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目的探讨胶质细胞源性神经营养因子(GDNF)和神经营养素3(NT-3)双基因修饰诱导的大鼠骨髓间充质干细胞(BMSCs)分化为神经样细胞的可行性以及GDNF和NT-3的表达变化。方法全骨髓法分离培养BMSCs,流式细胞术检测BMSCs标志CD90和造血干细胞标志CD45。转染带荧光的GDNF和NT-3基因,在荧光显微镜下观察绿色荧光蛋白(GFP)的表达及细胞形态变化;免疫荧光检测神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF)和神经胶质酸性蛋白(GFAP)表达;Western blot检测细胞中GDNF及NT-3蛋白表达。对照组为未转染GDNF和NT-3基因的BMSCs。结果 BMSCs能在体外成功分离培养,细胞高表达CD90(92.7%),不表达CD45。诱导分化后,BMSCs胞体变圆,伸出明显突起,并可见多数细胞相互交织成网状结构,呈神经细胞样形态。免疫荧光标记检测可见实验组细胞表达NSE和NF,而不表达GFAP。而对照组阴性。Western blot检测可见细胞GDNF及NT-3蛋白表达增强。结论 GDNF和NT-3双基因修饰诱导的BMSCs可分化为神经样细胞并表达神经元的标志,为基因治疗神经系统疾病如先天性巨结肠提供实验基础。
Objective To investigate the feasibility of differentiation of rat bone marrow mesenchymal stem cells (BMSCs) induced by glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) into neuron- 3 expression changes. Methods BMSCs were isolated and cultured by whole bone marrow method. The BMSCs marker CD90 and hematopoietic stem cell marker CD45 were detected by flow cytometry. Fluorescent GDNF and NT-3 gene were transfected, and the expression of green fluorescent protein (GFP) and morphological changes of cells were observed under a fluorescence microscope. The levels of neuron-specific enolase (NSE), neurofilament (NF) And glial acidic protein (GFAP) expression; Western blot detection of GDNF and NT-3 protein expression. The control group was BMSCs without GDNF and NT-3 gene transfection. Results BMSCs could be successfully isolated and cultured in vitro. CD90 (92.7%) cells were highly expressed but not CD45. After induction of differentiation, BMSCs became round and protruded obviously, showing that most of the cells were intertwined into a network structure with a neuron-like morphology. Immunofluorescence staining showed that the experimental group of cells expressed NSE and NF, but not expressed GFAP. The control group was negative. Western blot results showed that the expression of GDNF and NT-3 protein increased. Conclusion The BMSCs induced by GDNF and NT-3 double gene modification can differentiate into neuron-like cells and express neurons, which provide experimental basis for gene therapy of neurological diseases such as Hirschsprung’s disease.