MiR-124-1促进大鼠骨髓间充质干细胞神经分化的实验研究

miR-124-1 promotes neural differentiation of rat bone marrow mesenchymal stem cells

摘要:

目的：探讨miR-124-1在大鼠骨髓间充质干细胞（MSCs）神经分化中的作用。方法：构建大鼠miR-124-1慢病毒载体及Anti-rno-miR-124* Inhibitor载体，体外感染大鼠MSCs，筛选最适感染复数（MOI）。实验分为对照组（未行感/转染组）、miR-124-1+组（感染miR-124-1慢病毒）及miR-124-1-组（转染Anti-rno-miR-124* Inhibitor）。采用β-巯基乙醇诱导MSCs分化为神经细胞。倒置荧光显微镜下观察感染后荧光表达情况，MTT法检测感/转染后各组细胞存活率；免疫细胞化学法、RT-PCR法、Western blot法检测各组诱导6 d后神经细胞标记物β3微管蛋白（β3 tubulin）、神经微管结合蛋白（MAP-2）、胶质纤维酸性蛋白（GFAP）的表达变化。结果：倒置荧光显微镜下观察大鼠miR-124-1慢病毒感染成功，MOI值为30。miR-124-1+组慢病毒载体感染MSCs 2 d后，实时定量PCR显示miR-124-1的表达显著上调（P<0.01）；Anti-rno-miR-124* Inhibitor载体转染24 h后，细胞存活率下降，实时荧光定量PCR显示转染后miR-124-1的表达显著下降（P<0.01）。β-巯基乙醇可诱导MSCs分化为神经细胞，miR-124-1+组诱导6 d后细胞的β3 tubulin、MAP-2表达率显著高于其他两组（P<0.01），miR-124-1-组β3 tubulin、MAP-2的表达率显著低于对照组（P<0.01）；各组GFAP表达率均较低（<1%）。结论：miR-124可能促进大鼠MSCs的神经分化。

Abstract:

OBJECTIVE: To study the effects of miR-124-1 on neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs). METHODS: MSCs cells were assigned into three groups: control (uninfected and untransfected), miR-124-1+ (infected with miR-124-1), and miR-124-1- (transfected with Anti-rno-miR-124* Inhibitor). MSCs were induced by β-mercaptoethanol (β-ME) to differentiate into neurons. The fluorescence expressed by infected MSCs was observed under an inverted fluorescence microscope. MTT method was used to measure cell survival rate after transfection or infection. Immunocytochemistry, RT-PCR and Western blot methods were used to detect the expression of β3 tubulin, MAP-2 and GFAP 6 days after β-ME induction. RESULTS: The expression of miR-124-1 in the miR-124-1+ group was significantly higher 2 days after infection of lentivirus vector compared with the control group (P<0.01). In the miR-124-1- group, the cell survival rate and the miR-124-1 expression level decreased significantly 24 hrs after transfection of anti-rno-miR-124* inhibitor (P<0.01). After 6 days of β-ME induction, the protein and mRNA expression levels of β3 tubulin and MAP-2 in the miR-124-1+ group were much higher than the other two groups (P<0.01); while the expression levels of β3 tubulin and MAP-2 in the miR-124-1-group were lower than the control group (P<0.01) . The expression of GFAP in the three groups was weak (<1%). CONCLUSIONS: miR-124 might promote neuronal differentiation of rat MSCs.