软骨细胞代谢影响骨关节炎的作用和机制研究

发布时间：2018-03-15 12:46

本文选题：骨关节炎　切入点：IL-17A　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：软骨细胞作为软骨组织内唯一的细胞类型,表达和分泌大量的细胞外基质(ECM)维持软骨正常功能。然而,当骨关节炎(OA)发生后,软骨细胞合成和分解代谢的平衡被破坏,加速了软骨的破坏。因此,本课题以OA病理过程中软骨细胞的代谢改变为研究对象,分别对肠道微生物通过白介素17A(IL-17A)促进软骨细胞分解代谢以及Kdm6b调控软骨细胞合成代谢的作用和机制进行了相关的研究。首先,我们发现了一类IL-17A表达升高的OA患者,提示了 IL-17A可能参与OA病理过程。通过对不同来源小鼠OA造模,发现肠道内缺少分节丝状菌(SFB)定植的小鼠IL-17A水平下降,OA进展减缓。接着使用抗生素破坏小鼠肠道微生物组成,发现OA造模后滑膜IL-17A表达下降,滑膜炎症和软骨破坏缓解。通过对肠道微生物组成分析后证明SFB的定植与小鼠OA病理中IL-17A水平相关,影响OA进展。进一步通过IL-17A刺激软骨细胞和关节腔注射IL-17A,证明IL-17A促进软骨细胞分解代谢相关的HIF-2α和MMP13表达,抑制COLII表达,加速软骨基质的降解。最后,使用体内IL-17A特异性升高的MINK1-/-转基因小鼠,证实IL-17A升高加速OA进展。炎症因子在OA病理过程中促进软骨细胞分解代谢,破坏软骨稳态,所以加强软骨细胞合成代谢成为治疗OA的可能途径。在这部分研究中,我们发现Kdm6b在成软骨过程中表达升高,通过诱导型软骨细胞特异性敲除Kdm6b小鼠模型(CKO),发现在软骨发育过程中敲除Kdm6b后,抑制软骨细胞增殖和软骨基质合成,软骨发育异常。而且,在软骨发育成熟后敲除Kdm6b,发现CKO小鼠在12月后出现软骨基质丢失与软骨破坏。体外分离原代软骨细胞后通过转录组测序发现,Kdm6b调控软骨细胞一系列合成代谢相关的基因,进一步使用ChIP-qPCR,证明Kdm6b直接作用于Col2a1和Acan启动子区域,调控软骨细胞的合成代谢。接着我们使用内侧半月板不稳(DMM)模型加速软骨细胞代谢紊乱,发现在软骨发育成熟后敲除Kdm6b,加速小鼠OA进展。结合骨软骨缺损修复模型,证明敲除Kdm6b抑制软骨细胞合成代谢,尤其是COLII及AGGRECAN的合成减少,导致OA病理过程中软骨破坏的加剧。分析人软骨样本后,发现Kdm6b与软骨细胞合成代谢相关蛋白表达关系紧密,且Kdm6b在OA患者软骨损伤处的表达明显下降。最后,我们发现Kdm6b在炎症因子刺激后迅速上调,提示可能参与软骨细胞对炎症环境的应答。我们的研究首次发现了肠道微生物通过IL-17A影响OA炎症环境,以及Kdm6b在调控OA病理中软骨合成代谢中的重要作用,为OA的治疗提供了新的思路。
[Abstract]:As the only cell type in cartilage tissue, chondrocytes express and secrete a large number of extracellular matrix ECM (ECM) to maintain the normal function of cartilage. However, when osteoarthritis occurs, the balance of synthesis and catabolism of chondrocytes is disrupted. Therefore, the metabolic changes of chondrocytes in the pathological process of OA were studied. The effects and mechanisms of intestinal microbes promoting chondrocyte catabolism and Kdm6b regulating chondrocyte catabolism through interleukin-17 (IL-17A) were studied. Firstly, we found a group of OA patients with elevated IL-17A expression. It was suggested that IL-17A might be involved in the pathological process of OA. By establishing models of mouse OA from different sources, it was found that the level of IL-17A decreased in mice with the absence of secundum filamentous bacteria in the intestinal tract, and the progression of OA was slowed down, and then the microorganism composition of intestinal tract was destroyed by antibiotics. It was found that the expression of IL-17A in synovial membrane was decreased, synovitis and cartilage damage were alleviated after OA modeling. It was proved that the colonization of SFB was related to the level of IL-17A in mouse OA by analyzing the composition of intestinal microorganism. It is proved that IL-17A promotes the expression of HIF-2 伪 and MMP13 related to catabolism of chondrocytes, inhibits the expression of COLII, and accelerates the degradation of cartilage matrix through IL-17A stimulation of chondrocytes and intraarticular injection of IL-17A. Using MINK1-r-transgenic mice with a specific increase in IL-17A in vivo, it was confirmed that the increase of IL-17A accelerates the progression of OA. Inflammatory factors promote the catabolism of chondrocytes and destroy cartilage homeostasis during the pathological process of OA. In this part of the study, we found that the expression of Kdm6b increased during the process of chondrogenesis. By inducing chondrocyte specific knockout Kdm6b mouse model, it was found that after knockout Kdm6b during cartilage development, chondrocyte proliferation and cartilage matrix synthesis were inhibited, and cartilage development was abnormal. Kdm6b was knocked out after cartilage maturation, and cartilage matrix loss and cartilage destruction were found in CKO mice after December. After isolation of primary chondrocytes in vitro, transcriptional sequencing revealed that Kdm6b regulates a series of genes related to chondrocyte synthesis and metabolism. ChIP-qPCR was further used to prove that Kdm6b acts directly on the promoter region of Col2a1 and Acan and regulates the synthesis and metabolism of chondrocytes. Then we use the model of medial meniscus instability to accelerate the metabolic disorder of chondrocytes. It was found that knockout Kdm6b after cartilage maturation accelerated the progress of OA in mice. Combined with osteochondral defect repair model, it was proved that knockout Kdm6b inhibited chondrocyte metabolism, especially the reduction of COLII and AGGRECAN synthesis. After analyzing the human cartilage samples, we found that the expression of Kdm6b was closely related to the expression of metabolism-related protein in chondrocytes, and the expression of Kdm6b in cartilage injury of OA patients was obviously decreased. We found that Kdm6b was upregulated rapidly after stimulation of inflammatory cytokines, suggesting that it might be involved in the response of chondrocytes to the inflammatory environment. Our study showed for the first time that intestinal microbes affect the inflammatory environment of OA through IL-17A. The important role of Kdm6b in the regulation of cartilage synthesis and metabolism in OA pathology provides a new idea for the treatment of OA.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R684.3

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