DDR2的相互作用分子—Vimentin在RA滑膜MMPs过分泌及FLS细胞侵袭、迁移中的作用研究

发布时间：2018-06-07 07:37

本文选题：类风湿性关节炎 + 成纤维样滑膜细胞　；参考：《第四军医大学》2015年硕士论文

【摘要】：类风湿性关节炎(Rheumatoid Arthritis,RA)是一种以侵袭性、慢性、进行性、致残性关节病变为主要表现的全身性自身免疫病[1]。若不及时治疗,病情会迅速发展,导致关节软骨损伤加剧,最终造成关节畸形、功能丧失,具有极高的致残率。因此,系统地阐明参与RA关节邻近骨、软骨损伤的相关信号通路,发现其中的关键分子及调控机制,既能为RA的发生、发展提出新的理论认识,还能为RA的临床治疗提供潜在的药物作用靶点。现有的研究表明:RA的基本病理特征是周身关节滑膜炎症的累积以及血管翳形成。血管翳中的免疫病理成分可分为免疫和侵蚀两部分[2]。其中侵蚀部分主要由邻近骨、软骨的破骨细胞(Osteoclast)和滑膜成纤维细胞(Synovial Fibroblast,SF)构成,滑膜成纤维细胞包括巨噬细胞样滑膜细胞和成纤维细胞样滑膜细胞(Fibroblast-Like Synovial Cells,FLS)。FLS过度分泌的基质金属蛋白酶(Matrix Metalloproteinase,MMPs)被认为是RA骨、软骨破坏的“罪魁祸首”。RA组织中的MMPs直接参与了对关节骨、软骨的降解,产生的Ⅱ型胶原(CollageⅡ)又能诱导FLS分泌MMPs,进而形成恶性循环,加剧关节骨、软骨的破坏。我们前期发现:①盘状结构域受体2(DDR2)在RA患者滑膜组织及FLS细胞中高表达,并呈持续活化状态[3,4];②原代培养的RA患者FLS细胞能够持续高水平分泌MMP1、MMP13等与RA关节邻近骨、软骨病理损伤直接相关的MMPs分子[5];③DDR2可通过调节AP1、Runx2的转录活性调控MMP1、MMP13等MMPs分子的表达[5];④通过对DDR2受体胞外区的研究,我们发现DDR2胞外区可以阻断Ⅱ型胶原与DDR2的结合并可抑制RA滑膜细胞分泌MMP-1[6];此外,我们还在针对(collagen-induced arthritis,CIA)大鼠关节炎模型的研究中发现,可溶性的DDR2可改善RA关节邻近骨、软骨的损伤[7]。尽管前期我们以DDR2在RA发生、发展进程中的功能研究为切入点,进行了一些研究并取得了一些成果,但是仍有许多问题值得深入探讨:1.DDR2是分布在FLS细胞膜表面的具有酪氨酸蛋白激酶活性的受体,Ⅱ型胶原使DDR2发生磷酸化激活之后,它会结合哪些分子?2.这些分子是磷酸化分子还是非磷酸化分子?3.它们的功能又是什么?能够调控MMPs的表达吗?对于上述问题的回答有利于阐明DDR2介导RA FLS细胞MMPS过分泌的分子机制,并系统揭示Ⅱ型胶原作用下的RA关节邻近骨、软骨损伤的机制。因此,我们在前期工作的基础之上,拟从以下三个方面展开研究:①利用DDR2过表达慢病毒上调FLS细胞中DDR2的表达水平,以DDR2的相互作用分子为突破口,利用免疫沉淀结合SDS-PAGE的方法获得DDR2的相互作用分子,并利用多肽质谱确定其身份。结合生物信息学分析和相关文献的研究结果,我们以筛选得到的DDR2相互作用分子--波形蛋白(Vimentin)为研究对象,利用免疫共沉淀确定DDR2与Vimentin的相互作用关系,利用激光共聚焦显微镜观察Vimentin与DDR2在FLS细胞中的表达及分布情况。②利用RNAi、Real-time PCR、Western Blot等实验手段,考察DDR2活化后对Vimentin的表达及磷酸化状态的影响。并以Vimentin的功能研究为切入点,考察Vimentin对MMPs及FLS细胞侵袭、迁移能力的影响。③最后,我们以骨性关节炎(OA)患者的滑膜组织为阴性对照,扩大样本数,系统考察Vimentin在RA患者滑膜组织中的表达、分布情况,进一步评价其临床意义。通过上述实验的开展,我们得到以下结果:①通过免疫沉淀结合SDS-PAGE分离的方法,得到了8个DDR2相互作用蛋白,经多肽质谱和生物信息学分析,确定了8个蛋白的身份,我们选择Vimentin作为下一步的研究对象;②在HEK293T细胞中,通过免疫共沉淀的手段,确认了活化的DDR2与Vimentin的相互作用关系;③利用激光共聚焦显微镜观察发现DDR2与Vimentin在RA FLS细胞中存在共定位关系;④用Ⅱ型胶原活化RA FLS细胞膜上的DDR2后,Vimentin的表达水平变化不大,但Vimentin的磷酸化水平显著升高;⑤Vimentin的磷酸化激活可以促进RA FLS中MMP13的表达;⑥Transwell侵袭实验和细胞划痕实验结果显示,Vimentin的磷酸化激活能够促进RA FLS细胞的侵袭、迁移;⑦在扩大样本量后,以OA患者滑膜组织为对照,RA患者滑膜组织中Vimentin的表达水平与磷酸化水平均高于OA滑膜组织。通过本研究,我们首次证实了Vimentin与活性型DDR2的相互作用关系,并且作为DDR2的相互作用分子,Vimentin在介导RA FLS细胞MMPs过分泌及促进RA FLS细胞侵袭、迁移和关节软骨破坏过程中扮演了重要角色。上述研究结果补充和完善了我们课题组提出的Ⅱ型胶原-DDR2-MMPs通路在介导RA关节软骨侵袭、破坏机制中的关键分子和重要环节,既为阐明RA中晚期病变的发病机制提供了新的思路,又为开发治疗或缓解RA关节邻近骨、软骨损伤的新药提供了潜在的靶点。
[Abstract]:Rheumatoid arthritis (Rheumatoid Arthritis, RA) is an invasive, chronic, progressive, and disabling joint lesion as the main manifestation of the systemic autoimmune disease, [1]., if it is not treated in time, the disease will develop rapidly, resulting in the aggravation of articular cartilage damage, resulting in joint deformity, loss of function, and high disability rate. The relevant signal pathways involved in the adjacent bone and cartilage damage in the RA joint are clarified, and the key molecules and regulatory mechanisms are found, which can not only provide new theoretical understanding for the development of RA, but also provide potential drug targets for the clinical treatment of RA. The basic pathological features of RA are synovial inflammation of the joints of the body. Accumulation of pannus and formation of pannus. The immune pathological components in pannus can be divided into two parts of [2]., immune and erosion. The erosive part is mainly composed of adjacent bone, osteoclast (Osteoclast) and synovial fibroblasts (Synovial Fibroblast, SF), and synovial fibroblasts including macrophage like synovial cells and fibroblasts. The matrix metalloproteinase (Matrix Metalloproteinase, MMPs) oversecreted by Fibroblast-Like Synovial Cells (FLS).FLS is believed to be a RA bone. The MMPs of the cartilage destruction of.RA tissue is directly involved in the degradation of the articular bone and cartilage, and the production of type II collagen (Collage II) can also induce secretion. The formation of a vicious cycle aggravates the destruction of the joint bone and cartilage. We have found that: (1) the disc like domain receptor 2 (DDR2) is highly expressed in the synovium and FLS cells of RA patients and has a continuous active state of [3,4]; and the FLS cells in the primary RA patients can continue to secrete MMP1 at a high level, MMP13 and so on with the RA joint, and the cartilage pathological injury is straight. MMPs molecule [5]; (3) DDR2 can regulate the expression of [5] by regulating AP1, Runx2 transcriptional activity, MMP1, MMP13 and other MMPs molecules; 4. By studying the extracellular domain of DDR2 receptor, we found that DDR2 extracellular domain could block the binding of type II collagen to DDR2 and inhibit the secretory cell secretion of synovium; furthermore, we are still targeting. In the study of uced arthritis, CIA) rat arthritis model, it was found that soluble DDR2 could improve the adjacent bone of RA joint and cartilage damage [7]., although we took DDR2 in RA and the function research in the development process as the breakthrough point in the earlier period, some studies have been made and some fruits have been obtained, but there are still a lot of problems to be discussed in depth: 1.DDR2 is A receptor with tyrosine kinase activity on the surface of the FLS cell membrane. After type II collagen activates the phosphorylation of DDR2, what molecules will it combine? 2. are these molecules or non phosphorylated molecules? 3. what are their functions? Can the expression of MMPs be regulated? The answer to the above questions is beneficial. This paper elucidates the molecular mechanism of DDR2 mediated MMPS over secretion of RA FLS cells, and systematically reveals the mechanism of cartilage damage in the adjacent bone of RA joint under the action of type II collagen. Therefore, on the basis of the earlier work, we should study the following three aspects: (1) the expression level of DDR2 in FLS cells by DDR2 overexpression of slow disease virus, and the phase of DDR2 in DDR2 The interaction molecule is a breakthrough, using immunoprecipitation and SDS-PAGE method to obtain the interaction molecules of DDR2 and determine its identity by peptide mass spectrometry. Combined with the results of bioinformatics analysis and related literature, we use the screened DDR2 interaction molecule, wave protein (Vimentin) as the research object, and use immunization. The interaction between DDR2 and Vimentin was determined by precipitation, and the expression and distribution of Vimentin and DDR2 in FLS cells were observed by laser confocal microscopy. (2) the effects of DDR2 activation on the expression of Vimentin and the state of phosphorylation were investigated by using RNAi, Real-time PCR, Western Blot and so on. To investigate the effect of Vimentin on the invasion and migration of MMPs and FLS cells. Thirdly, we take the synovial tissue of the patients with osteoarthritis (OA) as negative control, enlarge the number of samples, and systematically investigate the expression and distribution of Vimentin in the synovial tissue of RA patients, and evaluate the clinical significance step by step. To the following results: (1) 8 DDR2 interacting proteins were obtained by immunoprecipitation and SDS-PAGE separation. After peptide mass spectrometry and bioinformatics analysis, the identity of 8 proteins was determined. We chose Vimentin as the next research object. 2. In HEK293T cells, the activated DDR2 was confirmed by the means of immunoprecipitation. The interaction relationship with Vimentin was found. (3) the co localization relationship between DDR2 and Vimentin was found in RA FLS cells by laser confocal microscopy; (4) the expression level of Vimentin changed little after activating DDR2 on the membrane of RA FLS cell with type II collagen, but the phosphorylation level of Vimentin increased significantly, and the activation of phosphorylation of Vimentin was possible. Promote the expression of MMP13 in RA FLS; 6. The results of Transwell invasion experiment and cell scratch test show that the activation of Vimentin can promote the invasion and migration of RA FLS cells, and the expression level of Vimentin in the synovial tissues of RA patients and the average of phosphorylated water are higher than that of the OA synovium group in the synovial tissue of OA patients after the enlargement of the sample size. Through this study, we have confirmed the interaction between Vimentin and active DDR2 for the first time, and as a interacting molecule of DDR2, Vimentin plays an important role in mediating MMPs over secretion of RA FLS cells and promoting RA FLS cell invasion, migration and articular cartilage destruction. The results complement and perfected our course. The type II collagen -DDR2-MMPs pathway proposed by the question group mediates the invasion of RA articular cartilage and destroys key molecules and important links in the mechanism. It not only provides a new idea for clarifying the pathogenesis of RA, but also provides potential targets for the development of new drugs for the treatment or mitigation of the adjacent bone of the RA joint and cartilage damage.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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