DMOG通过Rho/ROCK与Wnt信号cross-talk调控BMSCs成骨成脂分化治疗早期激素性股骨头坏死机制研究

The mechanism of the steroid-induced necrosis of femoral head (SANFH) is unclear and the treatment for early-stage SANFH is lacking. Dimethyloxalylglycine(DMOG) ,which inhibit the degradation of hypoxia inducible factor(HIF) and activate the HIF signaling pathway, is one of the prolyl hydroxylase inhibitors. Our previous study revealed that DMOG promoted MSCs osteogenesis via Rho/ROCK signaling and facilitated the steroid-induced necrosis of femoral head (SANFH) repair of rabbits,while the wnt inhibitor factor(WIF),which inhibited the canonical Wnt pathway,influenced the BMSCs osteogenesis induced by DMOG. Literature showed that DMOG activated the Rho/ROCK pathway independent of HIF and our study also showed that DMOG and hypoxia had different effect on the osteogenesis of BMSCs. The above studies suggest that DMOG may regulate the BMSCs balance of osteogenesis and adipogenesis through the cross-talk between Rho/ROCK and Wnt signaling in the manner of HIF independence for SANFH repair. Our item plan, at molecular,cellular and body level,to discuss the exact effect of the HIF and the cross talk between Rho/ROCK and Wnt pathway during the BMSCs regulation of osteogenesis and adipogenesis induced by DMOG using molecular biology and imaging techniques, and novel therapeutic target spot for the prophylaxis and treatment of SANFH will be raised through our research.

激素性股骨头坏死（SANFH）发病机制不明，缺乏有效的早期治疗手段。二甲基乙二酰基甘氨酸（DMOG）是脯氨酸羟化酶抑制剂，能抑制低氧诱导因子（HIF）降解而激活低氧通路。我们研究发现DMOG通过Rho/ROCK通路促进骨髓间充质干细胞（BMSCs）成骨分化且促进兔SANFH修复，同时Wnt通路抑制剂WIF影响DMOG介导的BMSCs成骨分化；研究显示DMOG能不依赖HIF激活Rho/ROCK通路且我们也发现DMOG与低氧对BMSCs成骨分化的作用不一致。以上研究提示DMOG可能以HIF非依赖的方式通过Rho/ROCK与Wnt间cross-talk调控BMSCs成骨成脂分化并促进SANFH修复。本项目拟在分子、细胞及整体动物水平，采用分子生物学及影像学技术探讨Rho/ROCK与Wnt间cross-talk及HIF在DMOG介导的BMSCs成骨成脂分化调控中作用，为SANFH的防治提供新靶点。

按所确定和进一步完善的技术路线，我们把整个实验研究分成十个部分开展。（一）小鼠骨髓间充质干细胞的原代培养与鉴定；（二）DMOG影响BMSCs细胞成骨成脂分化最佳时间的探讨；（三）HIF-1α、β-actin、RhoA低表达慢病毒的构建；（四）DMOG与si-NC对BMSCs成骨成脂分化的影响；（五）Wnt/β-catenin在DMOG对BMSCs成骨成脂分化及Rho/ROCK通路活化的影响；（六）Rho/ROCK通路在DMOG对BMSCs成骨成脂分化及Wnt/β-catenin通路活化的影响；（七）观察同时阻断Rho/ROCK与Wnt/β-catenin通路后，DMOG对BMSCs成骨成脂分化的影响；（八）阻断HIF-1α后，DMOG对BMSCs成骨成脂分化及Rho/ROCK与Wnt/β-catenin信号通路的影响；（九）阻断HIF-1α后，分别或同时阻断Rho/ROCK与Wnt/β-catenin通路，DMOG对BMSCs成骨成脂分化及Rho/ROCK与Wnt/β-catenin信号通路的影响。（十）DMOG及DMOG联合BMSCs经髓芯减压移植对兔激素性股骨头坏死的修复作用。我们的实验研究明确了DMOG能够促进间充质干细胞成骨分化而抑制其成脂分化。DMOG是通过激活RhoA/ROCK与经典Wnt/β-catenin信号通路发挥上述作用，且两条通路之间存在cross-talk; HIF-1α也参与了DMOG介导的间充质干细胞的成骨与成脂分化调控，但是DMOG介导的RhoA/ROCK与Wnt/β-catenin信号通路的活化是不依赖于HIF-1α的参与; DMOG及DMOG联合BMSCs经髓芯减压移植对兔早期激素性股骨头坏死的修复具有明显的促进和改善作用。尽管由于疫情影响了实验进程，推迟了论文的撰写进程，但是也顺利完成了任务书既定的所有实验，培养了两名硕士研究生，发表一篇北大核心期刊论文，另外有二篇SCI论文及一篇北大中文核心期刊论文正在投稿中，一人晋升高级职称，一人入选云南省医学学科后备人才。