脑源性神经营养因子基因转染骨髓间充质干细胞体外诱导分化为神经元样细胞初步观察

A Preliminary Study on the Differentiation of Bone-marrow Mesenchymal Stem Cells into Neuron-like Cells in Vitro after Transfected by Brain-derived Neurotrophic Factor Gene

目的探讨骨髓间充质干细胞(bone-marrow mesenchymal stem cells,BMSCs)转染人脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因后在体外分化为神经元样细胞的功能。方法克隆人BDNF基因并构建其真核表达载体;分离培养5只豚鼠BMSCs,观测其形态并用流式细胞仪鉴定;将人BDNF基因电穿孔法转染BMSCs,G418筛选后用维甲酸诱导分化,免疫细胞化学法对分化细胞进行鉴定。结果分离培养的细胞具有典型的BMSCs形态和标志,转染诱导后的细胞表达神经元特异性烯醇化酶、巢蛋白(Nestin)和胶质纤维酸性蛋白,并能分泌BDNF。结论BDNF基因转染的BMSCs在体外能分化为神经元样细胞,电穿孔法能提高转染率,RA有促诱导作用。

Objective To investigate the function of bone--marrow mesenchymal stem cells（BMSCs） differ- entiating into neuron--like cells in vitro after transfected by human brain--derived neurotrophic factor （BDNF） gene. Methods Human BDNF genes were cloned and recombinant peDNA3.1（--）--BDNF plasmids were construc- ted. BMSCs from five guinea pigs were isolated and cultured while their morphologies were observed by microscope. The surface antigen was detected by flowcytometry. BDNF genes were transfected into BMSCs with electroporation, and the transfected BMSCs were induced by ratinoic acid（RA）after bolted by Geneticin--418（G418）, then the dif- ferentiated BMSCs were identified by immunocytochemistry. Results The culture cells demonstroted the typical mor- phology and surface antigen of BMSCs. The transfected cells expressed neuron-- specific enolase（NSE）, Nestin and glial fi- brillary acid protein（GFAP） also secreted BDNF. Conclusion BMSCs transfected by BDNF genes can differentiate into neu- ron- like cells in vitro, electroporation can enhame the transfection efficiency, RA can promote cell induction.