壳聚糖导管复合自体骨髓间充质干细胞修复大鼠13mm坐骨神经缺损  

Chitosan conduits combined with autologous mesenchymal stem cells to repair 13-mm sciatic nerve defects

作　　者：廖文[1,2] 刘淑红[3] 张世强[4] 李章华[5] 焦建宝[1] 张玉富[6] 赵永岐[3] 王聪[1] 范明[3] 

机构地区：[1]河北大学附属医院骨科 [2]解放军军事医学科学院基础医学研究所,北京市100850 [3]解放军军事医学科学院基础医学研究所 [4]河北医科大学第三医院骨科 [5]武汉大学附属人民医院骨科 [6]北京大学医学部积水潭医院

出　　处：《中国组织工程研究与临床康复》2007年第18期3517-3522,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：河北省科技攻关项目(062761837);国家八六三生物技术领域课题(2001AA216031);北京市二四八工程重大创新项目(H010210190123)~~

摘　　要：目的:观察壳聚糖神经导管复合自体骨髓间充质干细胞,构建组织工程化人工神经,修复大鼠13mm坐骨神经缺损的效果。方法:实验于2002-10/2004-08在北京军事医学科学院基础医学研究所九室及河北大学附属医院中心实验室完成。实验分组:Wistar大鼠30只按随机数字表法分成3组,实验组、细胞外基质凝胶组和生理盐水对照组,每组10只。壳聚糖神经导管桥接大鼠右侧13mm坐骨神经缺损。实验组无菌条件下抽取股骨髓腔内骨髓组织,贴壁分离法纯化、增殖骨髓间充质干细胞,按1×109L-1细胞浓度与细胞外基质凝胶混合,植入自体神经再生室内;细胞外基质凝胶组神经再生室内植入细胞外基质凝胶,生理盐水对照组神经再生室内植入生理盐水。实验评估:术后12周观察以下指标:①大体观察:观察再生神经。②坐骨神经功能指数测定:选择印迹清晰的足印分别测量正常足(N)和伤侧足(E)的3个指标:足印长度(PL):足尖到足跟的最大距离;足趾宽度(TS):第1～5趾的距离;中间足趾宽度(IT):第2～4趾的距离。结果精确到0.1mm。坐骨神经指数=-38.3[(EPL-NPL)/NPL]+109.5[(ETS-NTS)/NTS]]+13.3[(EIT-NIT)/NIT]-8.8。坐骨神经指数值为0～-11%表示神经功能完全正常,-100%表示神经功能完全丧失,-11%～-100%表示部分神经功能恢复。③电生理检测:检测患侧小腿三头肌肌电图,检测再生神经的运动传导速度和波幅。④腓肠肌湿质量恢复率:腓肠肌湿质量恢复率(%)=手术侧腓肠肌湿质量/对侧正常腓肠肌湿质量×100%。⑤神经组织学检查:苏木精-伊红及Loyez苏木精髓鞘染色,光镜下观察再生神经横断面再生神经髓鞘的形成;Loyez苏木精髓鞘染色及Bielschowsky改良镀银染色,观察纵向切片再生神经神经纤维;神经细丝蛋白免疫组化染色,观察再生的神经轴突染色。随机选取部分正常坐骨神经作为正常对照。⑥透射电镜观察:再生神经的超微AIM： To evaluate the effectiveness of chitcoan conduits seeded with autologous mesenchymal stem cells to bridge 13-mm sciatic nerve defect by tissue engineering technique in rats. METHODS： The experiment was completed in the Ninth Room, Institute of Basic Medical Sciences, the Academy of Military Medical Sciences and the Central Laboratory of Hebei University Affiliated Hospital from October 2002 to August 2004. Totally 30 adult Wistar rats were randomly divided into 3 groups： experimental group, extracellular matrix （ECM） group and saline control group, each group contained 10 rats. A 13-mm-long nerve defect was caused in the right sciatic nerve and then was bridged by chitosan conduit. The thigh bone marrow was taken out from animals of experimental group under sterile condition, mesenchymal stem cells were purified using adherence method, cultivated and proliferated, then grafted into nerve regenerating room by 1 ×10^9 L^-1 cell density after mixed with ECM; In ECM and control groups, ECM and physiological saline were grafted into nerve regenerating room, respectively. Twelve weeks after operation, the experimental evaluation was made, including：（1）gross observation for regenerative nerve.（2）Sciatic nerve function index （SFI）： Clear footprint of both normal feet （N） and experimental feet （E） were measured in three indices： podogram length （PL）： the maximal distance from tiptoe to heel; toe spreadn （TS）： the distance between the first and the frith toes; inter-toes distance （IT）： the distance between the second and the fourth toes. All the results were calculated with the fidelity of 0.1 mm. SFI=-38.3[（EPL-NPL）/NPL]＋109.5[（ETS-NTS）/NTS]]＋13.3[（EIT-NIT）/NIT]-8.8. And SFI score of 0 to -11% stood for normal functional nerve; -100% for complete loss of nerve function; -11% to -100% SFI for partly recovery of nerve function. （3）Electrophysiological detection： The motor nerve conduction velocity and wave amplitude were determined using tric

关 键 词：组织工程 神经 再生 骨髓间充质干细胞 

分 类 号：R318[医药卫生—生物医学工程]