淫羊藿苷对骨关节炎滑膜细胞中MMP14表达和细胞增殖调控的研究

发布时间：2018-04-22 18:15

本文选题：骨关节炎 + 淫羊藿苷　；参考：《重庆大学》2016年硕士论文

【摘要】：骨关节炎(Osteoarthritis,OA)是一种以骨关节软骨的退行性病变和继发骨质增生为特点的慢性关节疾病,其病理性进程中滑膜的病变会导致关节腔微环境稳态发生紊乱进而使疾病恶化,其病理特征主要包括:组织中蛋白酶活性的增加导致胞外基质(ECM)的降解,细胞分泌大量炎症因子促进OA的发生进程,细胞的凋亡恶化关节腔微环境等。因此,OA疾病中ECM的降解、细胞因子的分泌及细胞凋亡是研究其发病机制的重要靶点。在OA的治疗方面,药物治疗的方式具有使用方便、治疗效果较好、成本较低等特点,寻找安全性更高药效更好的药物对OA进行治疗受到了极大的关注。作为一种传统中药类物质淫羊藿苷能够促进软骨细胞增殖,成骨细胞分化,降低炎症因子及基质金属蛋白酶等。OA疾病发展过程中滑膜组织会产生炎症反应并继发性增生,而淫羊藿苷是否能够通过对OA滑膜细胞进行调控从而达到治疗OA的目的尚不清楚。因此,本文以OA滑膜细胞为研究对象,以细胞活力、增殖、迁移以及不同细胞因子表达情况为指标,探索淫羊藿苷对OA滑膜细胞的调控作用及其机制,为OA的药物治疗途径提供参考。本课题主要研究工作和结果如下:(1)采用组织块培养法进行OA滑膜细胞的提取,并对所提细胞进行传代培养。(2)淫羊藿苷处理OA滑膜细胞后运用MTS方法检测不同浓度的淫羊藿苷对于细胞的活性影响及探讨该药物的毒性作用,并筛选出最佳药物作用浓度:0nM,100nM,500nM,1μM;采用流式细胞术检测淫羊藿苷处理OA滑膜细胞后细胞周期的变化,结果发现进入S期和G2/M期的细胞量增多,细胞出现增殖;采用EdU法检测淫羊藿苷处理OA滑膜细胞后细胞增殖的情况,结果显示在一定浓度范围内淫羊藿苷能够促进细胞增殖。(3)淫羊藿苷处理OA滑膜细胞后收集总RNA和总蛋白,采用Real-time PCR法检测MMP14和GRP78 mRNA表达量的变化;采用Western blot法检测MMP14和GRP78蛋白表达量的变化;采用免疫荧光法检测MMP14和IL-1β的表达。结果发现淫羊藿苷能够抑制MMP14和GRP78的基因以及蛋白的表达,并且也能降低IL-1β的表达水平。(4)淫羊藿苷处理细胞后运用Transwell法检测细胞迁移的情况,结果显示淫羊藿苷能够抑制OA滑膜细胞的迁移。并且抑制作用随着药物浓度升高而增强。综上所述,我们证实在一定浓度范围内淫羊藿苷对OA滑膜细胞无毒性,并且可以降低MMP14、GRP78和IL-1β的表达,从而积极调控OA滑膜细胞,进而改变关节腔微环境,对骨关节炎的治疗具有积极作用。
[Abstract]:Osteoarthritis Osteoarthritis (OAA) is a chronic joint disease characterized by degenerative lesions of osteoarticular cartilage and secondary hyperosteogeny. The histopathological features include the degradation of extracellular matrix ECM (ECM) due to the increase of protease activity, the secretion of a large number of inflammatory factors to promote the process of OA, the deterioration of apoptosis in the articular microenvironment and so on. Therefore, the degradation of ECM, cytokine secretion and apoptosis are important targets to study the pathogenesis of OA. In the treatment of OA, the drug therapy has the characteristics of convenient use, good therapeutic effect, low cost, and so on, so it has been paid great attention to find more safe and effective drugs to treat OA. As a traditional Chinese medicine, icariin can promote the proliferation of chondrocytes, osteoblast differentiation, and reduce inflammation factors and matrix metalloproteinases. It is not clear whether icariin can regulate OA synoviocytes to treat OA. Therefore, in this study, OA synoviocytes were used as the targets of cell viability, proliferation, migration and expression of different cytokines to explore the regulatory effect of icariin on OA synovial cells and its mechanism. To provide a reference for the drug treatment of OA. The main research work and results are as follows: (1) tissue block culture method was used to extract OA synovial cells. After treated OA synoviocytes with icariin, the effects of different concentrations of icariin on cell activity and toxicity of icariin were detected by MTS method. Flow cytometry was used to detect the changes of cell cycle in OA synovial cells treated with icariin. The results showed that the number of cells entering S phase and G 2 / M phase increased and cell proliferation appeared. The proliferation of OA synovial cells treated with icariin was detected by EdU method. The results showed that icariin could promote the proliferation of OA synoviocytes in a certain concentration range. The results showed that icariin could promote the proliferation of OA synoviocytes and collect total RNA and total protein after treated with icariin. MMP14 and GRP78 mRNA were detected by Real-time PCR, MMP14 and GRP78 by Western blot, MMP14 and IL-1 尾 by immunofluorescence. The results showed that icariin could inhibit the expression of gene and protein of MMP14 and GRP78, and decrease the expression level of IL-1 尾. The results showed that icariin could inhibit the migration of OA synovial cells. And the inhibitory effect increased with the increase of drug concentration. In conclusion, we prove that icariin is not toxic to OA synoviocytes in a certain concentration range, and can reduce the expression of MMP14, GRP78 and IL-1 尾, thus actively regulate the synovial cells of OA, and then change the microenvironment of articular cavity. It has a positive effect on the treatment of osteoarthritis.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R274.9

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