机械应力促进软骨前体细胞实现软骨再生修复的机制研究

Osteoarthritis is the fourth leading cause of human disability. Cartilage regenerative repair is the well-accepted core of treatment. Our previous study has found that mechanical stress could significantly promote the regeneration ability of cartilage progenitor cells which were recently found and considered as the main reparative cells in the cartilage.However,this promotion effect declined under the osteoarthritis microenvironment.The mechanism behind this phenomenon remains unresolved, which is also a bottleneck problem for improving the result of osteoarthritis treated by mechanical stress clinically. According to extensive literature review and our previous studies, MAPK and PI3K-Akt signal pathways are the major mechanism of chondrocyte repair. In addition, cartilage progenitor cells can be defferentiated toward chondrocyte phenotype.T herefore, we propose the following scientific hypothesis: MAPK and PI3K-Akt signal pathways are also the mechanotransduction mechanism of regeneration oriented cartilage repair mediated by cartilage progenitor cells, and the activation level and balance of the key molecules involved in the above-mentioned pathways directly affect the result of cartilage repair. In order to test our hypothesis,the well-proved osteoarthritis microenvironment experimental models in vitro and in vivo were established in our lab and further works are needed. We plan to do mechanical loading on the experimental models and to observe the expression of the above molecules by immunohistochemisty,gene transfection,RNA interference, immunoblotting, and so on. Our study will reveal the function of these molecules and the mechanism of the contribution of mechanical stress on cartilage repair. In summary, the results of our study would provide theoretical basis for improving the treatment result of osteoarthritis and developing a new therapeutic method targeting at the molecules.

骨关节炎是造成人类残疾的第四大原因，软骨再生修复是公认的治疗重点。我们的研究发现机械应力可以显著促进软骨中新发现的主要修复者软骨前体细胞的再生能力，但在骨关节炎微环境下其促进作用明显下降，说明炎症对软骨前体细胞的再生有抑制作用。这也是临床应用机械应力治疗骨关节炎的疗效难以提高的理论瓶颈。目前认为，MAPK和PI3K-Akt信号通路是软骨细胞修复的主要信号转导通路，而在软骨前体细胞能向软骨细胞表型分化的过程中，以上信号通路也起到关键作用。但骨关节炎症微环境是如何通过抑制这一信号通路来降低软骨前体细胞的再生能力的，机制仍然不清楚，我们的目的，就是借助于公认的模拟骨关节炎微环境的体外和体内模型，通过加载机械应力，对以上通路中关键分子进行研究，采用免疫定位、转染、干预和免疫印迹等方法进一步阐明关键分子在机械应力促进软骨再生修复中的作用机制，为提高骨关节炎的疗效和指导筛选新的治疗靶点提供理论依据。

第一部分 机械应力通过YAP促进炎性环境中的软骨前体细胞增殖.【目的】探索在含有IL-1β或骨关节炎（osteoarthritis,OA）关节液的炎性环境中施加周期性静水压(intermittent hydrostatic pressure ，IHP)对软骨前体细胞(（chondrogenic progenitor cells, CPCs）)增殖的影响。【主要研究内容】提取OA患者膝关节软骨中的CPCs，培养扩增后在海藻酸构建的支架上三维培养，在IL-1β与关节液塑造的炎症环境中施加IHP，western-blot验证信号通路蛋白的变化。【重要结果】0.01ng/ml IL1-β与20%关节液均抑制CPCs的增殖，施加IHP后可对抗其对细胞的抑制，流式细胞仪检测见G2+S期细胞增多。其机制可能为IHP激活HIPPO信号通路中的YAP所致，而MAPK、PI3K-AKT可能不是炎性环境下机械应力传导的主要通道。施加IHP后的免疫荧光观察见YAP在细胞核内的表达增多,电镜观察见细胞空泡化减少，在IL-1β中促进增殖基因表达,降低RUNX-2基因表达；在20%关节液中促进sox-9及CTGF基因表达，降低COLL IA1、RUNX-2的表达。【结论】在炎性环境下，通过适宜的机械应力可通过YAP促进CPCs的增殖，表现出更好的活性状态...第二部分 机械应力通过Hippo-YAP通路促进OA软骨的修复.【目的】探索适宜的机械应力与异常增高的机械应力对关节软骨的影响。【主要研究内容】切断大鼠的前交叉韧带构建OA模型，以微型外固定支架行膝关节牵开为关节牵引组，以微型外固定支架固定膝关节而不让其活动为关节固定组。【结果】关节牵引促进OA的软骨修复，而关节固定促进OA软骨损伤，组织学切片的Mankin评分显示牵引组评分最低，而固定组最高。免疫组化染色见关节牵引减少P-YAP，增加YAP的表达，而关节固定增加P-YAP,减少YAP的表达。【结论】关节牵引可提供适宜的机械应力，通过YAP来促进OA的软骨修复。..第三部分 P-YAP、TRPV4在OA与正常软骨中的差异表达 .OA软骨中P-YAP、TRPV4表达升高,从而认为Hippo通路被抑制，TRPV4的激活可能促进OA的进展。