignant tumors from benignones,but also for improving the detection of small ill-defined tumors onconventional grayscale imaging.UCA are important potentiators forincreasing the tissue coagulation range following HIFU.They may alsoserve as ultrasound cavitation cores,which may increase tissueabsorption of ultrasonic wave energy and rise tissue temperature.UCAmay enhance the thermal effect and cavitation of HIFU,while reducingthe tissue injury threshold to HIFU.To further investigate the propertiesof nanobubble,we evaluated the effectiveness of combiningnanobubble with HIFU on the treatment of breast cancer VX2tumorsin rabbits with ablation.Methods:Eighty female rabbits with10mmVX2breast tumor(per side),weighing2.5–3.0kg,were used in thestudy,randomized into groups.The JC-200focused ultrasound system(Haifu Technology) was used for tumor treatment.Underanesthesia,the breast tumor rabbit was immobilized on the treatmentcouch.Tumors were located,PBS(1ml) or nanobubble(0.2ml/kg) wereinjected into the ear rim vein10s prior to HIFU irradiation.The focusof the treatment system was within the tumor,and the ultrasoundimages were acquired immediately following HIFU irradiations withdifferent settings (150W for3s,150W for5s,120W for3s,120W for5s and100W for5s).Images demonstrating changes of the grayscale inthe target area before and after HIFU irradiation were stored in thecomputer for further analysis.Pathologic examination after2days.Statistical analysis data of grayscale value and area ratio ofnecrosis.Results:The increase of grayscale value of ultrasoundimaging is the result of HIFU exposure induced cavitation and bubbleformation,which causes a rapid temperature rise in tissues andstructural changes in coagulative necrotic tissues.The grayscale changein the target area was significantly higher in the HIFU+NB group thanthe HIFU+PBS group after5s irradiation (120W or150W)(p<0.01),butthere was no significant difference between both groups after5s at100W irradiation.Macroscopic observation,the necrotic area wassignificantly larger in the HIFU+NB group than in the HIFU+PBSgroup(5s,150W).HE staining showed the area of coagulation necrosiswas significantly larger in the HIFU+NB group than the HIFU+PBS group(p<0.001).These findings demonstrated that nanobubbleincreased the HIFU effectiveness.In the HIFU+NB group,the area ofdamage after irradiation at150W was larger than after irradiation at120W,given the same irradiation time.The area of damage after HIFUfor5s was larger than that after HIFU for3s,given the same irradiationpower.These findings suggested that the area of coagulation necrosiswas positively correlated with the dose and duration of HIFUirradiation.Conclusions:Poliachik et al. found that microbubbles serveas cavitation cores in blood and reduce the cavitation threshold, thuspromoting cavitation.Bailey et al.found that microbubbles affected themorphology of the HIFU treated area in vivo experiments involving ahydrostatic pressure chamber.Yu et al.conducted HIFU treatment inrabbit kidneys and found that its efficacy was significantly enhanced inthe microbubble group when compared to the control group; the tissuenecrosis rate increased by a factor of3.1–3.4and no viable tissues wereidentified pathologically in the focused area in either group.Sokka etal.performed an in vitro study to investigate the thermal effect offocused ultrasound plus microbubbles in rabbits,and found thatmicrobubbles significantly accelerated tissue heating and expanded thearea of tissue damage by a factor of2–3.In the present study,it wasdemonstrated that HIFU-induced lesions were larger in tumors thatcontained nanobubble than in those that contained PBS.The area of tissue damage in VX2breast tumors in rabbits was increased,and thestrength and time of HIFU irradiation were reduced significantly in theHIFU+NB group.Therefore,the nanobubble could function as anenhancer of HIFU ablation.During HIFU treatment,real-timeevaluation of the grayscale value determines whether coagulativenecrosis has occurred.When the grayscale value reaches a certainvalue,it represents coagulative necrosis.We analyzed and calculated thedifference in the grayscale values between pre-HIFU and post-HIFUexposure.As a result, the difference in grayscale value in theHIFU+NB group was higher than in the HIFU+PBS group.Thissuggested that the effectiveness of treatment by means of the size anddegree of coagulative necrosis was significantly different between theHIFU+NB group and the HIFU+PBS group.Therefore,the utilization ofreal-time ultrasound to guide,locate, monitor and evaluate therapeuticefficacy of HIFU with nanobubble should be encouraged.Themechanism of the heating effect produced by microbubbles andnanobubbles presenting in the ultrasound field remains unclear.Twopossible factors are: Heating by oscillation and/or explosion ofmicrobubble contrast agents exposed to HIFU,and cavitation bubblesgenerated by HIFU exposure.However,in terms of generatingheat,which one plays more important role than the other betweencavitation and microbubble oscillation is still unknown.To understand the interactions between ultrasound and contrast agents,it is importantto compare the effectiveness of microbubbles with nanobubbles inHIFU treatment.Consequently,our study suggested that a nanoscaleultrasound contrast agents appears to be a useful enhancer not only forincreasing the detection of tumors,but also for improving theeffectiveness of treatment of tumors with HIFU.
Objective:Texture analysis and correlation analysis are used to judgecoagulative necrosis after HIFU irradiation with nanobubble,andcompare with grey-scale values.Methods:One hundred and twentyhealthy female VX2breast tumor rabbits,weighing2.5–3.0kg,wereused in the study,randomized into groups.The JC-200focusedultrasound system was used for tumor treatment.Images demonstratingchanges of the grayscale in the target area before and after HIFU withnanobubble irradiation were stored in the computer for furtheranalysis.In Matlab software,extract four texture feature parametersfrom the wavelet ultrasonographic(kurtosis,skewness,mean,variance)as a Texture1.Another Tamura texture feature parameters extraction ofcoarseness,contrast and directionality,as a Texture2,and the correlation of the target area was computed.Establish decision surfaces by meansof support vector machine(SVM),and analyzing samples.Histology andmicroscopic examination were harvested2days after HIFU.Statisticalanalysis,compare with the gray scale.Results:Texture analysis ofcoagulative necrosis judgment,accuracy and sensitivity are better thangray evaluation(P<0.05).Accuracy rate of Correlation analysisis higherthan the accuracy rate of gray scale(P<0.05).Conclusions:Theaccuracy and sensitivity judgment of coagulation necrosis by means ofwavelet transformation texture analysis is higher than grey scaleevaluation.Correlation analysis data can be used to predict coagulationnecrosis of target tissues after HIFU irradiation with nanobubble,it ismore exact and sensitive than traditional gray-scale evaluation method.The next step,more samples should be use to confirmed these results.
引文
[1]Voigt JU. Ultrasound molecular imaging[J].Methods.2009,48(2):92-97
[2]de Groot-de Laat LE,Krenning BJ,ten Cate FJ,et al.Usefulness of contrastechocardiography for diagnosis of left ventricular noncompaction[J].Am JCardiol2005,95(9):1131–4
[3]Longmire M,Choyke PL,Kobayashi H.Dendrimer-based contrast agents formolecular imaging[J].Curr Top Med Chem.2008,8(14):1180-1186
[4]G rg C.The forgotten organ:contrast enhanced sonography of the Spleen[J].EurJ Radiol.2007,64(2):189–201
[5]Lencioni R,Piscaglia F,Bolondi L.Contrast-enhanced ultrasound in the diagnosisof hepatocellular carcinoma[J].J Hepatol.2008,48(5):848-57
[6]Thijssen JM, de Korte CL. Modeling ultrasound contrast measurement of bloodflow and perfusion in biological tissue[J].Ultrasound MedBiol.2005,31(2):279-285
[7]王志刚.超声分子影像学研究进展[J].中国医学影像技术.2009,25:921-924
[8]Lindner JR.Contrast ultrasound molecular imaging of inflammation incardiovascular disease[J].Cardiovasc Res.2009,84(2):182–9
[9]Ferrara KW,Borden MA,Zhang H.Lipid-shelled vehicles:engineering forultrasound molecular imaging and drug delivery[J].Acc ChemRes.2009,42(7):881–92
[10]Kaufmann BA.Ultrasound molecular imaging of atherosclerosis[J].CardiovascRes.2009,83(4):617–25
[11]XingSheng Li,ZhiGang Wang,et al.Experimental research on therapeuticangiogenesis induced by Hepatocyte Growth Factor directed byultrasound-targeted microbubble destruction in rats[J].J UltrasoundMed2008.27:439-446
[12]Lin Gong,Zhi Gang Wang,Hai Tao Ren,et al.Relationship between myocardialultrasonic integrated backscatter and mitochondria of the myocardium indogs[J].Clinical Imaging.2006,30:402-408
[13]Zhang Q,Wang Z,Ran H,et al.Enhanced gene delivery into skeletal muscleswith ultrasound and microbubble techniques[J].AcadRadiol.2006,13(3):363-367
[14]Wang Zhigang,Ling Zhiyu,Ran Haitao,et al.Ultrasound-mediated microbubbledestruction enhances VEGF gene delivery to the infarcted myocardium inrats[J].Clinical Imaging.2004,28(6):395-398
[15]Dong Wang,Ke Yang,Yun-Hua Gao,et al.Preparation and characterization of ananoscale ultrasound contrast agent[J].Clinical Imaging.2010,34(4):288-292
[16]Klibanov AL．Ligand-carrying gas-filled micr0bubbles：ultrasound contrastagents for targeted molecular imaging[J].Bioconjug Chem.2005,16(1):9-17
[17]Patel RM.Microbubble: a potential ultrasound tool in molecularimaging[J].Curr Pharm Biotechnol.2008,9(5):406-410
[18]杨钰楠，高云华.靶向超声造影剂制备的方法学研究[J].中华超声影像学杂志.2006,15(1):65-67
[19]李奇林,全学模.超声微泡造影剂的临床应用及研究进展[J].国外医学临床放射学分册.2006,29(6):419-421
[20]周永昌，郭万学.超声医学(第六版)[M].北京,科学技术文献出版社.2011,78-89
[21]Lozano MM,Longo ML.Microbubbles coated with disaturated lipids andDSPE-PEG2000:phase behavior,collapse transitions,andpermeability[J].Langmuir.2009,25(6):3705–12
[22]Wheatley MA,Forsberg F,Dube N,et al. Surfactant-stabilized contrast agent onthe nanoscale for diagnostic ultrasound imaging[J]. Ultrasound MedBiol.2006,32(1):83–93.
[23]Oeffinger BE,Wheatley MA.Development and characterization of a nano-scalecontrast agent[J].Ultrasonics.2004,42:343-7
[24]王冬,杨珂,闵加艳,等.高强度聚焦超声联合纳米微泡造影剂对兔VX2乳腺移植瘤辐照效果的影响[J].中国介入影像与治疗.2012,9(3):213-216
[1]Tewari M,Krishnamurthy A,Shukla HS.Breast conservation in locally advancedbreast cancer in developing countries:wise or waste[J].Surgicaloncology.2009,18(1):3-13
[2]Lencioni R,Piscaglia F,Bolondi L.Contrast-enhanced ultrasound in the diagnosisof hepatocellular carcinoma[J].J Hepatol.2008,48(5):848-57
[3]G rg C.The forgotten organ:contrast enhanced sonography of the spleen[J].EurJ Radiol.2007,64(2):189-201
[4]de Groot-de Laat LE,Krenning BJ,ten Cate FJ,et al.Usefulness of contrastechocardiography for diagnosis of left ventricular noncompaction[J].Am JCardiol.2005,95(9):1131-4
[5]Pisani E,Tsapis N,Paris J,et al.Polymeric nano/microcapsules of liquidperfluorocarbons for ultrasonic imaging:physicalcharacterization[J].Langmuir.2006,22(9):4397-4402
[6]Hauff P,Reinhardt M,Briel A,et al.Molecular targeting of lymph nodes withL-selectin ligand-specific US contrast agent:a feasibility study in mice anddogs[J].Radiology.2004,231(3):667-673
[7]Klibanov AL.Preparation of targeted microbubbles:ultrasound contrast agentsfor molecular imaging[J].Med Biol Eng Comput.2009,47(8):875-882
[8]Toya Nath Baral,Stefan Magez,Beno t Stijlemans,et al.Experimental therapy ofAfrican trypanosomiasis with a nanobody-conjugated human trypanolyticfactor[J].Nature Medicine.2006,12(5):580-584
[9]Stijlemans B,Conrath K,Cortez-Retamozo V,et al.Efficient targeting ofconserved cryptic epitopes of infectious agents by single-domainantibodies[J].African trypanosomes as a paradigm.J BiolChem.2004,279(2):1256-1261
[10]杨珂,王冬.纳米抗体及其应用[J].分子与细胞免疫杂志.2008,24(4):425-427
[11]Zhang J,Tanha J,Hirama T,et al.Pentamerization of single-domain antibodiesfrom phage libraries:a novel strategy for the rapid generation of high-avidityantibody reagents[J].J Mol Biol.2004,335(1):49-56
[12]Hughes MS,Marsh JN,Ha1l CS,et a1.Acoustic characterization in whole bloodand plasma of site-targeted nanoparticle ultrasound contrast agent formolecular imaging[J].Acoust Soc Am.2005,117(12):1964-1972
[13]Marsh JN,Hall CS,Scott MJ,et al.Improvements in the ultrasonic contrast oftargeted perfluorocarbon nanoparticles using an acoustic transmission linemodel[J].IEEE Trans Ultrason Ferroelectr Freq Control.2002,49(1):29-38
[14]Marsh JN, Partlow KC, Abendschein DR,et al.Molecular imaging withtargeted perfluorocarbon nanoparticles:quantification of the concentrationdependence of contrast enhancement for binding to sparse cellularepitopes[J].Ultrasound Med Biol.2007,33(6):950-958
[15]《乳腺癌HER2检测指南》编写组.乳腺癌HER2检测指南[J].中华病理学杂志,2006,35(10):631-633
[16]刘扬,吕万良,张强.脂质体及纳米粒药物递送系统的研究进展[J].中国医学科学院学报,2006,28(4):583-589
[17]杨莉,谭开彬,左松,等.阴离子脂膜超声微泡的制备及评价[J].临床超声医学杂志,2006,8(4):193-195
[18]杨钰楠,高云华,谭开彬,等.携RGDS超声造影剂的制备及体外靶向血栓研究[J].第三军医大学学报,2006,6(28):562－564
[19]杨钰楠,高云华.靶向超声造影剂制备的方法学研究[J].中华超声影像学杂志,2006,15(1):65-67
[20]Dong Wang,Ke Yang,Yun-Hua Gao,et al.Preparation and characterization of ananoscale ultrasound contrast agent[J].Clinical Imaging.2010,34(4):288-292
[1]Tewari M,Krishnamurthy A,Shukla HS.Breast conservation in locally advancedbreast cancer in developing countries: wise or waste[J].Surgicaloncology.2009,18(1):3-13
[2]Melodelima D,Chapelon JY,Theillere Y,et al.Combination of thermal andcavitation effects to generate deep lesions with an endocavitary applicator usinga planet transducer: ex vivo studies[J].Ultrasound Med Biol.2004,30:103–11
[3]Zhou Q,Zhu XQ,Zhang J,et al.Changes in circulating immunosuppressivecytokine levels of cancer patients after high intensity focused ultrasoundtreatment[J].Ultrasound Med Biol.2008,34(1):81-87
[4]Garnier C,Lafon C,Dillenseger JL.3-d modeling of the thermal coagulationnecrosis induced by an interstitial ultrasonic transducer[J].IEEE Trans BiomedEng.2008,55(2):833-837
[5]Haar GT,Coussios C.High intensity focused ultrasound: physical principles anddevices[J].Int J Hyperthermia.2007,23(2):89-104
[6]Cheng SQ,Zhou XD,Tang ZY,et al. Iodized oil enhances the thermal effect ofhigh-intensity focused ultrasound on ablating experimental liver cancer[J].JCancer Res Clin Oncol.1997,123(11-12):639-644
[7]Rubio Briones J,Collado Serra A,Gómez-Ferrer Lozano A,et al. High-intensityfocused ultrasound in small renal masses[J].Adv Urol.2008:809-45
[8]Wang Z,Li F,Bai J.A study of acoustic environment in tissue of high intensityfocused ultrasound.3rd International Symposium on TherapeuticUltrasound[C].INSERM U.556,2003,6:68
[9]Zderic V,Brayman AA,Sharar SR,et al.Microbubble-enhanced hemorrhagecontrol using high intensity focusedultrasound[J].Ultrasonics.2006,45(1-4):113-120
[10]沙卫红,李瑜元,聂玉强,等.高强度聚焦超声联用超声造影剂对治疗兔肝焦域效应的影响[J].中华超声影像学杂志.2004,13(4):299-302
[11]Tung YS,Liu HL,Wu CC,et al.Contrast-agent-enhanced ultrasound thermalablation[J].Ultrasound Med Biol.2006,32(7):1103-1110
[12]Moran CM,Watson RJ,Fox KAA,et al.In vitro acoustic characterisation of fourintravenous ultrasonic contrast agents at30MHz[J].Ultrasound MedBiol.2002,28:785-91
[13]Poliachik SL,Chandler WL,Mourad PD,et al.Effect of high-intensity focusedultrasound on whole blood with and without microbubble contrastagent[J].Ultrasound Med Biol.1999,25:991-998
[14]Bailey MR,Couret LN,Sapozhnikov OA,et al.Use of overpressure to assess therole of bubbles in focused ultrasound lesion shape in vitro[J].Ultrasound MedBiol.2001,27:695-708
[15]Yu T,Wang G,Hu K,et al.A micro-bubble agent improves the therapeuticefficiency of High Intensity Focused Ultrasound:a rabbit kidneystudy[J].Urological Research.2004,32:14-19
[16]Yu T,Xiong S,Mason TJ,Wang Z.The use of a micro-bubble agent to enhancerabbit liver destruction using high intensity focused ultrasound[J].UltrasonSonochem.2006,13(2):143-149
[17]Sokka SD,King R,Hynynen K.MRI-guided gas bubble enhanced ultrasoundheating in vivo rabbit t high[J].Phys Med Biol.2003,48(2):223-241
[18]Li P,Cao LQ,Dou CY,et al.Impact of myocardial contrast echocardiographyonvascular permealbility:an in vivo dose response study of deliverymode,pressure amplitude and contrast dose[J].Ultrasound MedBiol.2003,29(9):1341-1349
[19]王冬,杨珂,闵加艳,等.高强度聚焦超声联合纳米微泡造影剂对兔VX2乳腺移植瘤辐照效果的影响[J].中国介入影像与治疗.2012,9(3):213-216
[20]Takegami K,Kaneko Y,Watanabe T,et al.Heating and coagulation volumeobtained withhigh-intensity focused ultrasound therapy:comparison ofperflutren protein-type A microspheres and MRX-133inrabbits[J].Radiology.2005,237(1):132-136
[21]Oeffinger BE,Wheatley MA.Development and characterization of a nano-scalecontrast agent[J].Ultrasonics.2004,42:343-7
[22]Lindner JR.Contrast ultrasound molecular imaging of inflammation incardiovascular disease[J].Cardiovasc Res.2009,84(2):182-9
[23]Ferrara KW,Borden MA,Zhang H.Lipid-shelled vehicles:engineering forultrasound molecular imaging and drug delivery[J].Acc ChemRes.2009,42(7):881-92
[24]Kaufmann BA.Ultrasound molecular imaging of atherosclerosis[J].CardiovascRes.2009,83(4):617-25
[25]Dong Wang,Lu Li,Jiayan Min,et al.Combination of High Intensity FocusedUltrasound with Nanoscale Ultrasound Contrast Agent in Treatment of RabbitBreast VX2Tumors:A Pilot Study[J].Clinical Imaging.2012(Accept)
[1]刘宝琴,熊树华,王智彪,等.超声对高强度聚焦超声生物学焦域的监控研究[J].中华超声影像学杂志,2002,11(11):687-689
[2]邹建中,龚晓波,贺雪梅,等.高强度聚焦超声辐照离体组织靶区无灰度变化有坏死现象的研究[J].临床超声医学杂志.2008,10(9):581-583
[3]Liu BQ,Xiong SH,Wang ZB.The study on biological focal region of highintensity focused ultrasound under ultrasonography monitoring[J].ChinUltrasonogr,2002(11):687-688
[4]李雁,邹建中,王华,等.基于二维超声图像纹理分析判断HIFU凝固性坏死[J].中国医学影像技术.2010,26(6):1147-1149
[5]冯玉洁,邹建中,王华,等.Tamura纹理分析应用于HIFU监控的离体研究[J].中国医科大学学报.2010,39(8):610-612
[6]胡昌华,张军波,夏军,等.基于MATLAB的系统分析与设计—小波分析[M].西安:西安电子科技大学出版社.1999:16-19
[7]Lee JS,Sun YN,Lin XZ.A new approach to ultrasonic liver imageclassification[J].IEICE transactions on information andsystems.2000,E83-D(6):1301-1308
[8]Arivazhagan S,Ganesan L.Texture classification using wardertransform[J].Pattern Recognition etters.2003,24(9-10):1513-1521
[9]闵加艳,邹建中,郭宇,等.基于小波变换的高强度聚焦超声消融兔VX2乳腺肿瘤声像图纹理分析[J].中国医科大学学报.2012,41(1):14-17
[10]Hsu C,Lin C.A comparison of methods for multi-class support vectormachines[J].IEEE Trans on Neural Networks.2002,13(2):415-442
[11]李昆仑,黄厚宽,田盛丰,等.模糊多类支持向量机及其在入侵检测中的应用[J].计算机学报.2005,28(2):276-280
[12]刘清，林土胜，王晓军.基于系数关系共生矩阵和SVM的纹理分析[J].光电工程.2009,36(4):128-131
[13]刘俊梅.基于小波变换的图像纹理特征提取技术[J].计算机工程与设计.28(13):3141-3144
[14]JP Marques de sa著,吴译飞译.模式识别-原理、方法及应用[M].北京：清华大学出版社,2002:265-278
[15]Sapina R.Computing textural features based on co-occurrence matrix forinfrared images[A].In：Proceeding of the2nd International Symposium onImage and Signa l Process—ing and Analysis[C].Pula，Croatia:2001,373—376
[16]Manjunath B S,Ma W Y.Textures features for browsing and retrieval ofimage data[J].IEEE Trans on PAMI,1996,(8):837—842
[17]Chen Lianping．Effects of diferent Gabor filters parameters on image retrievalby texture[A].In:Proc lOth Int Conference on MultimediaModelling[C].Brisbane,Australia:2004,273—278
[18]郑伟花,朱鸿茂.超声散斑数字干涉法测量物体位移的研究[J].力学与实践.2008,30(1):75-78
[19]冯玉洁,李雁,王华,等.灰度相关函数应用于HIFU监控的离体研究[J].中国超声医学杂志.2010,26(6):485-8
[1]周永昌，郭万学.超声医学(第六版)[M].北京,科学技术文献出版社.2011,78-89
[2]李奇林,全学模.超声微泡造影剂的临床应用及研究进展[J].国外医学临床放射学分册.2006,29(6):419-421
[3]Deng CX,Lizzi FL.Areview of physical phenomena associated with ultrasoniccontrast agents and illustrative clinical applications[J].Ultrasound MedBiol.2002,28(3):277-286
[4]Longmire M,Choyke PL,Kobayashi H.Dendrimer-based contrast agents formolecular imaging[J].Curr Top Med Chem.2008,8(14):1180-1186
[5]Thijssen JM, de Korte CL. Modeling ultrasound contrast measurement of bloodflow and perfusion in biological tissue[J].Ultrasound MedBiol.2005,31(2):279-285
[6]Lencioni R,Piscaglia F,Bolondi L.Contrast-enhanced ultrasound in the diagnosisof hepatocellular carcinoma[J].J Hepatol.2008,48(5):848-57
[7]G rg C.The forgotten organ:contrast enhanced sonography of theSpleen[J].Eur J Radiol.2007,64(2):189–201
[8]Wang Zhigang,Ling Zhiyu,Ran Haitao,et al.Ultrasound-mediated microbubbledestruction enhances VEGF gene delivery to the infarcted myocardium inrats[J].Clinical Imaging.2004,28(6):395-398
[9]Zhang Q,Wang Z,Ran H,et al.Enhanced gene delivery into skeletal muscles withultrasound and microbubble techniques[J].Acad Radiol.2006,13(3):363-367
[10]Lin Gong,Zhi Gang Wang,Hai Tao Ren,et al.Relationship between myocardialultrasonic integrated backscatter and mitochondria of the myocardium indogs[J].Clinical Imaging.2006,30:402-408
[11]XingSheng Li,ZhiGang Wang,et al.Experimental research on therapeuticangiogenesis induced by Hepatocyte Growth Factor directed byultrasound-targeted microbubble destruction in rats[J].J UltrasoundMed2008.27:439-446
[12]Nishioka T,Luo H,Fishbein MC,et al.Dissolution of thrombotic arterialocclusion by high intensity,low frequency ultrasound and dodecaffuoropentaneemulsion:an in vitro and in vjvo study[J].J Am CollCardiol.1997,30(2):558-561
[13]Ward AC,Dowth GP,Pitsillides AA.Hyaluronan in joint cavitation[J].BiochemSoc Trans.1999,27(2):128-135
[14]Haar GT,Coussios C.High intensity focused ultrasound: physical principles anddevices[J].Int J Hyperthermia.2007,23(2):89-104
[15]Garnier C,Lafon C,Dillenseger JL.3-d modeling of the thermal coagulationnecrosis induced by an interstitial ultrasonic transducer[J].IEEE Trans BiomedEng.2008,55(2):833-837
[16]Zhou Q,Zhu XQ,Zhang J,et al.Changes in circulating immunosuppressivecytokine levels of cancer patients after high intensity focused ultrasoundtreatment[J].Ultrasound Med Biol.2008,34(1):81-87
[17]Kono Y,Moriyasu F,Nada T,et al.Gray scale second harmonic imaging of theliver:a preliminary animal study[J].Ultrasound Med Biol.1997,23:719-726
[18]Sedelaar J P,Aarnink RG,van Leenders GJ,et al.The application ofthree-dimensional contrast2enhanced ultrasound to measure volume of affectedtissue after HIFU treatment for localized prostate cancer[J].EurUrol.2000,37:559-568
[19]Moran CM,Watson RJ,Fox KAA,et al.In vitro acoustic characterisation of fourintravenous ultrasonic contrast agents at30MHz[J].Ultrasound MedBiol.2002,28:785-91
[20]Takegami K,Kaneko Y,Watanabe T,et al.Heating and coagulation volumeobtained withhigh-intensity focused ultrasound therapy:comparison ofperflutren protein-type A microspheres and MRX-133inrabbits[J].Radiology.2005,237(1):132-136
[21]Li P,Cao LQ,Dou CY,et al.Impact of myocardial contrast echocardiographyonvascular permealbility:an in vivo dose response study of deliverymode,pressure amplitude and contrast dose[J].Ultrasound MedBiol.2003,29(9):1341-1349
[22]Tran BC,Seo J,Hall TL,et al.Microbubble-enhanced cavitation for noninvasiveultrasound surgery[J].IEEE Trans Ultrason Ferroelectr FreqControl.2003,50(10):1296-304
[23]Poliachik SL,Chandler WL,Ollos RJ,et al.The relation between cavitation andplatelet aggregation during exposure to high-intensity focusedultrasoung[J].Ultrasound Med Biol.2004,30(2):261-269
[24]Shin-ichiro Umemura,Ken-ichi Kawabata. Narendra Sanghvi, et al.In vivoacceleration of ultrasonic tissue heating by microbubble agent[J].UltrasonicsSymposium.2002,l2:1407-1410
[25]Kaneko Y,Maruyama T,Takegami K,et al. Use of a microbubble agent toincrease the effects of high intensity focused ultrasound on liver tissue[J].EurRadiol,2005,15(7):1415-1420
[26]Bailey MR,Couret LN,Sapozhnikov OA,et al.Use of overpressure to assess therole of bubbles in focused ultrasound lesion shape in vitro[J].Ultrasound MedBiol.2001,27:695-708
[27]Klibanov AL.Preparation of targeted microbubbles:ultrasound contrast agentsfor molecular imaging[J].Med Biol Eng Comput.2009,47(8):875-882
[28]Hauff P,Reinhardt M,Briel A,et al.Molecular targeting of lymph nodes withL-selectin ligand-specific US contrast agent:a feasibility study in mice anddogs[J].Radiology.2004,231(3):667-673
[29]Pisani E,Tsapis N,Paris J,et al.Polymeric nano/microcapsules of liquidperfluorocarbons for ultrasonic imaging:physicalcharacterization[J].Langmuir.2006,22(9):4397-4402
[30]Toya Nath Baral,Stefan Magez,Beno t Stijlemans,et al.Experimental therapy ofAfrican trypanosomiasis with a nanobody-conjugated human trypanolyticfactor[J].Nature Medicine.2006,12(5):580-584
[31]Stijlemans B,Conrath K,Cortez-Retamozo V,et al.Efficient targeting ofconserved cryptic epitopes of infectious agents by single-domainantibodies[J].African trypanosomes as a paradigm.J BiolChem.2004,279(2):1256-1261
[32]杨珂,王冬.纳米抗体及其应用[J].分子与细胞免疫杂志.2008,24(4):425-427
[33]Marsh JN, Partlow KC, Abendschein DR,et al.Molecular imaging withtargeted perfluorocarbon nanoparticles:quantification of the concentrationdependence of contrast enhancement for binding to sparse cellularepitopes[J].Ultrasound Med Biol.2007,33(6):950-958
[34]Marsh JN,Hall CS,Scott MJ,et al.Improvements in the ultrasonic contrast oftargeted perfluorocarbon nanoparticles using an acoustic transmission linemodel[J].IEEE Trans Ultrason Ferroelectr Freq Control.2002,49(1):29-38
[35]Hughes MS,Marsh JN,Ha1l CS,et a1.Acoustic characterization in whole bloodand plasma of site-targeted nanoparticle ultrasound contrast agent formolecular imaging[J].Acoust Soc Am.2005,117(12):1964-1972
[2]de Groot-de Laat LE,Krenning BJ,ten Cate FJ,et al.Usefulness of contrastechocardiography for diagnosis of left ventricular noncompaction[J].Am JCardiol2005,95(9):1131–4
[3]Longmire M,Choyke PL,Kobayashi H.Dendrimer-based contrast agents formolecular imaging[J].Curr Top Med Chem.2008,8(14):1180-1186
[4]G rg C.The forgotten organ:contrast enhanced sonography of the Spleen[J].EurJ Radiol.2007,64(2):189–201
[5]Lencioni R,Piscaglia F,Bolondi L.Contrast-enhanced ultrasound in the diagnosisof hepatocellular carcinoma[J].J Hepatol.2008,48(5):848-57
[6]Thijssen JM, de Korte CL. Modeling ultrasound contrast measurement of bloodflow and perfusion in biological tissue[J].Ultrasound MedBiol.2005,31(2):279-285
[7]王志刚.超声分子影像学研究进展[J].中国医学影像技术.2009,25:921-924
[8]Lindner JR.Contrast ultrasound molecular imaging of inflammation incardiovascular disease[J].Cardiovasc Res.2009,84(2):182–9
[9]Ferrara KW,Borden MA,Zhang H.Lipid-shelled vehicles:engineering forultrasound molecular imaging and drug delivery[J].Acc ChemRes.2009,42(7):881–92
[10]Kaufmann BA.Ultrasound molecular imaging of atherosclerosis[J].CardiovascRes.2009,83(4):617–25
[11]XingSheng Li,ZhiGang Wang,et al.Experimental research on therapeuticangiogenesis induced by Hepatocyte Growth Factor directed byultrasound-targeted microbubble destruction in rats[J].J UltrasoundMed2008.27:439-446
[12]Lin Gong,Zhi Gang Wang,Hai Tao Ren,et al.Relationship between myocardialultrasonic integrated backscatter and mitochondria of the myocardium indogs[J].Clinical Imaging.2006,30:402-408
[13]Zhang Q,Wang Z,Ran H,et al.Enhanced gene delivery into skeletal muscleswith ultrasound and microbubble techniques[J].AcadRadiol.2006,13(3):363-367
[14]Wang Zhigang,Ling Zhiyu,Ran Haitao,et al.Ultrasound-mediated microbubbledestruction enhances VEGF gene delivery to the infarcted myocardium inrats[J].Clinical Imaging.2004,28(6):395-398
[15]Dong Wang,Ke Yang,Yun-Hua Gao,et al.Preparation and characterization of ananoscale ultrasound contrast agent[J].Clinical Imaging.2010,34(4):288-292
[16]Klibanov AL．Ligand-carrying gas-filled micr0bubbles：ultrasound contrastagents for targeted molecular imaging[J].Bioconjug Chem.2005,16(1):9-17
[17]Patel RM.Microbubble: a potential ultrasound tool in molecularimaging[J].Curr Pharm Biotechnol.2008,9(5):406-410
[18]杨钰楠，高云华.靶向超声造影剂制备的方法学研究[J].中华超声影像学杂志.2006,15(1):65-67
[19]李奇林,全学模.超声微泡造影剂的临床应用及研究进展[J].国外医学临床放射学分册.2006,29(6):419-421
[20]周永昌，郭万学.超声医学(第六版)[M].北京,科学技术文献出版社.2011,78-89
[21]Lozano MM,Longo ML.Microbubbles coated with disaturated lipids andDSPE-PEG2000:phase behavior,collapse transitions,andpermeability[J].Langmuir.2009,25(6):3705–12
[22]Wheatley MA,Forsberg F,Dube N,et al. Surfactant-stabilized contrast agent onthe nanoscale for diagnostic ultrasound imaging[J]. Ultrasound MedBiol.2006,32(1):83–93.
[23]Oeffinger BE,Wheatley MA.Development and characterization of a nano-scalecontrast agent[J].Ultrasonics.2004,42:343-7
[24]王冬,杨珂,闵加艳,等.高强度聚焦超声联合纳米微泡造影剂对兔VX2乳腺移植瘤辐照效果的影响[J].中国介入影像与治疗.2012,9(3):213-216
[1]Tewari M,Krishnamurthy A,Shukla HS.Breast conservation in locally advancedbreast cancer in developing countries:wise or waste[J].Surgicaloncology.2009,18(1):3-13
[2]Lencioni R,Piscaglia F,Bolondi L.Contrast-enhanced ultrasound in the diagnosisof hepatocellular carcinoma[J].J Hepatol.2008,48(5):848-57
[3]G rg C.The forgotten organ:contrast enhanced sonography of the spleen[J].EurJ Radiol.2007,64(2):189-201
[4]de Groot-de Laat LE,Krenning BJ,ten Cate FJ,et al.Usefulness of contrastechocardiography for diagnosis of left ventricular noncompaction[J].Am JCardiol.2005,95(9):1131-4
[5]Pisani E,Tsapis N,Paris J,et al.Polymeric nano/microcapsules of liquidperfluorocarbons for ultrasonic imaging:physicalcharacterization[J].Langmuir.2006,22(9):4397-4402
[6]Hauff P,Reinhardt M,Briel A,et al.Molecular targeting of lymph nodes withL-selectin ligand-specific US contrast agent:a feasibility study in mice anddogs[J].Radiology.2004,231(3):667-673
[7]Klibanov AL.Preparation of targeted microbubbles:ultrasound contrast agentsfor molecular imaging[J].Med Biol Eng Comput.2009,47(8):875-882
[8]Toya Nath Baral,Stefan Magez,Beno t Stijlemans,et al.Experimental therapy ofAfrican trypanosomiasis with a nanobody-conjugated human trypanolyticfactor[J].Nature Medicine.2006,12(5):580-584
[9]Stijlemans B,Conrath K,Cortez-Retamozo V,et al.Efficient targeting ofconserved cryptic epitopes of infectious agents by single-domainantibodies[J].African trypanosomes as a paradigm.J BiolChem.2004,279(2):1256-1261
[10]杨珂,王冬.纳米抗体及其应用[J].分子与细胞免疫杂志.2008,24(4):425-427
[11]Zhang J,Tanha J,Hirama T,et al.Pentamerization of single-domain antibodiesfrom phage libraries:a novel strategy for the rapid generation of high-avidityantibody reagents[J].J Mol Biol.2004,335(1):49-56
[12]Hughes MS,Marsh JN,Ha1l CS,et a1.Acoustic characterization in whole bloodand plasma of site-targeted nanoparticle ultrasound contrast agent formolecular imaging[J].Acoust Soc Am.2005,117(12):1964-1972
[13]Marsh JN,Hall CS,Scott MJ,et al.Improvements in the ultrasonic contrast oftargeted perfluorocarbon nanoparticles using an acoustic transmission linemodel[J].IEEE Trans Ultrason Ferroelectr Freq Control.2002,49(1):29-38
[14]Marsh JN, Partlow KC, Abendschein DR,et al.Molecular imaging withtargeted perfluorocarbon nanoparticles:quantification of the concentrationdependence of contrast enhancement for binding to sparse cellularepitopes[J].Ultrasound Med Biol.2007,33(6):950-958
[15]《乳腺癌HER2检测指南》编写组.乳腺癌HER2检测指南[J].中华病理学杂志,2006,35(10):631-633
[16]刘扬,吕万良,张强.脂质体及纳米粒药物递送系统的研究进展[J].中国医学科学院学报,2006,28(4):583-589
[17]杨莉,谭开彬,左松,等.阴离子脂膜超声微泡的制备及评价[J].临床超声医学杂志,2006,8(4):193-195
[18]杨钰楠,高云华,谭开彬,等.携RGDS超声造影剂的制备及体外靶向血栓研究[J].第三军医大学学报,2006,6(28):562－564
[19]杨钰楠,高云华.靶向超声造影剂制备的方法学研究[J].中华超声影像学杂志,2006,15(1):65-67
[20]Dong Wang,Ke Yang,Yun-Hua Gao,et al.Preparation and characterization of ananoscale ultrasound contrast agent[J].Clinical Imaging.2010,34(4):288-292
[1]Tewari M,Krishnamurthy A,Shukla HS.Breast conservation in locally advancedbreast cancer in developing countries: wise or waste[J].Surgicaloncology.2009,18(1):3-13
[2]Melodelima D,Chapelon JY,Theillere Y,et al.Combination of thermal andcavitation effects to generate deep lesions with an endocavitary applicator usinga planet transducer: ex vivo studies[J].Ultrasound Med Biol.2004,30:103–11
[3]Zhou Q,Zhu XQ,Zhang J,et al.Changes in circulating immunosuppressivecytokine levels of cancer patients after high intensity focused ultrasoundtreatment[J].Ultrasound Med Biol.2008,34(1):81-87
[4]Garnier C,Lafon C,Dillenseger JL.3-d modeling of the thermal coagulationnecrosis induced by an interstitial ultrasonic transducer[J].IEEE Trans BiomedEng.2008,55(2):833-837
[5]Haar GT,Coussios C.High intensity focused ultrasound: physical principles anddevices[J].Int J Hyperthermia.2007,23(2):89-104
[6]Cheng SQ,Zhou XD,Tang ZY,et al. Iodized oil enhances the thermal effect ofhigh-intensity focused ultrasound on ablating experimental liver cancer[J].JCancer Res Clin Oncol.1997,123(11-12):639-644
[7]Rubio Briones J,Collado Serra A,Gómez-Ferrer Lozano A,et al. High-intensityfocused ultrasound in small renal masses[J].Adv Urol.2008:809-45
[8]Wang Z,Li F,Bai J.A study of acoustic environment in tissue of high intensityfocused ultrasound.3rd International Symposium on TherapeuticUltrasound[C].INSERM U.556,2003,6:68
[9]Zderic V,Brayman AA,Sharar SR,et al.Microbubble-enhanced hemorrhagecontrol using high intensity focusedultrasound[J].Ultrasonics.2006,45(1-4):113-120
[10]沙卫红,李瑜元,聂玉强,等.高强度聚焦超声联用超声造影剂对治疗兔肝焦域效应的影响[J].中华超声影像学杂志.2004,13(4):299-302
[11]Tung YS,Liu HL,Wu CC,et al.Contrast-agent-enhanced ultrasound thermalablation[J].Ultrasound Med Biol.2006,32(7):1103-1110
[12]Moran CM,Watson RJ,Fox KAA,et al.In vitro acoustic characterisation of fourintravenous ultrasonic contrast agents at30MHz[J].Ultrasound MedBiol.2002,28:785-91
[13]Poliachik SL,Chandler WL,Mourad PD,et al.Effect of high-intensity focusedultrasound on whole blood with and without microbubble contrastagent[J].Ultrasound Med Biol.1999,25:991-998
[14]Bailey MR,Couret LN,Sapozhnikov OA,et al.Use of overpressure to assess therole of bubbles in focused ultrasound lesion shape in vitro[J].Ultrasound MedBiol.2001,27:695-708
[15]Yu T,Wang G,Hu K,et al.A micro-bubble agent improves the therapeuticefficiency of High Intensity Focused Ultrasound:a rabbit kidneystudy[J].Urological Research.2004,32:14-19
[16]Yu T,Xiong S,Mason TJ,Wang Z.The use of a micro-bubble agent to enhancerabbit liver destruction using high intensity focused ultrasound[J].UltrasonSonochem.2006,13(2):143-149
[17]Sokka SD,King R,Hynynen K.MRI-guided gas bubble enhanced ultrasoundheating in vivo rabbit t high[J].Phys Med Biol.2003,48(2):223-241
[18]Li P,Cao LQ,Dou CY,et al.Impact of myocardial contrast echocardiographyonvascular permealbility:an in vivo dose response study of deliverymode,pressure amplitude and contrast dose[J].Ultrasound MedBiol.2003,29(9):1341-1349
[19]王冬,杨珂,闵加艳,等.高强度聚焦超声联合纳米微泡造影剂对兔VX2乳腺移植瘤辐照效果的影响[J].中国介入影像与治疗.2012,9(3):213-216
[20]Takegami K,Kaneko Y,Watanabe T,et al.Heating and coagulation volumeobtained withhigh-intensity focused ultrasound therapy:comparison ofperflutren protein-type A microspheres and MRX-133inrabbits[J].Radiology.2005,237(1):132-136
[21]Oeffinger BE,Wheatley MA.Development and characterization of a nano-scalecontrast agent[J].Ultrasonics.2004,42:343-7
[22]Lindner JR.Contrast ultrasound molecular imaging of inflammation incardiovascular disease[J].Cardiovasc Res.2009,84(2):182-9
[23]Ferrara KW,Borden MA,Zhang H.Lipid-shelled vehicles:engineering forultrasound molecular imaging and drug delivery[J].Acc ChemRes.2009,42(7):881-92
[24]Kaufmann BA.Ultrasound molecular imaging of atherosclerosis[J].CardiovascRes.2009,83(4):617-25
[25]Dong Wang,Lu Li,Jiayan Min,et al.Combination of High Intensity FocusedUltrasound with Nanoscale Ultrasound Contrast Agent in Treatment of RabbitBreast VX2Tumors:A Pilot Study[J].Clinical Imaging.2012(Accept)
[1]刘宝琴,熊树华,王智彪,等.超声对高强度聚焦超声生物学焦域的监控研究[J].中华超声影像学杂志,2002,11(11):687-689
[2]邹建中,龚晓波,贺雪梅,等.高强度聚焦超声辐照离体组织靶区无灰度变化有坏死现象的研究[J].临床超声医学杂志.2008,10(9):581-583
[3]Liu BQ,Xiong SH,Wang ZB.The study on biological focal region of highintensity focused ultrasound under ultrasonography monitoring[J].ChinUltrasonogr,2002(11):687-688
[4]李雁,邹建中,王华,等.基于二维超声图像纹理分析判断HIFU凝固性坏死[J].中国医学影像技术.2010,26(6):1147-1149
[5]冯玉洁,邹建中,王华,等.Tamura纹理分析应用于HIFU监控的离体研究[J].中国医科大学学报.2010,39(8):610-612
[6]胡昌华,张军波,夏军,等.基于MATLAB的系统分析与设计—小波分析[M].西安:西安电子科技大学出版社.1999:16-19
[7]Lee JS,Sun YN,Lin XZ.A new approach to ultrasonic liver imageclassification[J].IEICE transactions on information andsystems.2000,E83-D(6):1301-1308
[8]Arivazhagan S,Ganesan L.Texture classification using wardertransform[J].Pattern Recognition etters.2003,24(9-10):1513-1521
[9]闵加艳,邹建中,郭宇,等.基于小波变换的高强度聚焦超声消融兔VX2乳腺肿瘤声像图纹理分析[J].中国医科大学学报.2012,41(1):14-17
[10]Hsu C,Lin C.A comparison of methods for multi-class support vectormachines[J].IEEE Trans on Neural Networks.2002,13(2):415-442
[11]李昆仑,黄厚宽,田盛丰,等.模糊多类支持向量机及其在入侵检测中的应用[J].计算机学报.2005,28(2):276-280
[12]刘清，林土胜，王晓军.基于系数关系共生矩阵和SVM的纹理分析[J].光电工程.2009,36(4):128-131
[13]刘俊梅.基于小波变换的图像纹理特征提取技术[J].计算机工程与设计.28(13):3141-3144
[14]JP Marques de sa著,吴译飞译.模式识别-原理、方法及应用[M].北京：清华大学出版社,2002:265-278
[15]Sapina R.Computing textural features based on co-occurrence matrix forinfrared images[A].In：Proceeding of the2nd International Symposium onImage and Signa l Process—ing and Analysis[C].Pula，Croatia:2001,373—376
[16]Manjunath B S,Ma W Y.Textures features for browsing and retrieval ofimage data[J].IEEE Trans on PAMI,1996,(8):837—842
[17]Chen Lianping．Effects of diferent Gabor filters parameters on image retrievalby texture[A].In:Proc lOth Int Conference on MultimediaModelling[C].Brisbane,Australia:2004,273—278
[18]郑伟花,朱鸿茂.超声散斑数字干涉法测量物体位移的研究[J].力学与实践.2008,30(1):75-78
[19]冯玉洁,李雁,王华,等.灰度相关函数应用于HIFU监控的离体研究[J].中国超声医学杂志.2010,26(6):485-8
[1]周永昌，郭万学.超声医学(第六版)[M].北京,科学技术文献出版社.2011,78-89
[2]李奇林,全学模.超声微泡造影剂的临床应用及研究进展[J].国外医学临床放射学分册.2006,29(6):419-421
[3]Deng CX,Lizzi FL.Areview of physical phenomena associated with ultrasoniccontrast agents and illustrative clinical applications[J].Ultrasound MedBiol.2002,28(3):277-286
[4]Longmire M,Choyke PL,Kobayashi H.Dendrimer-based contrast agents formolecular imaging[J].Curr Top Med Chem.2008,8(14):1180-1186
[5]Thijssen JM, de Korte CL. Modeling ultrasound contrast measurement of bloodflow and perfusion in biological tissue[J].Ultrasound MedBiol.2005,31(2):279-285
[6]Lencioni R,Piscaglia F,Bolondi L.Contrast-enhanced ultrasound in the diagnosisof hepatocellular carcinoma[J].J Hepatol.2008,48(5):848-57
[7]G rg C.The forgotten organ:contrast enhanced sonography of theSpleen[J].Eur J Radiol.2007,64(2):189–201
[8]Wang Zhigang,Ling Zhiyu,Ran Haitao,et al.Ultrasound-mediated microbubbledestruction enhances VEGF gene delivery to the infarcted myocardium inrats[J].Clinical Imaging.2004,28(6):395-398
[9]Zhang Q,Wang Z,Ran H,et al.Enhanced gene delivery into skeletal muscles withultrasound and microbubble techniques[J].Acad Radiol.2006,13(3):363-367
[10]Lin Gong,Zhi Gang Wang,Hai Tao Ren,et al.Relationship between myocardialultrasonic integrated backscatter and mitochondria of the myocardium indogs[J].Clinical Imaging.2006,30:402-408
[11]XingSheng Li,ZhiGang Wang,et al.Experimental research on therapeuticangiogenesis induced by Hepatocyte Growth Factor directed byultrasound-targeted microbubble destruction in rats[J].J UltrasoundMed2008.27:439-446
[12]Nishioka T,Luo H,Fishbein MC,et al.Dissolution of thrombotic arterialocclusion by high intensity,low frequency ultrasound and dodecaffuoropentaneemulsion:an in vitro and in vjvo study[J].J Am CollCardiol.1997,30(2):558-561
[13]Ward AC,Dowth GP,Pitsillides AA.Hyaluronan in joint cavitation[J].BiochemSoc Trans.1999,27(2):128-135
[14]Haar GT,Coussios C.High intensity focused ultrasound: physical principles anddevices[J].Int J Hyperthermia.2007,23(2):89-104
[15]Garnier C,Lafon C,Dillenseger JL.3-d modeling of the thermal coagulationnecrosis induced by an interstitial ultrasonic transducer[J].IEEE Trans BiomedEng.2008,55(2):833-837
[16]Zhou Q,Zhu XQ,Zhang J,et al.Changes in circulating immunosuppressivecytokine levels of cancer patients after high intensity focused ultrasoundtreatment[J].Ultrasound Med Biol.2008,34(1):81-87
[17]Kono Y,Moriyasu F,Nada T,et al.Gray scale second harmonic imaging of theliver:a preliminary animal study[J].Ultrasound Med Biol.1997,23:719-726
[18]Sedelaar J P,Aarnink RG,van Leenders GJ,et al.The application ofthree-dimensional contrast2enhanced ultrasound to measure volume of affectedtissue after HIFU treatment for localized prostate cancer[J].EurUrol.2000,37:559-568
[19]Moran CM,Watson RJ,Fox KAA,et al.In vitro acoustic characterisation of fourintravenous ultrasonic contrast agents at30MHz[J].Ultrasound MedBiol.2002,28:785-91
[20]Takegami K,Kaneko Y,Watanabe T,et al.Heating and coagulation volumeobtained withhigh-intensity focused ultrasound therapy:comparison ofperflutren protein-type A microspheres and MRX-133inrabbits[J].Radiology.2005,237(1):132-136
[21]Li P,Cao LQ,Dou CY,et al.Impact of myocardial contrast echocardiographyonvascular permealbility:an in vivo dose response study of deliverymode,pressure amplitude and contrast dose[J].Ultrasound MedBiol.2003,29(9):1341-1349
[22]Tran BC,Seo J,Hall TL,et al.Microbubble-enhanced cavitation for noninvasiveultrasound surgery[J].IEEE Trans Ultrason Ferroelectr FreqControl.2003,50(10):1296-304
[23]Poliachik SL,Chandler WL,Ollos RJ,et al.The relation between cavitation andplatelet aggregation during exposure to high-intensity focusedultrasoung[J].Ultrasound Med Biol.2004,30(2):261-269
[24]Shin-ichiro Umemura,Ken-ichi Kawabata. Narendra Sanghvi, et al.In vivoacceleration of ultrasonic tissue heating by microbubble agent[J].UltrasonicsSymposium.2002,l2:1407-1410
[25]Kaneko Y,Maruyama T,Takegami K,et al. Use of a microbubble agent toincrease the effects of high intensity focused ultrasound on liver tissue[J].EurRadiol,2005,15(7):1415-1420
[26]Bailey MR,Couret LN,Sapozhnikov OA,et al.Use of overpressure to assess therole of bubbles in focused ultrasound lesion shape in vitro[J].Ultrasound MedBiol.2001,27:695-708
[27]Klibanov AL.Preparation of targeted microbubbles:ultrasound contrast agentsfor molecular imaging[J].Med Biol Eng Comput.2009,47(8):875-882
[28]Hauff P,Reinhardt M,Briel A,et al.Molecular targeting of lymph nodes withL-selectin ligand-specific US contrast agent:a feasibility study in mice anddogs[J].Radiology.2004,231(3):667-673
[29]Pisani E,Tsapis N,Paris J,et al.Polymeric nano/microcapsules of liquidperfluorocarbons for ultrasonic imaging:physicalcharacterization[J].Langmuir.2006,22(9):4397-4402
[30]Toya Nath Baral,Stefan Magez,Beno t Stijlemans,et al.Experimental therapy ofAfrican trypanosomiasis with a nanobody-conjugated human trypanolyticfactor[J].Nature Medicine.2006,12(5):580-584
[31]Stijlemans B,Conrath K,Cortez-Retamozo V,et al.Efficient targeting ofconserved cryptic epitopes of infectious agents by single-domainantibodies[J].African trypanosomes as a paradigm.J BiolChem.2004,279(2):1256-1261
[32]杨珂,王冬.纳米抗体及其应用[J].分子与细胞免疫杂志.2008,24(4):425-427
[33]Marsh JN, Partlow KC, Abendschein DR,et al.Molecular imaging withtargeted perfluorocarbon nanoparticles:quantification of the concentrationdependence of contrast enhancement for binding to sparse cellularepitopes[J].Ultrasound Med Biol.2007,33(6):950-958
[34]Marsh JN,Hall CS,Scott MJ,et al.Improvements in the ultrasonic contrast oftargeted perfluorocarbon nanoparticles using an acoustic transmission linemodel[J].IEEE Trans Ultrason Ferroelectr Freq Control.2002,49(1):29-38
[35]Hughes MS,Marsh JN,Ha1l CS,et a1.Acoustic characterization in whole bloodand plasma of site-targeted nanoparticle ultrasound contrast agent formolecular imaging[J].Acoust Soc Am.2005,117(12):1964-1972