载GDNF基因的骨髓间充质干细胞促周围神经再生的实验研究

发布时间：2018-12-05 15:01

【摘要】：目的：探讨骨髓间充质干细胞(BMSCs)经胶质细胞源性神经营养因子(GDNF)复合绿色荧光蛋白(GFP)定向诱导向神经细胞分化及其促进大鼠外周损伤神经的再生。方法：应用骨髓间充质干细胞的贴壁特性复合连续换液法获取原代细胞,培养获取纯度及活力较高、形态较为统一的第三代细胞,第三代骨髓间充质干细胞(BMSCs)经载GDNF基因复合绿色荧光蛋白(GFP)的质粒以脂质体为介导转染诱导分化,记录对比细胞活力及形态学方面的变化,通过免疫荧光鉴定神经元特异性烯醇化酶(NSE)的荧光表达确定转染结果。钳夹法统一建立坐骨神经损伤模型,将诱导培养后形态学较为良好的细胞浓缩后植入周围神经再生室模型,术后定期观察统计坐骨神经功能指数(SFI)、完整解剖实验大鼠双侧腓肠肌称重,测量靶肌肉湿质量率、损伤神经再生室段解剖出神经组织行自动病理图文系统分析神经再生纤维、靶肌肉冰冻切片行DAPI染色观察细胞萎缩、核凝聚状态及形态学变化、神经损伤再生室组织取材电镜观察神经显微结构变化。结果：实验结果证实骨髓间充质干细胞经胶质细胞源性神经营养因子转染诱导分化组相关数据均较未经诱导对照组显著,这充分肯定了GDNF基因的确切作用,经胶质细胞源性神经营养因子转染的骨髓间充质干细胞生长活跃分化良好,神经元特异性烯醇化酶(NSE)特异表达,神经营养因子转染诱导组坐骨神经扫描电镜下超微结构再生更加明显,细胞核染色可见实验组能明显抑制靶肌肉的萎缩。结论：骨髓间充质干细胞(BMSCs)经胶质细胞源性神经营养因子(GDNF)复合绿色荧光蛋白(GFP)诱导后可向神经细胞分化,特异表达神经细胞相关表面标志。定向诱导分化后的细胞注入大鼠坐骨神经损伤模型可较对照组明显改善坐骨神经相关指数。
[Abstract]:Aim: to investigate the differentiation of bone marrow mesenchymal stem cells (BMSCs) into nerve cells induced by glial cell-derived neurotrophic factor (GDNF) combined with green fluorescent protein (GFP) and promote the regeneration of peripheral injured nerves in rats. Methods: the adherent characteristics of bone marrow mesenchymal stem cells (BMSCs) were used to obtain primary cells by continuous fluid exchange. The third generation cells with high purity and activity were obtained. The third generation of bone marrow mesenchymal stem cells (BMSCs) (BMSCs) was induced to differentiate by liposome mediated transfection with GDNF gene and green fluorescent protein (GFP). The changes of cell viability and morphology were recorded. The fluorescent expression of neuron-specific enolase (NSE) was confirmed by immunofluorescence. The sciatic nerve injury model was established by clamp method, and the model of sciatic nerve function index (SFI),) was observed regularly after operation. The model of sciatic nerve injury was implanted into peripheral nerve regeneration chamber with better morphology after induction and culture. The sciatic nerve function index (SFI),) was observed regularly after operation. In the complete anatomical experiment, the bilateral gastrocnemius muscle was weighed, the wet mass rate of target muscle was measured, and the nerve tissue was dissected to analyze the nerve regeneration fiber by automatic pathological graph and text system. The atrophy was observed by DAPI staining in frozen section of target muscle. Nuclear condensation and morphological changes were observed. The microstructure of nerve was observed by electron microscope. Results: the data of bone marrow mesenchymal stem cells induced by glial cell derived neurotrophic factor transfection were significantly higher than that of uninduced control group, which fully confirmed the exact role of GDNF gene. Bone marrow mesenchymal stem cells transfected with glial cell-derived neurotrophic factor (GDNF) were well differentiated and expressed neuron-specific enolase (NSE). The ultrastructure regeneration of sciatic nerve was more obvious under scanning electron microscope in the group induced by neurotrophic factor, and the atrophy of target muscle was obviously inhibited in the experimental group by nuclear staining. Conclusion: bone marrow mesenchymal stem cells (BMSCs) induced by glial cell derived neurotrophic factor (GDNF) combined with green fluorescent protein (GFP) can differentiate into neural cells and specifically express neuron-related surface markers. The model of sciatic nerve injury induced by directional differentiation could improve the sciatic nerve related index significantly compared with the control group.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R688

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