【摘要】 目的了解骨形态发生蛋白4(BMP4)是否具有诱导成年大鼠脑神经干细胞(NSCs)向胆碱能细胞分化的效应。方法将从两月龄大鼠脑海马、纹状体等区域分离的细胞培养于含EGF、bFGF的DMEM/F12培养液中,在光镜下观察细胞的形态特征,并行Nestin细胞化学染色,24h后换成含有BMP4的培养液,培养8d时,行ChAT间接免疫荧光染色,在光镜下观察细胞形态的变化,并行FITC标记的流式细胞术检测NSCs分化。以脑立体定位术给成年大鼠右侧海马齿状回内注射BMP4或生理盐水0.5μL;14d后取大鼠脑组织切片,行ChAT免疫组织化学染色;以图像分析仪测量ChAT阳性细胞总面积。结果分离出的大鼠脑海马、纹状体等区的细胞约48%为Nestin阳性的NSCs。培养8d时,光镜下见加BMP4组约34%的细胞呈神经元形态特征;间接免疫荧光染色见呈较强绿色荧光的ChAT阳性细胞较多;FITC标记流式细胞术检测到16%的细胞呈ChAT阳性。而未加BMP4组呈ChAT阳性的细胞约7%,明显少于加BMP4组,且荧光较弱。给大鼠海马齿状回区脑组织中注射BMP4后14d时,可见该区细胞出现较高的胆碱能表达。结论 BMP4可在体外培养及体内注射条件下诱导大鼠脑神经干细胞向胆碱能细胞分化。更多还原

【Abstract】 Objective To explore the cholinergic-inducing effects of BMP4 for rat’s cerebral neural stem cells in vivo and in vitro.Methods Neural stem cells(NSC) which were isolated from two month’s old rat’s brain region were cultivated in a medium containing EGF and bFGF and then changed to cultivate with a BMP4-added medium after 24 hours.The expression of choline acetyltransferase(ChAT) of the cultivated cytes was observed by indirect immunofluorescence test and FITC-labeled Flow Cytometry at 8 days.In order to determine oriented induction of BMP4 in NSC differentiation in the rat hippocampus dentate gyrus,ChAT immunocytochemistry was performed at 14 days after BMP4 injection.Results After cultivated with BMP4-added medium,about 34% cells showed neuron-like character observed by microscope,and more positive cells were found in the experimental group cells.The fluorescence intensity was stronger,while less positive cells were found in the control group.The results of differentiational efficiency showed that about 16% cells in experimental group appeared ChAT-positive,while control group only 7%.The in vivo experiment showed that BMP4 injection into the rat hippocampus dentate gyrus increased cholinergic expression in the hippocampus.Conclusion BMP4 could induce cholinergic differentiation of neural stem cells in vitro and increase the number of cholinergic neurons in the adult rat hippocampus dentate gyrus.更多还原