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目的观察黄芪甲苷对Wnt/β-catenin信号通路活化的人滑膜细胞和人软骨细胞共培养体系基质金属蛋白酶-7(MMP-7)、Ⅱ型胶原羧基端肽(CTX-Ⅱ)、软骨寡聚基质蛋白(COMP)的调控作用,初步阐释黄芪甲苷治疗骨关节炎的分子作用机制。方法通过β-catenin慢病毒载体转染正常人滑膜细胞,激活其Wnt/β-catenin信号通路。Wnt/β-catenin信号通路活化的人滑膜细胞与正常人软骨细胞置于Transwell小室中共培养。倒置显微镜观察Wnt/β-catenin信号通路活化的人滑膜细胞对人软骨细胞形态学变化的影响。黄芪甲苷混悬液干预共培养体系,采用Western bolt方法检测黄芪甲苷对滑膜细胞β-catenin蛋白表达的影响;采用ELISA方法检测黄芪甲苷对滑膜细胞、软骨细胞培养上清液MMP-7、CTX-Ⅱ、COMP表达的影响。结果β-catenin慢病毒载体转染正常人滑膜细胞后成功激活Wnt信号通路并与软骨细胞共培养。随培养时间的延长,软骨细胞生长逐渐受到抑制,软骨细胞胞体边缘与板壁接触处发白,折光度增加,细胞贴壁强度降低。黄芪甲苷干预后,Western blot检测发现滑膜细胞中β-catenin蛋白的表达明显下调(P<0.01);ELISA法检测发现滑膜与软骨细胞上清液中MMP-7、CTX-Ⅱ、COMP表达与正常对照相比明显升高(P<0.01)。结论滑膜细胞Wnt/β-catenin信号通路的激活可以使软骨细胞生长受到抑制。黄芪甲苷可以降低滑膜细胞β-catenin表达,且与干预时间长短相关。黄芪甲苷可以抑制滑膜细胞Wnt/β-catenin信号通路,下调MMP-7、CTX-Ⅱ、COMP的表达。黄芪甲苷可能通过抑制OA滑膜Wnt/β-catenin信号通路,改善滑膜炎症、调控滑膜-软骨共同体系微环境、达到抑制软骨降解,促进软骨细胞功能恢复而治疗骨关节炎。
Objective To observe the effects of astragaloside Ⅳ on the expression of matrix metalloproteinase-7 (MMP-7), type Ⅱ collagen carboxyterminal peptide (CTX-Ⅱ) and cartilage of human synovial cells and human chondrocytes activated by Wnt / β-catenin signaling pathway Oligomeric matrix protein (COMP) regulation of the initial interpretation of the role of Astragaloside in the treatment of osteoarthritis molecular mechanism. Methods The normal human synovial cells were transfected with β-catenin lentiviral vector to activate the Wnt / β-catenin signaling pathway. Human synoviocytes activated by Wnt / β-catenin signaling pathway were co-cultured with normal human chondrocytes in Transwell chambers. The effect of Wnt / β-catenin signaling pathway on human chondrocyte morphological changes was observed with inverted microscope. Astragaloside suspension intervention co-culture system, Western blot was used to detect the effect of astragaloside IV on the expression of β-catenin protein in synovial cells; ELISA was used to detect the effect of astragaloside IV on synovium, chondrocyte supernatant MMP -7, CTX-Ⅱ, COMP expression. Results β-catenin lentiviral vector transfected normal human synovial cells successfully activated Wnt signaling pathway and co-cultured with chondrocytes. With the extension of culture time, the growth of chondrocytes was gradually inhibited. The chondrocytes had a whitish contact with the cell wall at the edge of the cell body, increasing the refraction and decreasing the cell attachment strength. Western blot showed that the expression ofβ-catenin in synovial cells was significantly down-regulated after astragaloside IV intervention (P <0.01). The levels of MMP-7, CTX-Ⅱ, COMP in the synovium and chondrocyte supernatant The expression was significantly higher than that of the normal control (P <0.01). Conclusion The activation of Wnt / β-catenin signaling pathway in synoviocytes inhibits the growth of chondrocytes. Astragaloside IV could decrease the expression of β-catenin in synovial cells, and was related to the duration of intervention. Astragaloside could inhibit Wnt / β-catenin signaling pathway and down-regulate the expression of MMP-7, CTX-Ⅱand COMP in synovial cells. Astragaloside IV may treat osteoarthritis by inhibiting the synovial Wnt / β-catenin signaling pathway, improving synovial inflammation, regulating the microenvironment of synovial-cartilage common system, inhibiting the cartilage degradation and promoting the recovery of chondrocyte function.