釉基质蛋白诱导MC3T3-E1细胞成骨分化过程中微小核糖核酸的表达

目的 探讨微小核糖核酸(micro ribonucleicacid,miRNA)是否参与到釉基质蛋白(EnamelMatrix Derivative,EMD)诱导的小鼠前成骨细胞系MC3T3-E1细胞成骨分化的过程,并对发生显著变化的miRNA进行分析.方法 将MC3T3-E1用含EMD(刺激组)/不含EMD(对照组)的培养液培养0天、7天和14天,检测碱性磷酸酶(alkaline phosphatase,ALP)活性,实时聚合酶链式反应(RT-PCR)技术分析成骨分化标记物ALP、骨涎蛋白(bone sialoprotein,BSP)和骨钙素(osteocalcin,OC)的信使RNA(mRNA)水平的表达变化.利用基因芯片技术分析miRNA的相对表达.结果 0天、7天、14天的ALP染色结果显示随着培养时间的增加,两组ALP活性均逐渐增强,EMD刺激组ALP活性增强较对照组更为明显.RT-PCR结果表明,与对照组相比,ALP、BSP、OC的mRNA表达量均显著增加,ALP与OC均于14天时表达量达到峰值,BSP则于7天时处于峰值表达,EMD刺激组MC3T3-E1成骨活性更强;各期11个miRNA表达上调,28个miRNA表达下调,其中miR-335-5p,miR-503已被证实可参与促进骨形成,而miR-30家族(miR-30a,-30b,-30c和-30d)则被证实参与抑制成骨.结论 miRNA参与EMD诱导的MC3T3-E1细胞的成骨分化过程,这一发现可以为了解EMD促进成骨分化的机理及临床应用提供指导.

Micro RNA expression during the osteogenic differentiation of MC3T3-E1 cells by Enamel Matrix Derivative induction

Objective In this study, we aimed to identify whether micro ribonucleicacid(micro RNA, mi RNA) participated in the process of osteogenic differentiation induced by Enamel Matrix Derivative( EMD) and to anlayze the significant change of mi RNAs in response to EMD. Methods The mouse pre-osteoblast cell line MC3T3-E1 was cultured with/without(the EMD stimulating /negative control group)EMD induction(0, 7 and 14 days), and osteogenic differentiation was detected by alkaline phosphatase(ALP) staining and real-time quantitative polymerase chain reaction( RT-PCR) analysis. Taq Man Micro RNA Arrays(0, 7 and 14 days) was used to analyse the relative expression of mi RNAs. Results The results of the ALP staining indicated that EMD could enhance osteogenic differentiation in MC3T3-E1 cells, the results of EMD group were more significant than the negative group. The results of RT-PCR showed that when compared to the negative group, the expression of ALP, bone sialoprotein(BSP) and osteocalcin(OC) increased significantly. In addition, both ALP and OC reached their peak levels at 14 days, while BSP reached its peak at 7 days. These findings indicate that EMD enhanced osteogenic differentiation in MC3T3-E1 cells. At 0, 7 and 14 days, 11 mi RNAs were upregulated and 28 mi RNAs were downregulated significantly during the process of osteogenic differentiation induced by EMD. And mi R-335-5p, mi R-503 have been confirmed to promote bone formation, while mi R-30 family have been confirmed to be negative regulators of osteogenic differentiation. Conclusion mi RNAs were involved in EMD induced MC3T3-E1 osteogenic differentiation. These findings could be used to understand the mechanism of the regulatory process of osteogenic differentiation induced by EMD and guide clinical application.