模拟微重力环境Hedgehog调控MSCs成软骨分化的信号串话机制

Mesenchymal stem cell (MSCs) is the most promising seed cell in cartilage tissue engineering, however until now its clinical application is still challenging and many technological problems are unsolved. The Hedgehog signal pathway has been reported playing an important role in regulation of multiple differentiation from MSCs, though its regulation mechanisms of chondrogenesis differentiation from MSCs in physiological behavior are not interpreted.Our previous research works found microgravity rotating culture system was more effective for the construction of tissue engineering cartilage in vitro, and the chondrogenic feature proteins expression were obviously up-regulated in MSCs modified with Hedgehog gene.Thus we make the hypothesis that Hedgehog's crosstalking with other signal pathways may happen and promote the process of chondrogenesis from MSCs under physiological conditions. This project basing on the MSCs' cultivating mode of the mimic microgravity three dimensions environment, will carry out profound studies, which utilize the techniques of transgene, siRNA, Realtime-PCR,FISH，Luciferase analysis and Multiplex western blot, on the signal transduction of Ihh and Shh during chondrocyte differentiation from MSCs，and on the mechanisms of Hedgehog's crosstalking with other signal passways of Wnt, TGF-β/BMP,FGF and IGF. Then the chondrocyte modified with Ihh/Shh or Ihh/Shh siRNA,and combined with chitosan will be transplanted to repair the cartilage defect of New Zealand rabbit. The effects of signal pathway of Hedgehog on chondrogenesis in vivo will be explored. The project may establish a foundation exploring the effects of Hedgehog signal pathway on the chondrogenesis from MSCs，and will provide a new view angle for research of cartilage tissue enineering.

Hedgehog信号对骨髓间充质干细胞（MSCs）多向分化起重要调控作用，但生理状态下Hedgehog对MSCs成软骨分化的调控机制尚未阐明。我们预实验结果提示模拟微重力环境促进MSCs成软骨分化；经Hedgehog修饰的MSCs软骨特征性蛋白表达增强。因此我们提出假说：生理环境下Hedeghog可能与多种信号通路串话协调完成MSCs的成软骨分化。本课题我们将于模拟微重力环境采用转基因、siRNA干扰、FISH、Realtime-PCR、Luciferase、Multiplex Westernblot等对Ihh、Shh在MSCs软骨分化中的调控及与Wnt、TGF-β/BMP、FGF、IGF的信号串话机制开展深入研究，后采用动物软骨缺损修复模型，研究Hedgehog在体内局部的作用。本课题将为研究Hedgehog对MSCs成软骨分化的调控机理奠定基础，为软骨组织工程基础研究提供新的思路。

关节软骨由于其损伤后修复能力非常有限，所以一直是骨科临床及实验研究的重点方向。骨髓间充质干细胞（bone marrow mesenchymal stem cell， BMSC）是目前公认的理想的软骨组织工程种子细胞来源，但影响其软骨分化的参与因素很多。研究表明，hedgehog、IGF-1、TGF-β及BMP等信号通路都有参与BMSCs软骨分化的调控，但具体机制仍不清楚。本课题的目的是探讨微重力环境下hedgehog对骨髓间充质干细胞成软骨分化影响及其与IGF-1、BMP通路在诱导成软骨方面的相互影响作用。BMSCs转染腺病毒72小时后荧光显微镜下可见明显绿色荧光，转染效率达 95%。实时荧光定量 PCR及ELISA检测转染后Ihh和Shh高表达，加入BMP-2及IGF-1后，可明显促进Ihh的表达。RCCS诱导环境下转染Ihh及Shh诱导10天及21天Wertern blot检测collagenⅡ，ANCN表达水平高于二维诱导环境，且Ihh与IGF-1及BMP-2共培养时collagenⅡ，ANCN表达水平也高于对照组。BMP-2通路可通过刺激Smo及Gli-1表达，从而刺激Hedgehog通路下游基因表达。IGF-1通路可刺激Gli-1表达，刺激Hedgehog通路下游基因表达。体内研究显示BMSCs共转染Ihh和Shh后，6周及8周后软骨缺损修复情况明显优于单转染Ihh和Shh组。模拟微重力环境下（RCCS），共转染Ihh和Shh可明显提高骨髓间充质干细胞成软骨能力及软骨损伤修复能力，BMP-2及IGF-1也能协同刺激Hedgehog通路促进骨髓间充质干细胞成软骨分化。