重组人S100钙结合蛋白A4诱导类风湿关节炎成纤维样滑膜细胞产生血管内皮生长因子的机制研究

Mechanism of recombinant human S100 calcium binding protein A4 inducing vascular endothelial growth factors in rheumatoid arthritis fibroblast-like synoviocytes

目的 探讨重组人S100钙结合蛋白A4（rhS100A4）诱导类风湿关节炎成纤维样滑膜细胞（RAFLSs）产生血管内皮生长因子（VEGF）的机制. 方法 取行膝关节置换术的类风湿关节炎（RA）患者关节滑膜组织行体外RAFLSs培养;CCK-8法检测rhS100A4及其与哺乳动物雷帕霉素靶蛋白复合物1 （mTORC1）信号通路抑制剂雷帕霉素（Rap）共同作用对RAFLSs增殖的影响;免疫荧光法检测rhS100A4及其与Rap共同作用对RAFLSs表达VEGF的影响;rhS100A4及其与Rap共同作用刺激RAFLSs形成条件培养基（CM）,观察CM刺激人脐静脉内皮细胞（HUVECs）体外形成管腔的作用,检测S100A4血管生成能力;Western blot检测rhS100A4对RAFLSs mTORC1信号通路下游蛋白核糖体蛋白S6（S6）磷酸化水平的影响,分析rhS 100A4与Rap共同作用对RAFLSs S6蛋白磷酸化水平及VEGF蛋白表达的影响. 结果 rhS100A4促进RAFLSs增殖及VEGF蛋白水平表达,rhS100A4所形成CM促进HUVECs体外形成血管,Rap可抑制rhS100A4的上述生物学效应;rhS100A4激活RAFLSs mTORC1信号通路下游蛋白S6,使其磷酸化水平升高;rhS 100A4增强RAFLSs细胞S6蛋白磷酸化水平及VEGF蛋白水平表达的作用可被Rap抑制.结论 S100A4通过激活mTORC1信号通路促进RAFLSs产生VEGF.

Objective To investigate the mechanism of inducing production of vascular endothelial growth factors （VEGF） by recombinant human S100 calcium binding protein A4 （rhS100A4） in rheumatoid arthritis fibroblast-like synoviocytes （RAFLSs）.Methods Synovial tissue was sampled from the patients with rheumatoid arthritis （RA） undergoing knee arthroplasty for in vitro culture of RAFLSs.CCK-8 assay was conducted to detect the effect of rhS100A4 and the effect of its interaction with Rapamycin （Rap）,an inhibitor of mammalian rapamycin target 1 （mTORC1） signaling pathway,on the proliferation of RAFLSs.The effects of rhS100A4 and its interaction with Rap on the expression of VEGF in RAFLSs were detected by immunofluorescence.After rhS100A4 and its cooperation with Rap stimulated the conditioned medium （CM）produced by RAFLSs,the effect of CM on formation of lumen in human unbilical vein endothelial cells （HUVECs） in vitro was observed to detect the angiogenic ability of rhS100A4.Western blot was used to detect the effect of rhS100A4 on the phosphorylation of downstream ribosomal protein S6 （S6） in the mTORC1 signaling pathway in RAFLSs and to analyze the effects of rhS100A4 and Rap on phosphorylation of S6 protein and expression of VEGF protein in RAFLSs.Results rhS100A4 promoted cell proliferation and expression of VEGF protein in RAFLSs,and the CM formed by rhS100A4 promoted HUVECs to form blood vessels in vitro.Rap inhibited the above biological effects of rhS100A4,rhS100A4 activated the downstream protein S6 in the mTORC1 signaling pathway in RAFLSs cells to increase their phosphorylation levels.The effects of rhS100A4 on the phosphorylation of S6 protein and on the expression of VEGF protein in RAFLSs were inhibited by Rap.Conclusion rhS10OA4 promotes production of VEGF in RAFLSs by activating the mTORC 1 signaling pathway.