AIM: To investigate the role of microRNA -9 in inducing bone marrow mesenchymal stem cell ( MSCs ) differentiation into neurons.METHODS : The lentiviral vector of microRNA - 9 - 1( microRNA - 9 - 1 - LV )was constructed and transfected into mouse MSCs.The cells were divided into non - transfected group, transfected group ( transfec:ted with microRNA -9 - 1 - LV ) and negative control group( transfected with FU - RNAi - NC - LV ).MSCs were treated with β - mercaptoethanol( β - ME ) as an inducer for triggering the cells to differentiate into neurons.The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope.The mRNA expression of microtublin - associated protein 2( MAP - 2 ) was detected by RT - PCR.The expression of neuron - specific markers , neuron - spec:ific enolase( NSE ), MAP - 2 and glial fibrillary acidic protein( GFAP ), were measured by immunocytochemical method.The viability of MSCs was determined by MTT method.RESULTS : The results of PCR confirmed successful construction of mouse microRNA -9 - 1 - LV.The virus titer was 1 × 1012 TU/L( TU, transduction unit ).The best transfection efficiency( up to 91.3％ ±4.2％ ) and survival rate appeared when multiply of infection( MOI )was 20 and on 4th day.β -ME induced MSCs to differentiate into neurons and the best efficiency of the induction was observed in transfected group.The expression levels of NSE and MAP - 2 in transfected cells were higher than those in the cells of other group( P ＜ 0.05 ).CONCLUSION : MicroRNA - 9 - 1 - LV has high transfection efficiency in mouse MSCs.Higher differentiation rate from mouse MSCs to neurons is induced by β - ME after the cells are transfected with microRNA -9 - 1 - LV.%目的:探讨microRNA-9-1在体外诱导小鼠骨髓间充质干细胞(MSCs)向神经细胞分化中的作用.方法:构建小鼠microRNA-9-1慢病毒载体(microRNA-9-1-LV)并感染小鼠MSCs,筛选最适感染复数(MOI);实验分为未感染组、感染组(感染microRNA-9-1-LV)、阴性对照组(感染FU-RNAi-NC-LV);采用β-巯基乙醇诱导感染后小鼠MSCs分化为神经细胞.倒置荧光显微镜下观察MSCs感染后荧光表达情况;采用免疫细胞化学染色检测神经元烯醇化酶(NSE)、神经微管结合蛋白(MAP-2)和胶质纤维酸性蛋白(GFAP)的表达变化;PT-PCR检测MAP-2 mRNA的表达变化;MTT方法检测细胞存活率.结果:(1)阳性克隆PCR证明小鼠microRNA-9-1慢病毒载体构建成功,孔稀释法测定病毒滴度为1×1012 TU/L.(2)倒置显微镜下观察小鼠microRNA-9-1慢病毒载体感染成功,MOI值为20,感染4 d时感染率最高,细胞存活率较高,感染率可达 91.3%±4.2%.(3)β-巯基乙醇可以诱导MSCs向神经细胞分化,其中以感染组诱导效果最佳,NSE和MAP-2的表达率显著高于其它各组(P＜0.05).结论:(1)MicroRNA-9-1-LV可高效感染小鼠MSCs.(2) 感染miRNA-9-1-LV后MSCs经β-巯基乙醇诱导向神经细胞分化比率增加.
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