摘要
目的：采用超顺磁性氧化铁纳米颗粒(SPIO)作为磁探针标记兔骨髓间充质干细胞(BMSCs),并将SPIO标记的兔BMSCs采用原位移植方式移植入兔股骨头坏死区,进行MRI和组织学检查,观察SPIO标记的BMSCs在MRI信号上的变化情况及其对兔股骨头坏死的修复效果,为SPIO标记的BMSCs活体示踪及其对股骨头坏死的治疗提供理论依据。
方法：1、采用红细胞裂解贴壁法培养扩增兔BMSCs,通过形态学观察及流式细胞仪鉴定证实得到的细胞为BMSCs。
2、采用含25ug/ml SPIO联合0.75ug/ml PLL的10%胎牛血清培养基(DMEM/F12)培养和标记兔BMSCs。用碱性磷酸酶检测法和茜素红染色法鉴定其成骨分化能力,油红O染色法鉴定其成脂分化能力。
3、采用液氮冷冻法建立兔股骨头坏死动物模型,通过形态学、组织学观察及MRI检测,表明股骨头坏死模型建立成功。
4、将SPIO标记的兔BMSCs采用原位移植方式移植入已建立的兔股骨头坏死区,行MRI检测,观察移植入坏死区的SPIO标记的BMSCs分别在SET2WI、FSE T2WI、GRE T2*WI三种扫描序列中信号变化情况；同时行组织学观察及高倍镜下缺损标本边缘新生骨小梁面积百分比行统计学分析。
结果：1、倒置相差显微镜可观察到活性及传代能力很强的大量梭形贴壁细胞,流式细胞仪鉴定结果表明所培养的细胞高表达BMSCs表面标志CD29、CD44、CD90；低表达CD34、CD45等造血细胞表面标志,证实所培养的细胞为兔BMSCs。
2、BMSCs在含25ug/ml SPIO联合0.75ug/ml PLL的10%胎牛血清培养基(DMEM/F12)中孵育,SPIO可安全有效的对BMSCs进行标记。成骨、成脂诱导分化实验显示,SPIO (25ug/ml)标记的BMSCs具备成骨、成脂分化能力。
3、液氮冷冻法造模术后4W,大体标本检查及MRI检测示冷冻区出现明显骨坏死现象；组织学检查示空骨陷窝明显增多,空骨陷窝率35%±2.2%,表明股骨头坏死模型建立成功。
4、SPIO标记的BMSCs原位移植侧,在SE T2WI、FSE T2WI、GRE T2*WI三种扫描序列中信号减低区即为实验中的靶点,MRI图像示靶点在三种扫描序列中信号强度均有不同程度的降低,而对照侧则无明显信号改变；移植术后6W,A*组(SPIO标记的BMSCs移植组)高倍镜下缺损标本边缘新生骨小梁面积百分比为(19.31±2.81)%,B*组(未标记的BMSCs移植组)为(20.87±1.55)%,两组间的差异无统计学意义(P>0.05)；较阴性对照组C*组(生理盐水移植组,骨小梁面积百分比(2.96±1.25)%)有显著差异(P<0.01)。
结论：SPIO标记的BMSCs原位移植治疗兔股骨头坏死与未标记的BMSCs一样有着同样的效果,MRI可对SPIO标记的BMSCs进行活体示踪检测,持续时间长达6周。
Object: SPIO(superparmagnetic iron oxide) were used to label rabbit BMSCs(bone marrow mesenchymal stem cells) as a magnetical probe. To observe the magnetic resonace tracing in vivo and the curative effects on femoral head necrosis with SPIO-labeled BMSCs plantation, and to provide the logical proofs for the magnetic resonace tracing in vivo and the treatment of femoral head necrosis.
Methods:1. Rabbit BMSCs were isolated and trained by Ficoll density gradient centrifugation and repetitive adherence to culture plate technique.And the cells were identified by morphology and flow cytometry technique.
2. BMSCs were incubated with DMEM/F12containing10%FBS supplanted with25ug/ml SPIO jointed0.75ug/ml PLL.To identify the osteogenic differentiation capacity by alkaline phosphatase assay and alizarin red vital staining and to identify the adipogenic differentiation capacity by oil red O dyeing.
3. Rabbit femoral head necrosis model was established by liquid nitrogen frozen method and confirmed by morphologica,histological and MRI detection.
4.After the SPIO-labeled rabbit BMSCs transplanted into the rabbit femoral head necrosis has been established via in situ way.Then,MRI tracing was conducted,the image changes of transplanted BMSCs marked by SPIO were observed among the three scanning sequences of SE T2WI,FSE T2WI and GRE T2*WI.Compared with control group by histology,to calculate the area percentage of newly formed bone trabecula in the defect samples through high power lens and make statistical analysis.
Results:1.We can detect a great quantity of adherent cells with great activity and generation capacity by inverted phase contrast microscope.Flow cytometric analysis results show that the cultured cells witn high level expression of CD29、 CD44、 CD90and low level expression of CD34、CD45. To confirm the cultured cells is rabbit BMSCs.
2.BMSCs were incubated with DMEM/F12containing10%FBS supplanted with25ug/ml SPIO jointed0.75ug/ml PLL,safely and effectively.The SPIO-labeled BMSCs and unlabeled BMSCs have the same osteogenic and adipogenic differentiation capacity.
3.Liquid nitrogen frozen method lead to obvious femoral head necrosis with empty lacunae rate of35%±2.2%in the4week,which suggests that Rabbit femoral head necrosis model was established successfully.
4. In situ cell transplantation group.the emerging and extinctive time of the decreased-signal region was different among the three scanning sequences of SE T2WI,FSE T2WI and GRE T2*WI.It was found that the decreased-signal region of the MRI scanning sequences was the target of the present experiment.No obvious signal change in the control side.The results of calculating the area percentage of newly formed bone trabecula in the defect samples through high power lens and statistical analysis showed that group A*(19.31±2.81)%has no significant difference with group B*(20.87±1.55)%(P>0.05),has significant differences compared with the negative control group C*(2.96±1.25)%(P＜0.01).
Conclusion:The SPIO-labeled BMSCs and unlabeled BMSCs have the same effect in the treatment of femoral head necrosis.The SPIO-labeled BMSCs can observe obviously by MRI detection in vitro and it lasted for six weeks.
引文
[1]Knippenberg M,Helder MN,Doulabi BZ,et al.Adipose Tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation[J].Tissue Eng,2005,(11-12):1780-788
[2]Gronthos S,Zannetcino AC,Hay SJ,et al.Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow[J].J Cell Sci,2003,116(Pt 9):1827-1835
[3]Studeny M,Marinj FC,Champlin RE,et al.Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors [J].Cancer Ras,2002,62(13):3603-3608
[4]Potapova I,Plotnikov A,Lu Z,et al.Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers [J]. Circ Res ,2004,94(7):952-959
[5]Frank JA,Kalish H,Jordan EK,et al.Color transformation and fluorescence of Prussian blue-positive cells:implications for histologic verification of cells labeled with superparamagnetic iron oxide nanoparticles[J].Mol Imaging,2007 May-Jun,6(3):212-218
[6]Friedenstein AJ,Chailakhyan RK,Gerasimov UV.Bone marrow osteogenic stem cells:in vitro cultivation and transplantation in diffusion chambers[J].Cell Tissue Kinet,1987,20(3):263-272
[7]Weissleder R.Molecular imaging:exploring the next frontier. Radiology,1999,212(3):609-614
[8]Shyu WC,Chen CP,Lin SZ,et al.Efficient tracking of non-iron-labeled mesenchymal stem cells with serial MRI in chronic stroke rats[J]. Stroke,2007;38(2):367-374
[9]Urdzikova L,Jendelová P,Glogarova K,et al.Transplantation of bone marrow stem cells as well as mobilization by granulocyte-colony stimulating factor promotes recovery after spinal cord injury in rats[J]. Neurotrauma,2006;23(9):1379-1391
[10]Park BH,Jung JC,Lee GH,et al.Comparison of labeling efficiency of different magnetic nanopartieles into stem cell[J].Colloids Surf A Physicochem Eng Asp,2008;313-314:145-149
[11]Bulte JW,Kostura L,Mackay A,et al.Feridex-labeled mesenchymal stem cells:cellular differentiation and MR assessment in a canine myocardial infarction model[J].Acad Radiol, 2005;12Suppl 1:S2-6
[12]Arbab AS,Bashaw LA,Miller BR,et al.Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular MR imaging[J].Radiology,2003;229(3):838-846
[13]Song M,Moon WK,Kim Y,et al.Labeling efficacy of superparamagnetic iron oxide nanoparticles to human neural stem cells:comparison of ferumoxides, monocrystalline iron oxide, cross-linked iron oxide (CLIO)-NH2 and tat-CLIO[J].Korean J Radiol,2007;8(5):365-371
[14]Babic M,Horak D,Trchova M,et al.Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling[J].Bioconjug Chem,2008;19(3):740-750
[15]Wang YJ,Hussain SM,Krestin GP,et al.Superparamagnetic iron oxide contrast agents:physcoehemical characteristics and application in MR imaging[J].Eur Radiology,2001,1 1(7):2319-2331
[16]Baklanov DV,Demuinck ED,Thompson CA,et al.Novel double contrast MRI technique for intramyocardial detection of percutaneously transplanted autologous cells[J].Mag Res Med,2004,52(6):1438-1442
[17]Muldoon LL,Sandor M,Pinkston KE,et al.Imaging,distribution,and toxicity of superparamagnetic iron oxide magnetic resonance nanoparticles in the rat brain and intra cerebral tumor[J].Neurosurgery,2005,57(4):785-796
[18]Arbab AS,Bashaw LA,Miller BR,et al.Intracytoplasmic tagging of cells with ferumoxides and transfection agent for cellular magnetic resonance imaging after cell transplantation:methods and techniques[J].Transplantation,2003,76(7):1123-1130
[19]Himes N,Min JY,Lee R,et al.In vivo MRI of embryonic stem cells in amousemodel of myocardial infarction[J].Magn Reson Med.2004,52(5):1214-1219
[20]孟增东,邱伟,胡彪,等.超顺磁性氧化铁纳米颗粒体外标记兔骨髓间充质干细胞的安全性以及MR]成像特征[J].中国组织工程研究,2012,6(16)：951-957
[21]Bartonicek J,Fric V,et al.Avascular Necrosis of the Femoral Head in Pertrochanteric Fractures:A Reporl of 8 Cases and a Review of the Literature[J].J Orthop Trauma,2007;21(4):229-236
[22]Mont MA,Eihnomr AT,SPonseller AP,et al.The trapdoor Procedure using Cortical and cancellous bone grafting of osteonecrosis of the femoral head[J].Bone Joint Surg,1998;80(1): 56-62
[23]Manggold J,Segri C,Backer K,et al.A new animal model of femoral head necrosis induced by introasseous injection of ethanol[J].Laboratory Animal,2002;36(2):173-180
[24]周强,李起鸿,杨柳,等.糖皮质激素诱导性股骨头坏死模型的血管改变[J].中华外科杂志,2000,38(3)：212-215
[25]彭吾训,龚跃昆,李世和,等.液氮冷冻建立犬股骨头缺损坏死模型的实验研究[J].中国康复医学,2005,5(20)：76-79
[26]王江泳,王保芝,崔慧先,等.改良液氮冷冻法制备家兔股骨头坏死模型的形态学研究[J].河北医科大学学报,2008,29(1)：5-8
[27]龚跃昆,彭吾训,李世和,等.股骨头缺血坏死动物模型的建立[J].昆明医学院学报,2004,25(2)：106-109
[28]Mont MA,Hungerford DS.Non-traumatic avascular necrosis of the femoral head[J].J Bone Joint Surg Am,1995:77(3):459-474
[29]Takaoka T,Yashioka T,Hosoya T, et al.The repair process experimental induced avascular necrosis of the femoral head in dogs[J].Arch Othop,1981;99:109-115
[30]王卫明,赵德伟.氮冷冻法制备股骨头缺血性坏死动物模型研究探讨[J].骨与关节损伤杂志,2001,16(6)：444-445
[31]Homan NE,Bischof JC.The cryobiology of cryosurgical injury [J].Urology,2002,60(2 Suppl 1):40-49
[32]Hernigou P,Beaujean F,Lambotte JC. Decrease of mesenchymal stem cell pool in the upper femoral extremity of patients with osteonecrosis related to corticosteroid therapy[J].J Bone Jiont Surg,1999,81B:349-355
[33]孙伟,李子荣,史振才,等.骨髓间充质干细胞对股骨头坏死缺损模型修复的组织学观察[J].中国康复理论与实践,2005,11(1)：4-6
[34]Peter J.Morris cell transplantation immunobiology.Foreign Med Sci Immunology.1994,17(5):264
[35]Henning TD,Boddington S,Daldrup-Link HE,et al.Labeling hESCs and hMSCs with iron oxide nanoparticles for non-invasive in vivo tracking with MR imaging[J].J Vis Exp,2008,31;(13), 685.
[36]Yamada M,Yang P.In vitro labeling of human embryonic stem cells for magnetic resonance imaging[J].J Vis Exp,2008,3(17),827.
[37]Saldanha KJ,Piper SL,Ainslie KM,et al.Magnetic resonance imaging of iron oxide labeled stem cells:applications to tissue engineering based regeneration of the intervertebral disc[J].Eur Cell Mater,2008,16:17-25.
[38]Homna T,Honmou O,Iihoshi S,et al.Intravenous infusion of immortalized human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat[J].Exp Neurol,2006,199(1):16-19.
[39]Jin XH,Yang L,Duan XJ,et al.In vivo MR imaging tracking of magnetic iron oxide nanoparticle labeled,engineered,autologous bone marrow mesenchymal stem cells following intra-articular injection[J].Joint Bone Spine.2008,75(4):432-438.
[40]薛静,高培毅,李晋,等.胎鼠神经干细胞超顺磁性氧化铁颗粒标记移植后MRI研究[J].放射学实践,2006,2：110-114.
[41]徐锋,吴惺,朱剑虹,等.骨髓基质干细胞靶向胶质瘤迁徙的MRI示踪研究[J].中华神经外科杂志,2007,2：684-689.
[42]王平,王建华,颜志平,等.超顺磁性氧化铁标记大鼠骨髓基质细胞经门静脉移植MRI活体示踪的研究[J].中华放射学杂志,2006,40：133-139.
[43]Ittrich H,Lange C,T?gel F,et al.In vivo magnetic resonance imaging of iron oxide-labeled,arterially-injected mesenchymal stem cells in kidneys of rats with acute ischemic kidney injury:detection and monitoring at 3T[J].J Magn Reson Imaging.2007,25(6):1 179-1191.
[1]Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone marrow osteogenic stem cels:in vitro cultivation and transplantation in diffusion chambers[J]. Cell Tissue Kinet,1987,20: 263─272.
[2]Weissleder R. Molecular imaging:exploring the next frontier[J]. Radiology,1999,212(3): 609─614.
[3]Buhe JW, Kraitehman DL. Monitoring cell therapy using iron oxide MR contrast agents [J]. Curt Pharm Biotechnol,2004,5(6):567-584.
[4]Corot C, Robert P, Idee JM, et al. Recent advances in iron oxide nanocrystal technology for medical imaging[J]. Adv Drug Deliv Rev,2006,58(14):1471─1504.
[5]Farrell E, Wielopolski P, Pavljasevic P,et al. Efects of iron oxide incorporation for long term cell tracking on MSC diferentiation in vitro and in vivo .Biochem Biophys Res Commun.2008;369(4):1076-1081.
[6]Shyu WC, Chen CP, Lin SZ, et al. Eficient tracking of non-iron-labeled mesenchymal stem cells with serial MRI in chronic stroke rats [J]. Stroke,2007,38(2):367-374.
[7]Urdzikova L, Jendelova P, Glogarova K, et al. Transplantation of bone marrow stem cells as well as mobilization by granulocyte-colony stimulating factor promotes recovery after spinal cord injury in rats[J].J Neurotrauma,2006,23(9):1379─1391.
[8]Park BH, Jung JC, Lee GH, et al. Comparison of labeling efficiency of different magnetic nanopartieles into stem cell[J].Colloids Surf A Physicochem Eng Asp,2008,313─314:145─149.
[9]Bulte JW, Kostura L, Mackay A, et al. Feridex─Labeled mesenchymal stem cells:cellular diferentiation and MR assessment in a canine myocardial infarction model [J]. Acad Radiol,2005, 12(1):2─6.
[10]Arbab AS, Bashaw LA, MillerBR, et al. Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular MR imaging[J]. Radiology,2003,229(3):838─846.
[11]Song M, Moon WK, Kim Y,et al.Labeling efficacy of superparamagnetic iron oxide nanoparticles to human neural stem cells:comparison of ferumoxides,monocrystalline iron oxide,cross-linked iron oxide (CLIO)-NH2 and tat-CLIO. Korean J Radiol,2007,8(5):365-371.
[12]Babic M, Horak D, Trchova M,et al.Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling. Bioconjugate Chemistry.2008,19(3):740-750.
[13]Yeh TC, Zhang W, Ildstad ST, et al.In vivo dynamic MRI tracking of rat T-cells labeled with superparamagnetic iron-oxide particles. [J].Magn Reson Med,1995,33(2):200-208.
[14]Ittrich H, Lange C, Dahnke H, et al. Labeling of mesenehymal stem cels with diferent superparamagnetie particles of iron oxide and detectability with MRI at 3T[J]. Rofo,2005, 177(8):1151─1163.
[15]Ko IK, Song HT, Cho EJ, et al. In vivo MR imaging of tissue─engineered human mesenehymal stem cells transplanted to mouse:a preliminary study[J]. Ann Biomed Eng,2007, 35(1):101-108.
[16]Arbab AS, Bashaw LA, Miler BR, et al. Intraeytoplasmie tagging of cells with fernmoxides and transfeetion agent for cellular magnetic resonance imaging after cel transplantation:methods and techniques[J]. Transplantation,2003,76(7):1123─1130.
[17]Himes N, Min JY, Lee R, et al. In vivo MRI of embryonic stem cells in a mouse model of myocardial infarction[J]. Magn Reson Med,2004,52(5):1214-1219.
[18]Bos C, Delmas Y, Desmouliere A, et al. In vivo MR imaging Of intravaseularly injeeted magnetically labeled mesenehymal stem cells in rat kidney and liver[J]. Radiology,2004,233(3): 781─789.
[19]Dai GH, Xiu JG, Zhou ZJ, et al. Efect of superparamagnetie iron oxide labeling on neural stem cell survival and proliferation[J]. Nan Fang Yi Ke Da Xue Xue Bao,2007,27(1):49─51, 55.
[20]Arbab AS, Yocum GT, Rad AM, et al. Labeling of cels with ferumoxides-protamine sulfate complexes does not inhibit function or diferentiation capacity Of hematopoietic or mesenchymal stem cells[J]. NMR Biomed,2005,18(8):553─559.
[21]刘永浩,郭亮,陈剑华,等.超顺磁性氧化铁标记猪骨髓间充质干细胞的体外MR成像研究.实用放射学杂志,2008,24(3)：390-395.
[22]Namur J, Chapot R, Pelage JP,et al.MR imaging detection of superparamagnetic iron oxide loaded tris-acryl embolization microspheres. J Vasc Interv Radiol,2007,18(10):1287-1295.
[23]王常珺,卢姗,冯铭, 等.超顺磁性氧化铁标记人Flk-1+CD31-CD34-间充质干细胞对细胞增殖及向神经细胞分化的影响[J].基础医学与临床,2008,28(9)：930-935.
[24]Kostura L, Kraitehman D L, Mackay A M, et al. Feridexlabeling of mcsenchymal stem cells inhibits chondrogenesis but not adipogenesis or osteogenesis[J]. NMR Biomad,2004,17(7): 513─517.
[25]陈小伍,方驰华,刘胜军,等.超顺磁性纳米铁粒子标记骨髓基质干细胞及对其分化能力的影响[J].中华神经医学杂志,2006,5(3)：253—257.
[26]Jendelova P, Herynek V, Urdzikova L, et al. Magnetic resonance tracking of transplanted bone marrow and embryonic stem cells labeled by iron oxide nanoparticles in rat brain and spinal cord. J Neurosci Res,2004,76:232-243.
[27]Sykova E, Jendelova P. Magnetic resonance tracking of implanted adult and embryonic stem cells in injured brain and spinal cord. Ann NY Acad Sci,2005,1049:146-160.
[28]魏俊吉,王任直,陆菁菁,等.超顺磁性氧化铁标记骨髓间充质干细胞治疗大鼠脑卒中的磁共振活体追踪[J].中国医学科学院学报,2007,29(1)：73-77.
[29]冯铭,王任直,朱华,等Ferumoxide标记Flk1+CD31-CD34-人骨髓间充质干细胞及其在食蟹猴脑内移植示踪观察[J].中国医学科学院学报,2008,30(5)：559-563.
[30]刘宇,邓宇斌,张德胜,等.蛛网膜下腔移植SPIO标记的MSCs在大鼠脊髓损伤模型的MR活体示踪研究.中国医学影像技术,2009,25(11)：1933-1936.
[31]Kraitchman DL, Heldman AW, Atalar E, Amado L, et al. In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infaretion. Circulation.2003.107:2290 ─2293.
[32]黄浙勇, 葛均波,杨姗,等.超顺磁性氧化铁标记间充质干细胞心肌移植的磁共振成像研究.中华心血管病杂志,2007,35(4)：344-349.
[33]顾玉梅,朱伟,魏盟.超顺磁性氧化铁纳米粒子标记心脏内骨髓问充质干细胞及活体磁共振示踪的实验研究.实用医学杂志,2009,25(2)：185-187.
[34]居胜红,滕皋军,陆海华,等.大鼠间充质干细胞超顺磁性氧化铁标记和经脾移植后的MR示踪研究.中华放射学杂志,2006,40(2)：127-132.
[35]孙军辉,滕皋军,居胜红,等.肾功能衰竭大鼠超顺磁性氧化铁标记骨髓间充质干细胞肾脏移植MR活体示踪的研究.中华放射学杂志,2006,40(2)：144-148.
[36]Saldanha KJ, Piper SL, Ainslie KM, et al.Magnetic resonance imaging of iron oxide labeled stem cells:Applications to tissue engineering based regeneration of the Intervertebral disc[J].Eur Cell Mater,2008,16:17-25
[37]谭树森,贾长青,刘振宁,等.经超顺磁性氧化铁标记的自体BMSCs兔椎间盘内存活时间研究.中国修复重建外科杂志,2009,23(11)：1355-1359.
[38]曹烨,吴小涛,王运涛,等.骨髓间充质干细胞的体内示踪及其修复椎间盘退变的实验研究.东南大学学报(医学版),2009,28(4)：269-273.
[39]Sobajima S, Vadala G, Shimera A, et al. Feasibility of stem cell therapy for intervertebral disc degeneration[J]Spine,2008,8(6):888-896.
[40]金旭红,杨柳,段小军,等.应用超顺磁性氧化铁粒子标记组织工程关节软骨种子细胞及在体磁共振示踪的研究.中华医学杂志,2007,87(45)：3213-3218.