Let-7d慢病毒载体体外诱导大鼠骨髓间充质干细胞分化为神经细胞

Let-7d lentiviral vector induces the differentiation of rat bone marrow mesenchymal stem cells into neurons in vitro

背景:微小RNA在生命体生长、衰老的过程中起着重要的作用,了解微小RNA let-7d家族成员对骨髓间充质干细胞向神经细胞分化的影响可以对干细胞移植起促进作用。目的:探讨let-7d慢病毒载体在体外诱导大鼠骨髓间充质干细胞向神经细胞分化中的作用。方法:1构建大鼠慢病毒载体并感染大鼠骨髓间充质干细胞;实验分为未转染组、阴性对照组(感染空白慢病毒)、转染上调组(感染let-7d-LV)、转染下调组(感染let-7d-inhibition-LV)。2采用法舒地尔诱导转染后大鼠骨髓间充质干细胞分化为神经细胞,以免疫细胞化学染色检测NSE、MAP-2的表达变化;RT-PCR检测MAP-2mRNA的表达变化;MTT法检测细胞存活率。结果与结论:倒置荧光显微镜下观察let-7d慢病毒载体转染成功。法舒地尔可以诱导骨髓间充质干细胞向神经细胞分化,其中转染上调组的诱导效率与阴性对照组相比升高,NSE、MAP-2表达率上升(P<0.05);转染下调组的诱导效率与阴性对照组相比下降,NSE、MAP-2表达率下降(P<0.05)。结果表明let-7d慢病毒载体可以促进骨髓间充质干细胞向神经细胞的分化,通过控制let-7d的表达可以影响骨髓间充质干细胞向神经细胞的分化效率。

BACKGROUND：MicroRNA plays an important role in the process of growth and aging of living body. To know the role of let-7d in inducing bone marrow mesenchymal stem celldifferentiation into neurons can promote the stem celltransplantation. OBJECTIVE：To investigate the role of let-7d in inducing bone marrow mesenchymal stem celldifferentiation into neurons. METHODS：（1） The lentiviral vector of let-7d was constructed and transfected into rat bone marrow mesenchymal stem cells. The cells were divided into non-transfected group, negative control group （transfected with FU-RNAi-NC-LV）, transfected enhancement group （transfected with let-7d-LV）, transfected inhibition group （ transfected with let-7d-inhibition-LV）. （2） Rat bone marrow mesenchymal stem cells were treated with fasudil as an inducer for triggering the cells to differentiate into neurons. The expression of neuron-specific markers, neuron-specific enolase and microtubule-associated protein 2, were measured by immunocytochemical method. The mRNA expression of microtubule-associated protein 2 was detected by RT-PCR. The viability of bone marrow mesenchymal stem cells was determined by MTT method. RESULTS AND CONCLUSION：Under inverted fluorescence microscope, the cells were successful y transfected with let-7d. Fasudil induced bone marrow mesenchymal stem cells to differentiate into neurons. The transfection efficiency and expression levels of neuron-specific enolase and microtubule-associated protein 2 in transfected enhancement group were higher than those in the negative control group （P〈0.05）;while in the inhibition group, they were lower than those in the negative control group （P〈0.05）. These findings indicate that let-7d can promote the differentiation of bone marrow mesenchymal stem cells into neurons induced by fasudil, and by control ing the expression of let-7d we can influence the differentiation efficiency from bone marrow mesenchymal stem cells to neurons.