角蛋白17（K17）作為自身抗原銀屑病發(fā)病機(jī)制中的作用研究
發(fā)布時間：2019-04-09 06:53
【摘要】： 銀屑病是一種常見的,易反復(fù)發(fā)作的慢性炎癥性皮膚病,是Th1/ Th17細(xì)胞共同介導(dǎo)的自身免疫紊亂。白介素17(IL-17)是輔助性T細(xì)胞亞群之一Th17細(xì)胞分泌的主要細(xì)胞因子,在銀屑病的發(fā)病中占有重要的地位,它和IFN-γ都被認(rèn)為是“銀屑病的關(guān)鍵細(xì)胞因子”。IL-17信號轉(zhuǎn)導(dǎo)機(jī)制包括3條途徑:JAK/STAT、NF-κB和MAPK,其中,JAK/STAT與銀屑病角質(zhì)形成細(xì)胞(KC)關(guān)系密切。角蛋白17(K17)是“銀屑病相關(guān)性細(xì)胞角蛋白”,與鏈球菌M蛋白具有相同的序列ALEEAN,可特異刺激銀屑病T細(xì)胞,使之活化、釋放IFN-γ等炎癥因子,而IFN-γ又能夠經(jīng)STAT1信號通路上調(diào)K17的表達(dá),使機(jī)體產(chǎn)生針對K17的自身免疫反應(yīng),從而形成了一個惡性環(huán)路,導(dǎo)致炎癥反應(yīng)和KC異常增生等病理改變,成為銀屑病發(fā)病過程中的關(guān)鍵環(huán)節(jié)。因K17在銀屑病皮損的高表達(dá)以及其具有刺激T細(xì)胞活化的作用,被界定為銀屑病自身反應(yīng)性T細(xì)胞識別的候選靶抗原。我們以往的研究已經(jīng)證明在銀屑病的病理過程中存在“K17—T細(xì)胞─細(xì)胞因子環(huán)路”,該環(huán)路可能是銀屑病皮損持續(xù)存在和反復(fù)發(fā)作的重要基礎(chǔ)。 本研究在此基礎(chǔ)上首次提出并驗證IL-17是該環(huán)路中的重要細(xì)胞因子成員,觀察IL-17誘導(dǎo)表皮角質(zhì)形成細(xì)胞(KC)表達(dá)K17的作用及其調(diào)控的信號轉(zhuǎn)導(dǎo)機(jī)制,進(jìn)一步深入探討K17作為自身抗原在銀屑病發(fā)病過程中的意義,從而加深對銀屑病發(fā)病機(jī)理的認(rèn)識,并為銀屑病的治療提供新的思路。 主要實驗方法及結(jié)果如下: 1)使用DMEM/10%FBS培養(yǎng)基培養(yǎng)HaCaT細(xì)胞; 2)以10U/mL、50U/mL、250 U /mL和500 U /mL的IL-17分別作用于培養(yǎng)的HaCaT細(xì)胞,以IFN-γ處理的HaCaT細(xì)胞為陽性對照,不施加任何干預(yù)的HaCaT細(xì)胞作為空白對照。培養(yǎng)24~72小時后收獲細(xì)胞,并分別提取RNA和蛋白質(zhì)用于K17表達(dá)水平的檢測; 3)用實時熒光定量RT-PCR、ELISA、Western blot、共聚焦免疫熒光等方法從mRNA和蛋白水平測定K17的表達(dá)水平,篩選IL-17誘導(dǎo)K17表達(dá)的最佳濃度,觀察是否呈劑量依賴關(guān)系; 4)以篩選得出的最佳濃度的IL-17處理培養(yǎng)的HaCaT細(xì)胞; 5)用抗STAT1和STAT3及各自的磷酸化產(chǎn)物的抗體與HaCaT細(xì)胞共同孵育,ELISA、Western blot、免疫熒光方法篩選在HaCaT細(xì)胞表達(dá)的信號分子; 6)未處理的HaCaT細(xì)胞分別加入相應(yīng)的信號分子抑制劑Fludarabine和Piceatannol孵育2小時,再加入IL-17培養(yǎng)HaCaT細(xì)胞,12～24小時后利用實時定量RT-PCR、ELISA、Western blot、免疫熒光等方法觀察信號分子抑制劑各自對IL-17誘導(dǎo)K17表達(dá)的影響; 研究結(jié)果: IL-17以劑量依賴的方式誘導(dǎo)體外培養(yǎng)的HaCaT細(xì)胞表達(dá)K17,其調(diào)控K17表達(dá)的機(jī)制是經(jīng)STAT1和STAT3兩個信號轉(zhuǎn)導(dǎo)通路實現(xiàn)的。 本研究首次發(fā)現(xiàn)了調(diào)控K17表達(dá)的一個新的通路——IL-17誘導(dǎo)KC表達(dá)K17,明確了IL-17調(diào)控K17表達(dá)的主要信號轉(zhuǎn)導(dǎo)機(jī)制是經(jīng)STAT1和STAT3信號通路,進(jìn)一步完善了“K17─T細(xì)胞─細(xì)胞因子環(huán)路”及其在銀屑病發(fā)病中的意義,初步闡明了K17作為自身抗原在其中的作用。研究結(jié)果不僅深化了對銀屑病發(fā)病機(jī)制的認(rèn)識,也為銀屑病的治療提供了新的思路。
[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
本文編號：2454944
[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
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R758.63
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