摘要
随着超声造影诊断和超声造影剂制备技术的不断发展，探讨以超声微泡造影剂为载体进行超声分子影像和靶向治疗是当前研究的热点。实现靶向传输，首先需要通过一定的载体或方法将治疗性外源物质安全、高效、靶向性地导入靶细胞或组织内，释放到组织间隙的外源物质然后被细胞吞噬吸收或翻译或表达利用，达到靶向治疗的目的。目前用作靶向传输的载体在靶向性、安全性及效率等方面均存在各自的局限性。最近，国内外一些学者的研究表明：将表面黏附或内部包裹有外源物质的微泡造影剂经静脉注射，同时在靶器官或组织给予一定条件的超声照射后，使微泡破坏，其携带的外源物质在局部可以达到有效浓度，而到机体其他部位的外源物质随微泡代谢，不在局部停留，从而降低副作用，微泡造影剂在靶向传输方面显示出令人鼓舞的应用前景。
超声介导微泡空化效应靶向传输的原理是：在特定部位发射不同声强的超声，在超声作用下该部位的微泡发生破裂，发生空化，微泡的空化效应产生很大的压力和能量，对周围的组织产生生物学效应，使得血管内皮屏障损伤，血管通透性增加，从而使外源物质通过本来无法通过的血管内皮屏障到达特定的部位。
目前超声介导微泡空化效应靶向传输机制尚未完全阐明。不同的条件下，微泡空化对局部组织的生物学效应程度不同，使其可以用于实现不同的目的。在靶向传输时，需要损伤，既要保证外源物质可以通过损伤的细胞膜进入细胞，或通过受损的内皮屏障到达组织间隙，又要保证微泡空化所致的生物学效应不会产生严重的后果。
目的：
1、通过观察声学造影剂白蛋白微泡在不同能量的超声作用下，对大鼠脊斜肌微血管的损伤情况、伊文思蓝以及绿色荧光微球外溢情况的观察，评估微泡空化效应对脊斜肌微血管通透性的影响；2、在不同的时间点，用透射电镜及HE染色的方法观察微泡空化导致的微血管损伤情况，以及了解微血管壁的结构变化情况，初步探讨超声介导微泡空化效应靶向传输的机制；3、应用脊斜肌活体微循环标本，观察超声破坏微泡前后不同时间点平均动脉压、微血管口径、血流速度及血流量的变化，结合透射电镜及HE染色结果，评估微泡空化效应对局部微循环的影响以及探讨其在靶向传输应用时的安全性问题。
方法：
1、微泡空化效应对脊斜肌微血管通透性的影响
根据超声能量大小将56只健康的SD大鼠分为0.25W/cm~2，0.75W/cm~2和1.5W/cm~2三个超声微泡照射组和一个对照组，每组又细分为伊文思蓝和荧光微球输入亚组，共分8组，每组7只大鼠，大鼠按取动物顺序依次分别分配到各组。实验时经静脉输入含有伊文思蓝(Evans blue，EB)或荧光微球的微泡造影剂，分别采用0.25W/cm~2，0.75W/cm~2和1.5W/cm~2超声照射游离在体脊斜肌1分钟，破坏脊斜肌组织内造影剂，在体荧光显微镜观察微血管损伤情况和计数每100倍光镜视野出血点数目。通过比较红细胞、伊文思蓝及荧光微球的外溢情况，结合在超声照射破坏造影剂后取局部损伤组织作微血管电镜观察结果，初步评价微泡空化效应对脊斜肌微血管通透性的影响，并探讨超声介导微泡空化效应靶向传输机制。
2、微泡空化效应对脊斜肌微循环的影响
将72只健康的SD大鼠分为对照组、微泡组、超声组(超声能量为1.5W·cm~(-2))、高能量超声微泡组(超声能量为1.5W·cm~(-2))、中能量超声微泡组(超声能量为0.75W·cm~(-2))及低能量超声微泡组(超声能量为0.25W·cm~(-2))，每组12只。大鼠按取动物顺序依次分别分配到对照组、微泡组、超声组、高能量超声微泡组、中能量超声微泡组及低能量超声微泡组。按Gray法制备大鼠脊斜肌活体微循环观察标本，每组6只大鼠分别在超声破坏微泡前后不同时间点测量平均动脉压，用红细胞跟踪相关仪和电视测微仪测量微动静脉口径和血流速度，其余6只大鼠在不同时间点用电镜观察大鼠脊斜肌组织结构的变化。
结果：
1、用低能量的超声(0.25W/cm~2)照射含有微泡的脊斜肌后，在体荧光显微镜未观察到出血点及荧光微球外溢，但活体显微镜下脊斜肌可见到EB外溢至组织间隙(P＜0.05)。而用较大能量的超声(0.75W/cm~2和1.5W/cm~2)照射后，在体荧光显微镜均观察到出血点，与对照组比较有显著差异(0.75W/cm~2：P=0.006和0.016，1.5W/cm~2：P=0.016和(0.001)；EB外溢量与对照组比较有显著差异(0.75W/cm~2：P=0.002和1.5W/cm~2：P=0.006)，同时可见荧光微球外溢。
2、透射电镜观察结果显示低能量的超声(0.25W/cm~2)破坏微泡后，微血管结构完整，未见红细胞外溢到组织间隙，但可见微血管内皮间隙增宽；较大能量的(0.75W/cm~2和1.5W/cm~2)超声破坏微泡后即刻的出血点组织标本电镜结果显示：微血管内皮间隙明显增宽，同时内皮细胞上有破裂口，其中用1.5W/cm~2超声破坏微泡后可观察到组织空泡化和肌溶解等现象；2h后出血点组织标本电镜观察未发现内皮破口。
3、微泡空化效应不会对大鼠微血管口径产生显著的变化(P＞0.05)；但可致微血管血流速度减慢，血流量降低，血流速度及血流量在超声破坏微泡后1min下降到最低，然后呈上升趋势；微循环血流速度和血流量下降和恢复的程度与超声能量有关：低能量(0.25W/cm~2)的超声破坏微泡后血流速度及血流量下降的程度小，较高能量(0.75W/cm~2和1.5W/cm~2)的超声破坏微泡后血流速度及血流量下降的的程度明显；低能量组血流速度及血流量15min后可明显恢复，中能量组血流速度及血流量在2h可恢复，而高能量组血流速度及血流量在2h仍未完全恢复。
结论：
1、低能量超声介导微泡空化效应不会导致大鼠骨骼肌产生出血点，但可致微血管内皮间隙增宽，微血管通透性增加，小分子物质可外溢到组织间隙，这可能是低能量超声介导微泡空化效应靶向传输的机理。
2、高能量超声介导微泡空化，造成微血管内皮细胞损伤，在微血管内皮屏障上产生撕裂孔，微血管通透性明显增加，红细胞和100nm荧光微球等大分子物质可以通过这些的撕裂孔形成的通道外溢到组织间隙，这可能是高能量超声介导微泡空化靶向传输的机理。这种程度内皮细胞损伤具有可修复性。
3、微泡空化效应对局部微循环及微血管产生损伤的程度跟超声的能量有关，能量越小产生损伤越轻，局部微循环修复的能力越强。高能量声场的微泡空化对微血管造成的损伤较重，微循环血流速度和血流量下降作用明显，可能导致严重的病理、生理后果，不利于靶向传输。而低能量的超声照射由于损伤较轻，微循环所受的影响较小，而且超声破坏微泡后血流速度和血流量随着时间的推移有逐渐增加趋势，短时间内可恢复到接近正常的水平，在一个稳定局部微循环条件下可实现较佳的靶向传输效果。
With ongoing development of diagnose and manufacture technique of ultrasound contrast agent, ultrasound molecule image and target transmission using ultrasound microbubble as a vehicle is a popular research topic at present. Achievement of targeted delivery not only needs a particular vehicle to carry foreingn matter to targeted cell or tissue safely and effectively, but also need foreign matter can be used in defferent ways to achieve final purpose. Recently, researchs suggests that ultrasound microbubble show an encouraging application perspective in targeted delivery. The basic principle of ultrasound mediated microbubble cavitation target transmission: by applying different sound intensity ultransound to intravascular microbubble contrast agent in the definite position, microbubble is destroyed and then cavitation follow; the pressure and the energy created by cavitation exert a biological effect on the local cell or tissue, resulting in the rupture of endothelium cell and the increased permeability of cell membranes, thus foreign matter can pass endothelium barrier to tissue space.
Microbubble cavitation has different biological effect on local tissue or cell decided by different ultrasound microbubble condition. At present the mechanism of ultrasound mediated microbubble cavitation target transmission is not completely illuminated. During the target transmission, we need the endothelium injure which allow the foreign matter enter the cell through membrane, or pass through the endothelium barrier into tissue, but we should make injure as mild as possible in lest that caused serious consequence.
Objectives:
1、To evaluate influence of microbubble cavitation on the microvessel permeability of rat spinotrapezius and to approach the mechanism of target transmission by ultrasound microbubble cavitation. 2、To investigate influence of microbubble cavitation on the local microcirculation and safty with the application of targeted transmission.
Methods:
1、The effect of albumin microbubble cavitation on membrane permeability of rat spinotrapeziu
According to ultrasound power, fifty-six Sprague-Dawley rats were divided randomly into seven groups: 0.25W/cm~2, 0.75W/cm~2 and 1.5W/cm~2 ultrasound microbubble groups and control group and each group were divided into two subset groups: evans blue group and polymer microsphere group. After infusion of contrast agent mixed with evans blue or fluorescent polymer microspheres, ultrasound was applied to the exteriorized spinotrapeziu to destroy contrast agent. The changes of the structure of the spinotrapezius microvessel were observed by microscope under transillumination and counted petechia numbers per 100 times illumination field of view. After destruction of contrast agent by ultrasound, we observed the change of microvessel structure by looking into the petechia with electronic microscope and hematoxylin and eosin stain. The rats infused with mixture ofmicrobubble and evans blue were executed by the way of blood letting 2 hours after microbubble was destroyed, the extracted evans blue contents was measured with method of the standard curve and the absorption spectrometry.
2、The influence of microbubble cavitation on microcirculation of rat spinotrapeziu
Seventy -two Sprague-Dawley rats were divided randomly into 6 groups of 12 each: normal saline-treated control group, microbubble group, ultrasound group, high-energy ultrasound (1.5 W-cm~(-2)) microbubble group, medial-energy ultrasound (0.75 W-cm~(-2)) microbubble group and low-energy ultrasound (0.25 W-cm~(-2)) microbubble group. The diameter and the flow velocity of microvessel were measured by RBC tracking correlator and IV500 model viedo microscaler respectively in 6 Sprague-Dawley rats of each group. The structure of the injured microvessel was investigated by electron microscopy at different time before and after microbubble destruction in the other rats of each goup.
Results:
1、No petechiae and fluorescent polymer microspheres extraction were caused by albumin microbubble destructed by low power ultrasound (0.25 W-cm~(-2)) in rat spinotrapezius but evans blue can be saw to extracted to tissue in rat spinotrapezius. While by high power ultrasound (0.75W/cm~2 and 1.5W/cm~2) petechiae and extraction of fluorescent polymer microspheres and evans blue were caused. (P＜0.05)
2、No microvessel or tissue injure or petechiae but widen of endothelium interspace were caused under electronic microscope in low power ultrasound microbuble group. While in the high energy ultrasound groups, The rupture of endothelium cell, the widening of the endothelium interspace and the extraction of red cell to the tissue were revealed by electron microscopy. But endothelium cell rupture was not observed 2 hours after treatment
3、Microbubble cavitation had no effect on the mean arterial pressure and the diameter of microvessel, but had a adverse effect on the velocity and blood flow: flow velocity were slowed and blood flow of the microvessle were reduced which reach the bottom at 1 minute after microbubble destruction and then went up slowly. The effect on the microcirculation and the ability of flow velocity and blood flow to recover to normal condition were correlated with ultrasound energy. In the low ultrasound energy group, the flow velocity and blood flow ofveinule were found to recover significantly after about 15 minute, and those of arteriole were found to recover after about one hour. While in the high ultrasound energy groups (0.75W/cm~2 and 1.5W/cm~2), the flow velocity and blood flow ofmicrovessel were not found to recover after two hours completely.
Conclusions:
1、No petechiae were caused but endothelium interspace was widen by albumin microbubble destructed by low power ultrasound in rat spinotrapezius. The increased permeability, which allow small molecule matter enter to interstitial space, could be the mechanism of low power ultrasound induced microbubble cavitation target transmission.
2、the mechanism of the hige power ultrasound induced microbubble cavitation target transmission could be that the microbubble cavitation causes endothelium cell rupture and permeability remarkable increased which permit red cell and 100 nm fluorescent polymer mierospheres extract to the tissue.
3、The injure and the influence on the rnicrocirculation caused by microbubble cavitation is affected by ultrasound energy. The rupture is repairable. The repariable effect is correlated with the ultrasound energy.
引文
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