Let-7b 慢病毒载体构建及对大鼠骨髓间充质干细胞诱导分化为神经细胞的影响

摘要

目的：探讨Let‐7b过表达和Let‐7b抑制慢病毒载体在体外诱导大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Let‐7b过表达和Let‐7b抑制慢病毒载体并感染大鼠MSCs ，筛选最适感染复数（MOI）；实验分未感染组、感染组1（感染LV‐rno‐Let‐7b‐up）、感染组2（感染LV‐rno‐Let‐7b‐inhibition）、阴性对照组（感染空病毒）；采用法舒地尔诱导感染后大鼠M SCs分化为神经元细胞。倒置荧光显微镜下观察M SCs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）的表达变化；PT‐PCR检测MAP‐2 mRNA的表达变化；MTT 法检测细胞存活率。结果倒置显微镜下观察大鼠LV‐rno‐Let‐7b‐up和LV‐rno‐Let‐7b‐inhibition慢病毒载体感染成功，MOI值为10，感染3 d时大鼠LV‐rno‐Let‐7b‐up慢病毒感染率最高，细胞存活率较高，感染率可达（92.1±4.3）％；MOI为20，感染5 d时大鼠LV‐rno‐Let‐7b‐inhibition慢病毒感染率最高，细胞存活率较高，感染率可达（90.3±4.2）％。法舒地尔可以诱导MSCs向神经细胞分化，感染组1诱导MSCs为神经细胞显著高于阴性对照组和感染组2，NSE、MAP‐2表达率明显高于阴性对照组和感染组，差异有统计学意义（P＜0.05）。结论结论 LV‐rno‐Let‐7b‐up可更高效感染大鼠 MSCs ，感染LV‐rno‐Let‐7b‐up后大鼠MSCs经法舒地尔诱导向神经细胞分化比率增加。%Objective To investigate the role of Let‐7b in inducing bone marrow mesenchymal stem cell(MSCs) differenti‐ation into neurons.Methods The lentiviral vector of Let‐7b up (LV‐rno‐Let‐7b‐up) and inhibition (LV‐rno‐Let‐7b‐inhibition) was constructed and transfected into rat MSCs.The cells were divided into non‐transfected group ,transfected group 1(trans‐fected with LV‐rno‐Let‐7b‐up) ,transfected group 2 (transfected with LV‐rno‐Let‐7b‐inhibition) and negative control group (transfected with empty virus ).MSCs were treated with Fashudier 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‐specific enolase(NSE) and MAP‐2 were measured by immunocytochemical method.The viability of MSCs was deter‐mined by MTT method.Results In LV‐rno‐Let‐7b‐up the best transfection efficiency(up to 92.1% ± 4.3% ) and survival rate appeared when multiply of infection (MOI) was 10 and on 3rd day.In LV‐rno‐Let‐7b‐inhibition the best transfection efficiency (up to 90.3% ± 4.2% ) and survival rate appeared when MOI was 20 and on 5th day.Fashudier induced MSCs to differentiate into neurons and the best efficiency of the induction was observed in transfected group 1.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 LV‐rno‐Let‐7b‐up has high transfection efficiency in rat MSCs.Higher differentiation rate from rat MSCs to neurons is induced by Fashudier after the cells are transfected with LV‐rno‐Let‐7b‐up.