摘要
目的:探讨Wnt/β-catenin信号通路在骨关节炎发病中的作用,观察补肾活血方对异常的滑膜-软骨微环境的调控作用,初步阐释补肾活血方治疗骨关节炎的分子作用机制。方法:构建β-catenin过表达慢病毒载体转染正常人滑膜细胞,Transwell小室共培养人滑膜细胞与正常人软骨细胞模拟滑膜软骨关节体系内环境;各时间点不同浓度药物干预共培养体系,Western bolt方法检测补肾活血方对滑膜细胞β-catenin蛋白表达的影响;ELISA方法检测补肾活血方对共培养体系中滑膜细胞、软骨细胞培养上清液基质金属蛋白酶-7(MMP-7),人Ⅱ型胶原交联羧基端肽(CTX-Ⅱ),软骨寡聚基质蛋白(COMP)表达的影响。结果:Western blot检测发现β-catenin过表达慢病毒转染正常人滑膜细胞后滑膜细胞胞浆及胞核中β-catenin表达明显上调(P<0.01),补肾活血方能够明显下调共培养滑膜细胞中β-catenin蛋白的表达(P<0.01);同时,ELISA法检测发现补肾活血方能够明显下调滑膜细胞与软骨细胞培养上清液中MMP-7,CTX-Ⅱ,COMP的表达(P<0.01)。结论:β-catenin过表达慢病毒转染正常人滑膜细胞可成功激活Wnt/β-catenin信号通路,Wnt/β-catenin信号通路激活后共培养体系中MMP-7,CTX-Ⅱ,COMP的水平明显上升;补肾活血方可以抑制滑膜细胞Wnt/β-catenin信号通路,下调共培养体系中MMP-7,CTX-Ⅱ,COMP的表达,减轻异常滑膜细胞对软骨细胞的损害,减缓软骨基质的降解,有利于骨关节炎的缓解和恢复。
Objective：To research the contributions of Wnt/β-catenin signal pathway on osteoarthritis（OA）,to investigate the regulation of Bushenhuoxue decoction on the microenvironment of synovium-cartilage,and to clarify the molecular mechanism of Bushenhuoxue decoction in the treatment of OA.Methods：β-catenin over expressing lentivirus vector was constructed to transfect synovial cells of normal people.Synovial cells with and chondrocytes were cultured in transwell chamber to simulate the joint system environment between synovium and cartilage.Drug was intervening in co-culture system on different time points of different concentrations.The expression ofβ-catenin protein of synovial cells influenced by Bushenhuoxue decoction was detected by Western bolt method.The expression of MMP-7,CTX-Ⅱ,and COMP in the culture supernatant was detected by ELISA method.Results：The expression ofβ-catenin in the cytoplasm and nucleus of synovial cells increased significantly after transfected（P〈0.01）.The expression ofβ-catenin in synovial cells nucleus was significantly reduced by Bushenhuoxue decoction（P〈0.01）.The level of MMP-7,CTX-Ⅱ,and COMP was significantly regulated down by Bushenhuoxue decoction in co-culture cells supernatant（P〈0.01）.Conclusion：The Wnt/β-catenin signaling pathway can be activating by lentivirus withβ-catenin over expressing.Then,the level of MMP-7,CTX-Ⅱ,and COMPin co-cuture system is increasing significantly.Bushenhuoxue decoction can inhibit Wnt/β-catenin signaling pathway,regulate the expression level of MMP-7,CTX-Ⅱ,and COMP down,relieve harmful of abnormal synovial cells contributing to chondrocytes and degradation of cartilage matrix,and promote the restoration of OA.
出处
《中国中医骨伤科杂志》
CAS
2016年第4期1-6,共6页
Chinese Journal of Traditional Medical Traumatology & Orthopedics
基金
上海中医药大学预算内科研项目(2014YSN64)
上海市教委创新课题(14YZ058)
