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该文研究了在诱导小鼠间充质干细胞C3H10T1/2细胞成骨分化过程中miR-155的作用及其是否是通过调控BMP9/Smad(bonemorphogenetic protein 9/drosophila mothers against de-capentaplegic)信号通路发挥作用。在诱导C3H10T1/2细胞成骨分化过程中,采用实时定量PCR(Real-time quantitative PCR,q RT-PCR)检测miR-155水平的变化。转染miR-155模拟剂(miR-155)至C3H10T1/2细胞后,miR-155水平显著增高(P<0.001),而转染其抑制剂(anti-miR-155)至C3H10T1/2细胞后,miR-155水平显著降低(P<0.001)。转染后成骨诱导培养基诱导成骨7 d,碱性磷酸酶(alkaline phosphatase,ALP)活性及染色结果显示,miR-155能显著降低C3H10T1/2细胞成骨分化过程中的ALP活性(P<0.01)、减弱ALP染色,而anti-miR-155则能逆转其作用。成骨诱导14 d茜素红S染色结果显示,miR-155组钙盐结节较对照组少,下调miR-155的水平后,钙盐沉积结节增多。q RT-RCR检测结果显示,miR-155显著降低BMP9 m RNA水平(P<0.001),且miR-155组成骨基因Runx2和ALP表达均显著低于对照NC组(P<0.05、P<0.01)。Western blot检测BMP9、Runx2和p-Smad1/5/8蛋白质水平,结果显示,miR-155组蛋白质水平均显著降低(P<0.01、P<0.05、P<0.001)。该研究结果提示,miR-155对C3H10T1/2细胞成骨分化的抑制作用可能是通过抑制BMP9/Smad信号通路发挥作用的。
This study investigated the role of miR-155 in the induction of osteogenic differentiation of mouse mesenchymal stem cell line C3H10T1 / 2 and whether it exerted its effects through the regulation of the BMP9 / Smad / drosophila mothers versus de-capentaplegic signaling pathway effect. During the osteogenic differentiation induced by C3H10T1 / 2 cells, miR-155 levels were detected by real-time quantitative PCR (q RT-PCR). The level of miR-155 was significantly increased after transfected with miR-155 mimics (miR-155) to C3H10T1 / 2 cells (P <0.001), while the transfection of its anti-miR-155 to C3H10T1 / 2 cells , miR-155 levels were significantly lower (P <0.001). After transfection for 7 days, the activity of alkaline phosphatase (ALP) and the staining results showed that miR-155 could significantly decrease the ALP activity in osteogenic differentiation of C3H10T1 / 2 cells (P <0.01), weakened ALP staining, while anti-miR-155 reversed its effect. Alizarin red S staining induced osteogenic induction at 14 d showed that there were fewer calcium nodules in miR-155 group than those in control group. After the level of miR-155 was down-regulated, calcium deposition increased. q RT-RCR results showed that miR-155 significantly decreased the level of BMP9 mRNA (P <0.001), and the expressions of Runx2 and ALP in osteoblasts of miR-155 group were significantly lower than those in NC group (P <0.05, P <0.01) . The protein levels of BMP9, Runx2 and p-Smad1 / 5/8 were detected by Western blot. The results showed that the protein levels of miR-155 were significantly decreased (P <0.01, P <0.05, P <0.001). The results suggest that the inhibitory effect of miR-155 on osteogenic differentiation of C3H10T1 / 2 cells may be through the inhibition of BMP9 / Smad signaling pathway.