miR-let-7b慢病毒载体促进大鼠骨髓间充质干细胞向神经细胞分化

miR-let-7b Lentiviral Vector Promote the Differentiation of Bone Marrow Mesenchymal Stem Cells into Nerve Cells

微小RNA在生命体生长、衰老的过程中起着重要的作用,参与骨髓间充质干细胞(MSCs)重要的生物学进程,包括增殖、分化、信号转导和死亡等。该文探讨了miR-let-7b对MSCs来源神经细胞的调控作用。体外分离、扩增大鼠MSCs,通过细胞形态学观察、表面标志物的流式细胞仪检测进行鉴定。将MSCs分为miR-let-7b+组、miR-let-7b-组和对照组,分别转染miR-let-7b慢病毒载体、Anti-rno-miR-let-7b Inhibitor载体及不进行任何转染操作,实时荧光定量PCR(RTq PCR)检测各组细胞miR-let-7b的表达水平,确认转染情况。多因子联合法诱导各组MSCs向神经细胞分化,RT-q PCR检测神经细胞标志物MAP-2的表达,免疫细胞化学染色检测神经原特异性烯醇化酶(NSE)的表达和各组MSCs的神经细胞分化率。分离培养的MSCs在镜下呈长梭形或成纤维细胞样,CD90、CD44阳性表达率均大于90%,CD45的表达不足2%,证实得到的细胞即为MSCs。RTq PCR结果显示,与对照组相比,miR-let-7b+组的miR-let-7b表达水平升高,miR-let-7b-组几乎未检测到miR-let-7,提示miR-let-7b载体及miR-let-7b Inhibitor载体均成功转染了MSCs。经诱导分化后,与对照组相比,miR-let-7b+组神经细胞标志蛋白SIM312、Gap43、MBP和NSE与对照组相比表达水平显著升高(P<0.05);miR-let-7b-组SIM312、Gap43、MBP和NSE表达水平与miR-let-7b+组相比具有明显差异(P<0.05)。结果提示,miR-let-7b可以促进MSCs向神经细胞分化,通过控制miR-let-7b的水平可以调控MSCs的神经细胞分化率。

Micro RNAs play an important role in the process of growth and senescence of living organisms, and participate in the important biological processes of MSCs, including proliferation, differentiation, signal transduction and death. The effect of miR-let-7 family members on the differentiation of bone marrow mesenchymal stem cells into neurons can promote stem cell transplantation. To investigate the role of miR-let-7 b in promoting the differentiation of rat bone marrow mesenchymal stem cells into nerve cells, the rat miR-let-7 b lentiviral vector and Anti-rno-miR-let-7 b Inhibitor vector were transfected into rat bone marrow mesenchymal stem cells in vitro. The experiment was divided into control group（Mi R-let-7 b lentiviral） and miR-let-7 b-group（transfected with Anti-rno-miR-let-7 b Inhibitor）. Furthermore, the effects of all-trans retinoic acid（RA）, basic fibroblast growth factor（b FGF） and epidermal growth factor（EGF） were used to induce bone marrow. The expression of miR-let-7 b in three groups of cells was compared by Real-time quantitative PCR（RT-q PCR）. The expression of NSE was detected by immunocytochemical staining. The expression of MAP-2 m RNA was detected by RT-q PCR. The expression of CD90 and CD44 was more than 90% and the expression of CD45 was less than 2%. The results showed that the cells were MSCs. RT-q PCR results showed that the miR-let-7 b expression level in the miR-let-7 b＋ group was higher than that in the control group, and miR-let-7 was hardly detected in the miR-let-7 b-group, suggesting that miR-let-7 b vector and miR-let-7 b Inhibitor vector were successfully transfected with MSCs. The expression of SIM312, Gap43, MBP and NSE protein in miR-let-7 b＋ group was significantly higher than these in control group（P0.05）. Meanwhile, The expression of SIM312, Gap43, MBP and NSE protein had significantly differences in the groups with or without miR-let-7（P0.05）. The results suggest that miR-let-7 b can promote the differentiation of MSCs into neurons and regulate the differentiation rate of MSCs by controlling the level of miRlet-7 b.