miR17-92簇在BMP9诱导间充质干细胞成骨中的作用及机制研究

Bone morphogenetic protein is a cytokines commonly used in repairing bone defect. BMP9，as one of the members of the BMP family，since it was identified as the strongest osteogenic potential BMP，BMP9 has been applied to the bone research. But the specific mechanism of the BMP9-induced osteogenic differentiation of MSCs has not been fully elucidated. In recent years，many studies have shown that bone regulation small RNA molecules involved in the osteogenic differentiation of MSCs，which have brought a new dawn for the development of bone tissue engineering. Our study tries to find the miRNAs which participate in the BMP9-induced osteogenic differentiation of MSCs，in order to clarify the specific molecular mechanisms of BMP9-induced osteogenic differentiation of MSCs in miRNA level. MiRNA array was used to pre-screen the miRNAs which relate to BMP9-induced osteogenic differentiation of MSCs. The results show that miR17-92 cluster may be involved in the BMP9-induced osteogenic differentiation of MSCs.Next，we have test the function of all members of miR17-92 cluster which involve in the BMP9-induced osteogenic differentiation of MSCs，we have found that miR-17 could inhibit BMP9-induced osteogenic differentiation of MSCs in vitro and in vivo. On the basis of our previous study，we will determine the direct downstream target genes of miR-17 by bioinformatics combined with biological experiments，in order to futher explore the mechanism of BMP9-induced osteogenic differentiation of MSCs.In this study，by looking to the miRNAs participating in the BMP9-induced osteogenic differentiation of MSCs，we want to clarify the mechanism of BMP9-induced osteogenic differentiation of MSCs，and expect to enhance the BMP9-induced osteogenic differentiation of MSCs through sponge-17. Based on this，this study will provides the experimental fundamentals and theoretical basis in the application of BMP9 as a cytokines in repairing bone defect.

骨形态发生蛋白(BMP)是临床上修复骨缺损最为高效的生长因子。虽然BMP2、7已应用于临床，但因其副作用较多、患者经济负担重等缺点限制了二者的应用。课题组前期对人BMP家族14种因子(BMP2-BMP15) 成骨能力进行了系统比较，发现BMP9诱导间充质干细胞(MSCs)的成骨能力最强，但分子机制尚未完全阐明。我们前期通过微小RNA(miRNA)芯片筛选BMP9诱导MSCs成骨相关miRNAs，发现miR17-92簇多个成员均表达下调，由此提出miR17-92簇可能参与BMP9诱导MSCs成骨的假说。已通过体内、外实验初步表明该簇中miR-17抑制BMP9诱导MSCs成骨，后续拟通过生物学实验鉴定miR-17下游靶基因，进一步阐明BMP9诱导MSCs成骨的机制，试图通过靶向调节miR-17，增强BMP9诱导MSCs成骨的作用。期望通过本课题的研究，为研发更高效的生长因子提供弩实的实验基础。

本研究探讨了miR-17调控BMP9诱导MSCs成骨分化的现象及机制: .1.基于前期研究工作,本研究使用体内(裸鼠皮下成骨实验)、体外(ALP染色、ALP读数测定 ;茜素红染色)成骨实验正向(过表达miR-17)及反向(敲减miR-17,sponge miR-17) 对miR-17调控BMP9诱导的MSCs成骨分化的现象进行验证,结果发现miR-17能抑制BMP9诱导的MSCs成骨分化,而sponge miR-17能逆转此现象,说明miR-17在BMP9诱导的MSCs成骨分化的过程中起着重要的作用。.2.为了进一步探讨miR-17调控BMP9诱导MSCs成骨分化的机制,接下来通过双荧光素酶报告基因实验、real-time PCR实验筛选出Rb基因作为miR-17直接下游靶基因,并且通过体内(裸鼠皮下成骨实验)、体外(ALP染色、ALP读数测定;茜素红染色)成骨实验证实Rb能逆转miR-17抑制BMP9诱导MSCs成骨的作用。.骨再生机制是当前研究的热点,通过本项目的开展,在临床上可以通过运用Sponge miR-17来进一步增强BMP9成骨的作用,为临床治疗骨缺损类疾病奠定坚实的实验基础。