角蛋白17（K17）作为自身抗原银屑病发病机制中的作用研究

发布时间：2019-04-09 06:53

【摘要】： 银屑病是一种常见的,易反复发作的慢性炎症性皮肤病,是Th1/ Th17细胞共同介导的自身免疫紊乱。白介素17(IL-17)是辅助性T细胞亚群之一Th17细胞分泌的主要细胞因子,在银屑病的发病中占有重要的地位,它和IFN-γ都被认为是“银屑病的关键细胞因子”。IL-17信号转导机制包括3条途径:JAK/STAT、NF-κB和MAPK,其中,JAK/STAT与银屑病角质形成细胞(KC)关系密切。角蛋白17(K17)是“银屑病相关性细胞角蛋白”,与链球菌M蛋白具有相同的序列ALEEAN,可特异刺激银屑病T细胞,使之活化、释放IFN-γ等炎症因子,而IFN-γ又能够经STAT1信号通路上调K17的表达,使机体产生针对K17的自身免疫反应,从而形成了一个恶性环路,导致炎症反应和KC异常增生等病理改变,成为银屑病发病过程中的关键环节。因K17在银屑病皮损的高表达以及其具有刺激T细胞活化的作用,被界定为银屑病自身反应性T细胞识别的候选靶抗原。我们以往的研究已经证明在银屑病的病理过程中存在“K17—T细胞─细胞因子环路”,该环路可能是银屑病皮损持续存在和反复发作的重要基础。本研究在此基础上首次提出并验证IL-17是该环路中的重要细胞因子成员,观察IL-17诱导表皮角质形成细胞(KC)表达K17的作用及其调控的信号转导机制,进一步深入探讨K17作为自身抗原在银屑病发病过程中的意义,从而加深对银屑病发病机理的认识,并为银屑病的治疗提供新的思路。主要实验方法及结果如下: 1)使用DMEM/10%FBS培养基培养HaCaT细胞; 2)以10U/mL、50U/mL、250 U /mL和500 U /mL的IL-17分别作用于培养的HaCaT细胞,以IFN-γ处理的HaCaT细胞为阳性对照,不施加任何干预的HaCaT细胞作为空白对照。培养24~72小时后收获细胞,并分别提取RNA和蛋白质用于K17表达水平的检测; 3)用实时荧光定量RT-PCR、ELISA、Western blot、共聚焦免疫荧光等方法从mRNA和蛋白水平测定K17的表达水平,筛选IL-17诱导K17表达的最佳浓度,观察是否呈剂量依赖关系; 4)以筛选得出的最佳浓度的IL-17处理培养的HaCaT细胞; 5)用抗STAT1和STAT3及各自的磷酸化产物的抗体与HaCaT细胞共同孵育,ELISA、Western blot、免疫荧光方法筛选在HaCaT细胞表达的信号分子; 6)未处理的HaCaT细胞分别加入相应的信号分子抑制剂Fludarabine和Piceatannol孵育2小时,再加入IL-17培养HaCaT细胞,12～24小时后利用实时定量RT-PCR、ELISA、Western blot、免疫荧光等方法观察信号分子抑制剂各自对IL-17诱导K17表达的影响; 研究结果: IL-17以剂量依赖的方式诱导体外培养的HaCaT细胞表达K17,其调控K17表达的机制是经STAT1和STAT3两个信号转导通路实现的。本研究首次发现了调控K17表达的一个新的通路——IL-17诱导KC表达K17,明确了IL-17调控K17表达的主要信号转导机制是经STAT1和STAT3信号通路,进一步完善了“K17─T细胞─细胞因子环路”及其在银屑病发病中的意义,初步阐明了K17作为自身抗原在其中的作用。研究结果不仅深化了对银屑病发病机制的认识,也为银屑病的治疗提供了新的思路。
[Abstract]:Psoriasis is a common and recurrent chronic inflammatory skin disease, which is a co-mediated autoimmune disorder of Th1/ Th17 cells. Interleukin-17 (IL-17) is a major cytokine secreted by Th17 cells of T-cell subpopulations, and plays an important role in the pathogenesis of psoriasis, which is considered to be a "Key Cytokine in Psoriasis". The IL-17 signal transduction mechanism consists of three pathways: JAK/ STAT, NF-EMAB and MAPK, where JAK/ STAT is closely related to psoriatic keratinocytes (KC). The keratin 17 (K17) is a "psoriasis-related cytokeratin", and has the same sequence of ALEEAN as the Streptococcus M protein, can specifically stimulate the psoriasis T cells, activate and release the inflammatory factors such as the IFN-1 and the like, and the IFN-1 can increase the expression of the K17 through the STAT1 signal path, so that the body can generate the self-immune response to the K17, So as to form a malignant loop, which leads to the pathological changes of the inflammatory reaction and the abnormal proliferation of the KC, and is a key link in the pathogenesis of the psoriasis. The high expression of K17 in psoriatic lesions and its role in stimulating T cell activation are defined as candidate target antigens for the self-reactive T cell identification of psoriasis. Our previous studies have shown that there is a "K17-T cell-derived cytokine loop" in the pathological process of psoriasis, which may be an important basis for the persistence and recurrence of psoriatic lesions. This study first proposed and verified that IL-17 was an important cytokine in the loop, and the effect of IL-17 on the expression of K17 in the epidermal keratinocytes (KC) and the signal to be controlled by IL-17 were observed. To further study the significance of K17 as its own antigen in the pathogenesis of psoriasis, so as to deepen the understanding of the pathogenesis of psoriasis and to provide a new method for the treatment of psoriasis. The main experiment The method and results are as follows:1) DMEM/10% FBS culture is used The cultured HaCaT cells were cultured in a culture group;2) an IL-17 of 10 U/ mL,50 U/ mL,250 U/ mL and 500 U/ mL was applied to the cultured HaCaT cells, respectively, to IFN-1. The treated HaCaT cells were positive controls and did not apply any dry The pre-prepared HaCaT cells are used as the blank control, and the cells are harvested after the culture is cultured for 24 to 72 hours, and the RNA and the RNA are respectively extracted. the protein is used for detecting the expression level of K17;3) the expression level of K17 is determined from the mRNA and the protein level by using real-time fluorescence quantitative RT-PCR, ELISA, Western blot, confocal immunofluorescence and the like, Screening of IL-17-Induced K17 The best concentration of expression, observed for dose-dependent relationship;4) for screening The best concentration of IL-17 was used to treat the cultured HaCaT cells;5) the antibodies against STAT1 and STAT3 and the respective phosphorylated products were incubated with HaCaT cells, ELISA, Western b, And 6) the untreated HaCaT cells are respectively added with a corresponding signal molecule inhibitor, Fludarpine and Piceatanol for 2 hours, and then IL-17 is added for culturing the HaCaT cell, and after 12 to 24 hours, the real-time quantitative RT-PCR, the ELISA, the Western blot and the immunity are used. The effect of the signal molecule inhibitor on the expression of IL-17 on the expression of K17 was observed by light and the like. The results of the study: IL-17 induced the expression of K17 in the in vitro cultured HaCaT cells in a dose-dependent manner. The mechanism of regulation and control of K17 expression was realized by two signal transduction pathways of STAT1 and STAT3. The first time in this study, a new pathway _ IL-17, which regulates the expression of K17, was found to induce the expression of K17, and the main signal transduction mechanism of the expression of IL-17 in the regulation of K17 was confirmed by the STAT1 and STAT3 signaling pathways, and the

"K17-T cell was further improved. l-derived cytokine loop and its significance in the pathogenesis of psoriasis, and the preliminary elucidation of K17 as its own antigen
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R758.63

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