Notch信号在盐酸法舒地尔诱导大鼠骨髓间充质干细胞向神经元分化过程中的作用

Effect of notch signaling on differentiation of rat marrow mesenchymal stem cells into neurons induced by fasudil hydrochloride

目的:探讨Notch信号通路在盐酸法舒地尔诱导大鼠骨髓间充质干细胞(MSCs)向神经元分化中的作用。方法:实验分为未转染组、转染组(转染Rn-Notch1-siRNA)、阳性对照组(转染Rn-MAPK-1 Control siRNA)及阴性对照组(转染Negative Control siRNA)等4组。采用盐酸法舒地尔诱导大鼠MSCs分化为神经元。倒置荧光显微镜下观察MSCs转染后荧光表达情况;RT-PCR检测Notch1、Hes1和MAPK1 mRNA的表达变化;免疫细胞化学法检测Notch1、神经元烯醇化酶(NSE)、神经微丝蛋白亚单位(NF-M)和胶质纤维酸性蛋白(GFAP)的表达变化;MTT方法检测细胞存活率。结果:①siRNA转染72h,MSCs荧光表达最强,转染率可达91.3%±4.2%;同时,转染组MSCs的Notch1和Hes1 mRNA转录下降(P<0.05);MTT提示转染组细胞存活率也显著减少(P<0.05)。②盐酸法舒地尔可以诱导MSCs向神经元分化,其中以转染组诱导效果最佳,NSE、NF-M的表达率显著的高于其它各组(P<0.05)。结论:盐酸法舒地尔在诱导大鼠MSCs向神经元分化过程中,可能存在Notch信号通路与RhoA/Rho激酶通路信号的协同作用,共同促进MSCs向神经元分化。

Objective：To investigate the effect of notch signaling on differentiation of rat bone marrow mesenchymal stem cells （MSCs） into neurons induced by fasudil hydrochloride. Methods： The experiments were divided into non-transfected group,transfected group （transfected with Rn-Notch1-siRNA）,positive control group （transfected with Rn-MAPK-1 Control siRNA） and negative control group （transfected with negative control siRNA）. Fasudil hydrochloride induced MSCs differentiating into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The expression of notch1 mRNA,Hes1 mRNA and MAPK1 mRNA in MSCs was detected by RT-PCR. The expression of Notch1 protein,NSE,neurofilament M （NF-M） and glial fibrillary acidic protein（GFAP）was detected by immunocytochemical method. The viability of MSCs was detected by MTT. Results： ①The fluorescence of MSCs was mostly displayed after transfection for 72 h and the efficiency of transfection was up to 91.3%±4.2%. Meanwhile ,the notch1 mRNA and Hes1 mRNA expressed by MSCs of transfected group were significantly decreased（P0.05）and MTT displayed that the viability of MSCs was also significantly reduced（P0.05）. ②Fasudil hydrochloride could induce MSCs differentiate into neurons and the best efficiency of induction was observed in the transfected group. There was higher expression of NSE and neurofilament-M （NF-M） than the other groups（P0.05）.Conclusion： There may be notch1 signaling and Rho/Rho GTPase signaling synergy on differentiation of rat bone marrow stromal cell into neurons induced by fasudil hydrochloride and they jointly promote the differentiation of MSCs into neurons.