胶质细胞源性神经营养因子和神经营养素-3双基因转染大鼠骨髓间充质干细胞体外诱导分化为神经样细胞的研究

Investigation on Inducing Rat Bone Marrow Mesenchymal Cells Transfected by Glial Cell Line-Derived Neurotrophic Factor and Neurotrophin-3 Gene into Neuron-Like Cells in vitro

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 inducing rat bone marrow mesenchymal stem cells（BMSCs） which were transfected by glial cell line-derived neurotrophic factor（GDNF） and neurotrophin-3（NT-3） gene to differentiate into neuron-like cells in vitro and the change of expression of GDNF and NT-3.Methods BMSCs were isolated by the whole bone marrow culture,characterized by flow cytometry of CD90 and CD45.BMSCs with GDNF and NT-3 gene were transfected,and the expression of green fluorescent protein（GFP） and morphological changes were measured with fluorescence microscope.The expression of neuronalmarkers neural specific enolase（NSE）,neurofilament（NF） and glial cell marker glial fibrillary acidic protein（GFAP） were detected by immunofluorescence assay.The changes of GDNF and NT-3 protein expressions were quantified with Western blot.The control group was BMSCs without GDNF and NT-3 gene.Results BMSCs were cultured and purified in vitro.The cultured BMSCs were positive for CD90（92.7%） and negative for CD45 on flow cytometry.After the induction of differentiation,BMSCs became round or cone-shaped with distinctive outgrowth of protrusions.These cells revealed neuron-like morphological changes,and most of these cells were intertwined into a network structure.Immunofluorescence assay showed positive expressions of NSE and NF without the expression of GFAP in experimental group,while the cells showed no expressions of NSE,NF and GFAP in control group.The expressions of GDNF and NT-3 protein were highly increased.Conclusions Genetically modified BMSCs of co-expressing GDNF and NT-3 were able to differentiate into neuronal-like cells and express nerve markers.There provides an experimental basis for gene therapy to treat nervous system-related disorders,such as Hirschsprung′s disease.