27nt-miRNA对间充质干细胞向血管平滑肌细胞分化的影响

Effect of 27nt-miRNA on the differentiation of mesenchymal stem cells into vascular smooth muscle cells

为探讨27nt-miRNA对间充质干细胞向血管平滑肌细胞分化影响,构建27nt-miRNA过表达、反义序列Anti-27nt-miRNA以及阴性对照的表达质粒,慢病毒包装后分别转染人脐带间充质干细胞(hUCMSC),加入Ⅳ型胶原诱导hUCMSC定向分化为血管平滑肌细胞。四唑盐(MTT)比色法检测分化后细胞活力,免疫细胞化学染色法检测分化后细胞SM22α(兔抗平滑肌22α,smooth muscle 22α)的表达,Western印迹法和RT-PCR检测分化后细胞内的SMA (兔抗平滑肌肌动蛋白,smooth muscle actin) mRNA、SM 22α mRNA及其蛋白质表达情况。经检测,27nt-miRNA过表达分化组与阴性对照组相比,细胞活力下降20.48%(P<0.05),SMA mRNA、SM22α mRNA及其蛋白质表达量明显升高(P<0.05);而Anti-27nt-miRNA分化组细胞活力上升了18.07%(P<0.05),SMA mRNA、SM22α mRNA及其蛋白质表达量下降(P<0.05)。综上所述,27nt-miRNA能够促进间充质干细胞向血管平滑肌细胞分化,并且抑制分化后的细胞活力。

To investigate the effect of 27nt-miRNA on the differentiation of mesenchymal stem cells into vascular smooth muscle cells. The highly expression plasmids of 27nt-miRNA and anti-27nt-miRNA, and negative control plasmids were constructed, packaged with lentivirus and transfected into human umbilical cord mesenchymal stem cells(hUCMSCs). Collagen IV was added to induce hUCMSCs differentiation into blood vessel smooth muscle cells(VSMCs). The cell viability was measured by MTT assay. The expression of SMA, SM22α at mRNA and protein levels was determined by RT-PCR, immunocytochemical staining and Western blotting. Compared with the negative control group, the viability of the 27nt-miRNA overexpression group was decreased by 20.48%(P<0.05), and the expression of SMA mRNA and SM22α mRNA and protein was significantly increased(P<0.05);the viability of Anti-27nt-miRNA group was increased 18.07%(P<0.05), and the expression of SMA mRNA and SM22α mRNA and protein was decreased(P<0.05). In summary, 27nt-miRNA promotes mesenchymal stem cells differentiation into vascular smooth muscle cells and inhibits cells viability.