Vaspin通过PI3K/AKT通路介导BMSCs微环境改变在关节炎中的机制研究

Osteoarthritis(OA) is a very common chronic disease characterized by joint pain and hypofunction, but the etiology and pathogenesis of OA are not definite, there are no especially effective therapies to prevent or reverse the progression of OA, this depends on the study of pathogenesis. In our previous study, we found there were the functional abnormality of bone marrow mesenchymal stem cells (BMSCs) from OA patients in proliferative and differentiation capability. The functional state of BMSCs greatly depended on the bone marrow microenvironment. As one of the important factors in constituting the microenvironment, our studies have shown there were abnormal expression of Vaspin in OA, which appear to be higher in severe OA. The OA patients are candidates for knee arthroplasty procedures, bone marrows were aspirated from femur during surgeries, and we found there were Vaspin expression in bone marrows, which shown a negative relation with TNF-α, and a positive relationship with pAkt, the latter is a key molecule in PI3K/AKT signalling pathway. It should serve as a reminder that Vaspin might participate in the regulation of BMSCs microenvironment, whether this is done by PI3K/AKT signalling pathway is still waiting for further researching. Therefore, based on the preliminary experimental results, the purpose of this study is to establish OA animal model in rats, explore the effects of Vaspin on bone marrow microenvironment of BMSCs, illustrate the impact of Vaspin on the proliferation and chondrogenic differentiation abilities of BMSCs, and review the molecule mechanism and signal control.

骨关节炎（OA）是以关节疼痛和功能减退为特征的慢性病，其病因尚未完全明确，其治疗有赖于发病机制的研究。我们前期研究发现，OA来源的骨髓间充质干细胞（BMSCs），无论增殖能力还是向软骨细胞分化能力，都较正常人降低；BMSCs的功能状态，受骨髓微环境影响极大，脂肪细胞作为骨髓微环境的重要组成，我们研究发现其分泌的脂肪因子Vaspin在OA中存在异常表达，重度OA患者的Vaspin水平更高；OA患者行关节置换时，股骨髓腔中Vaspin的表达与TNF-α呈负相关，而与PI3K/AKT通路中pAkt呈正相关，提示Vaspin可能参与了BMSCs微环境调节，而是否是通过PI3K/AKT通路介导的仍需进一步研究。因此我们在前期工作基础上，拟通过构建OA动物模型，从在体和体外两方面探索Vaspin对BMSCs微环境的影响，阐述其对BMSCs增殖和分化能力的影响和相关分子信号机制。

骨髓微环境是由基质细胞及细胞因子组成的动态网络，骨髓间充质干细胞（BMSCs）是骨髓微环境中基质细胞的重要组成，除了自我更新外，还能分化为成骨细胞、脂肪细胞、软骨细胞等，是软骨和软骨下骨的重要前体细胞来源。BMSCs的增殖和分化受骨髓微环境的影响，在OA的发病中具有重要意义。Vaspin是脂肪细胞分泌的脂肪因子，具有调节机体代谢、炎症反应及成骨细胞分化和凋亡等作用。OA骨髓微环境中Vaspin的分泌特征，及其对BMSCs增殖、向软骨细胞分化和ECM分泌的影响，目前尚不清楚。.本研究的主要内容和结果：1.比较晚期OA和股骨颈骨折患者骨髓液中Vaspin和TGF-β1的表达差异，发现OA患者骨髓微环境中Vaspin和TGF-β1的表达水平均降低，且Vaspin和TGF-β1的表达间呈正相关；2.使用CCK-8方法检测Vaspin对BMSCs和软骨细胞增殖能力的影响，发现Vaspin刺激能促进BMSCs和软骨细胞的增殖能力，且呈剂量依赖性；3.使用Real-time PCR和Western Blot方法检测在上述过程中，Vaspin对Akt的表达和活化的影响，发现Vaspin能增加Akt的表达和活化，使用Akt特异性抑制剂API-2能抑制上述促进作用；4.探索在BMSCs成软骨细胞诱分化过程中，Vaspin刺激对软骨细胞分化和ECM分泌关键基因Sox9、COL2A1和COMP转录和表达的影响，发现Vaspin刺激能促进Sox9、COL2A1和COMP的转录和表达；5.Wistar大鼠腹腔注射Vaspin蛋白，检测Vaspin刺激后软骨和软骨下骨中Akt的表达，发现Vaspin蛋白腹腔注射可增加软骨下骨和软骨陷窝中Akt的表达。.本研究最终得出以下结论：1.OA骨髓微环境中Vaspin存在低表达特征，参与构成OA特定的骨髓微环境；2.Vaspin能增加Akt的表达和活化，促进BMSCs和软骨细胞的增殖，在OA的病理过程中具有保护作用；3.Vaspin对软骨细胞分化和ECM分泌过程中关键基因的转录和表达具有促进作用，有利于关节软骨稳态的维持；4.Wistar大鼠腹腔注射重组Vaspin蛋白，可增加软骨下骨和软骨陷窝中Akt的表达，提示重组Vaspin蛋白或通过转基因方法高表达内源性Vaspin，可影响PI3K/Akt信号通路，对BMSCs的增殖和向软骨细胞分化产生促进作用。