力学环境miRNA-365协同Ihh调控MSCs成软骨分化的分子机制

Mechanical loadings play essential roles in chondrogenic differentiation of MSCs by up-regulating expression of Ihh gene and miRNA-365, though their regulation mechanisms are not interpreted. Our previous research works found that the effects of Ihh over-expression on promoting generation of cartilage and inhibiting cartilage aging were dependent on the existence of mechanical environments of MSCs. Thus we make the hypothesis that mechano-sensitive miRNA-365 has synergistic effects with Ihh to regulate chondrogenic differentiation of MSCs in mechanical loadings environment.. In the study, cyclic mechanical loadings will be imposed on MSCs with Ihh over expression, and FACS of cell sorting technique will be used to indentify the most reasonable pattern of mechanical loadings inducing chondrogenic differentiation of MSCs. Experimental techniques including transfection, Realtime-PCR, FISH, Luciferase analysis and Multiplex western blot will be utilized to analyze the the crosstalking mechanisms of miRNA-365’s target gene HDAC4 with Ihh and other signal channels. Then MSCs transfected with miRNA-365 mimics/inhibitor and combined with chitosan will be transplanted to repair the cartilage defect of New Zealand rabbit in order to study the effects of miRNA-365-Ihh on chondrogenesis of MSCs in vivo.. The project may establish a foundation exploring the molecular mechanism of synergistic effects of miRNA-365 and Ihh during regulating chondrogenic differentiation of MSCs in mechanical loadings environment and will provide a new view angle for research of cartilage tissue engineering.

机械力上调Ihh基因及miRNA-365表达，对诱导MSCs成软骨分化起着重要作用，但其调控机制尚未阐明。我们预实验结果提示Ihh过表达对MSCs促进软骨生成、抑制软骨老化作用依赖力学环境存在。因此我们提出假说：力学环境下力学敏感miRNA-365协同Ihh调控MSCs成软骨分化。本课题我们将通过循环应力作用过表达Ihh的MSCs，FACS分选软骨细胞以确定诱导MSCs成软骨分化的最佳力学模式。采用转基因、FISH、Realtime-PCR、Luciferase、Multiplex Westernblot等解析miRNA-365 靶基因与Ihh及多信号通路的串话机制，后采用动物软骨缺损模型研究miRNA-365-Ihh修饰的MSCs在体内局部的作用。本项目为研究力学环境miRNA-365协同Ihh调控MSCs 成软骨分化的机制奠定基础，为MSCs构建软骨组织工程的研究提供新的思路。

关节软骨损伤是常见的临床疾病，软骨组织缺乏血运、神经及淋巴组织，一旦损伤后自我再生修复能力有限。骨髓间充质干细胞（bone marrow mesenchymal stem cell， BMSC）是目前常见应用于软骨组织工程的种子细胞，可以通过成软骨分化修复软骨损伤。与原本软骨相比，BMSCs成软骨分化所形成的软骨在形态、组织成分以及生物性能上存在一定差异。所以，如何使BMSCs更好的成软骨分化是软骨组织工程中亟需解决的难题。研究发现，适当的力学刺激在成软骨分化工程可以促进BMSCs成软骨分化，当前研究的力学刺激主要聚焦压缩于力和剪切力。截至目前，周期性牵张力对BMSCs成软骨分化的影响尚未知晓。本课题的目的是探讨周期性牵张力环境下miR-365对骨髓间充质干细胞成软骨分化影响以及对Ihh力学敏感基因促进成软骨的协同作用。通过比较比较二维培养环境，三维培养环境和CTS力学作用环境，发现周期性牵张力不仅能促进BMSCs成软骨分化，还能减缓软骨细胞肥大和老化。进一步发现，周期性牵张力刺激会引起miR-365表达升高，而通过抑制miR-365的表达可以减弱周期性牵张力促进骨髓间充质干细胞分化成软骨能力。随后我们通过生物信息分析以及荧光素酶基因报告实验确HDAC4是miR-365的潜在靶点，并且发现过表达miR-365在抑制HDAC4的同时可以提高HDAC4下游Ihh的表达。在大鼠膝关节缺损体内实验中，与单纯体外诱导分化的BMSCs作用相比，我们发现周期性牵张力作用下诱导分化的BMSCs和miR-365过表达诱导分化的BMSCs，具有更好的软骨缺损修复效果。 综上所述，我们发现周期性牵张力可以通过miR-365来调控HDAC4和Ihh的表达来促进BMSCs成软骨分化和修复软骨缺损。这为软骨组织的重建提供了新的思路。