本文選題：類風(fēng)濕性關(guān)節(jié)炎 + 成纖維樣滑膜細(xì)胞　； 參考：《第四軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：類風(fēng)濕性關(guān)節(jié)炎(Rheumatoid Arthritis,RA)是一種以侵襲性、慢性、進(jìn)行性、致殘性關(guān)節(jié)病變?yōu)橹饕憩F(xiàn)的全身性自身免疫病[1]。若不及時治療,病情會迅速發(fā)展,導(dǎo)致關(guān)節(jié)軟骨損傷加劇,最終造成關(guān)節(jié)畸形、功能喪失,具有極高的致殘率。因此,系統(tǒng)地闡明參與RA關(guān)節(jié)鄰近骨、軟骨損傷的相關(guān)信號通路,發(fā)現(xiàn)其中的關(guān)鍵分子及調(diào)控機(jī)制,既能為RA的發(fā)生、發(fā)展提出新的理論認(rèn)識,還能為RA的臨床治療提供潛在的藥物作用靶點(diǎn)。現(xiàn)有的研究表明:RA的基本病理特征是周身關(guān)節(jié)滑膜炎癥的累積以及血管翳形成。血管翳中的免疫病理成分可分為免疫和侵蝕兩部分[2]。其中侵蝕部分主要由鄰近骨、軟骨的破骨細(xì)胞(Osteoclast)和滑膜成纖維細(xì)胞(Synovial Fibroblast,SF)構(gòu)成,滑膜成纖維細(xì)胞包括巨噬細(xì)胞樣滑膜細(xì)胞和成纖維細(xì)胞樣滑膜細(xì)胞(Fibroblast-Like Synovial Cells,FLS)。FLS過度分泌的基質(zhì)金屬蛋白酶(Matrix Metalloproteinase,MMPs)被認(rèn)為是RA骨、軟骨破壞的“罪魁禍?zhǔn)住薄A組織中的MMPs直接參與了對關(guān)節(jié)骨、軟骨的降解,產(chǎn)生的Ⅱ型膠原(CollageⅡ)又能誘導(dǎo)FLS分泌MMPs,進(jìn)而形成惡性循環(huán),加劇關(guān)節(jié)骨、軟骨的破壞。我們前期發(fā)現(xiàn):①盤狀結(jié)構(gòu)域受體2(DDR2)在RA患者滑膜組織及FLS細(xì)胞中高表達(dá),并呈持續(xù)活化狀態(tài)[3,4];②原代培養(yǎng)的RA患者FLS細(xì)胞能夠持續(xù)高水平分泌MMP1、MMP13等與RA關(guān)節(jié)鄰近骨、軟骨病理損傷直接相關(guān)的MMPs分子[5];③DDR2可通過調(diào)節(jié)AP1、Runx2的轉(zhuǎn)錄活性調(diào)控MMP1、MMP13等MMPs分子的表達(dá)[5];④通過對DDR2受體胞外區(qū)的研究,我們發(fā)現(xiàn)DDR2胞外區(qū)可以阻斷Ⅱ型膠原與DDR2的結(jié)合并可抑制RA滑膜細(xì)胞分泌MMP-1[6];此外,我們還在針對(collagen-induced arthritis,CIA)大鼠關(guān)節(jié)炎模型的研究中發(fā)現(xiàn),可溶性的DDR2可改善RA關(guān)節(jié)鄰近骨、軟骨的損傷[7]。盡管前期我們以DDR2在RA發(fā)生、發(fā)展進(jìn)程中的功能研究為切入點(diǎn),進(jìn)行了一些研究并取得了一些成果,但是仍有許多問題值得深入探討:1.DDR2是分布在FLS細(xì)胞膜表面的具有酪氨酸蛋白激酶活性的受體,Ⅱ型膠原使DDR2發(fā)生磷酸化激活之后,它會結(jié)合哪些分子?2.這些分子是磷酸化分子還是非磷酸化分子?3.它們的功能又是什么?能夠調(diào)控MMPs的表達(dá)嗎?對于上述問題的回答有利于闡明DDR2介導(dǎo)RA FLS細(xì)胞MMPS過分泌的分子機(jī)制,并系統(tǒng)揭示Ⅱ型膠原作用下的RA關(guān)節(jié)鄰近骨、軟骨損傷的機(jī)制。因此,我們在前期工作的基礎(chǔ)之上,擬從以下三個方面展開研究:①利用DDR2過表達(dá)慢病毒上調(diào)FLS細(xì)胞中DDR2的表達(dá)水平,以DDR2的相互作用分子為突破口,利用免疫沉淀結(jié)合SDS-PAGE的方法獲得DDR2的相互作用分子,并利用多肽質(zhì)譜確定其身份。結(jié)合生物信息學(xué)分析和相關(guān)文獻(xiàn)的研究結(jié)果,我們以篩選得到的DDR2相互作用分子--波形蛋白(Vimentin)為研究對象,利用免疫共沉淀確定DDR2與Vimentin的相互作用關(guān)系,利用激光共聚焦顯微鏡觀察Vimentin與DDR2在FLS細(xì)胞中的表達(dá)及分布情況。②利用RNAi、Real-time PCR、Western Blot等實驗手段,考察DDR2活化后對Vimentin的表達(dá)及磷酸化狀態(tài)的影響。并以Vimentin的功能研究為切入點(diǎn),考察Vimentin對MMPs及FLS細(xì)胞侵襲、遷移能力的影響。③最后,我們以骨性關(guān)節(jié)炎(OA)患者的滑膜組織為陰性對照,擴(kuò)大樣本數(shù),系統(tǒng)考察Vimentin在RA患者滑膜組織中的表達(dá)、分布情況,進(jìn)一步評價其臨床意義。通過上述實驗的開展,我們得到以下結(jié)果:①通過免疫沉淀結(jié)合SDS-PAGE分離的方法,得到了8個DDR2相互作用蛋白,經(jīng)多肽質(zhì)譜和生物信息學(xué)分析,確定了8個蛋白的身份,我們選擇Vimentin作為下一步的研究對象;②在HEK293T細(xì)胞中,通過免疫共沉淀的手段,確認(rèn)了活化的DDR2與Vimentin的相互作用關(guān)系;③利用激光共聚焦顯微鏡觀察發(fā)現(xiàn)DDR2與Vimentin在RA FLS細(xì)胞中存在共定位關(guān)系;④用Ⅱ型膠原活化RA FLS細(xì)胞膜上的DDR2后,Vimentin的表達(dá)水平變化不大,但Vimentin的磷酸化水平顯著升高;⑤Vimentin的磷酸化激活可以促進(jìn)RA FLS中MMP13的表達(dá);⑥Transwell侵襲實驗和細(xì)胞劃痕實驗結(jié)果顯示,Vimentin的磷酸化激活能夠促進(jìn)RA FLS細(xì)胞的侵襲、遷移;⑦在擴(kuò)大樣本量后,以O(shè)A患者滑膜組織為對照,RA患者滑膜組織中Vimentin的表達(dá)水平與磷酸化水平均高于OA滑膜組織。通過本研究,我們首次證實了Vimentin與活性型DDR2的相互作用關(guān)系,并且作為DDR2的相互作用分子,Vimentin在介導(dǎo)RA FLS細(xì)胞MMPs過分泌及促進(jìn)RA FLS細(xì)胞侵襲、遷移和關(guān)節(jié)軟骨破壞過程中扮演了重要角色。上述研究結(jié)果補(bǔ)充和完善了我們課題組提出的Ⅱ型膠原-DDR2-MMPs通路在介導(dǎo)RA關(guān)節(jié)軟骨侵襲、破壞機(jī)制中的關(guān)鍵分子和重要環(huán)節(jié),既為闡明RA中晚期病變的發(fā)病機(jī)制提供了新的思路,又為開發(fā)治療或緩解RA關(guān)節(jié)鄰近骨、軟骨損傷的新藥提供了潛在的靶點(diǎn)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R593.22

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