氧化應激敏感的TRPM2活化NLRP3炎癥小體參與白癜風異常免疫應答的機制研究
發(fā)布時間：2018-07-16 07:31
【摘要】：背景:白癜風是一種常見的自身免疫性皮膚病,其黑素細胞破壞主要系CD8~+T細胞介導的免疫損傷所致。文獻及我們以往的研究表明,氧化應激是導致白癜風發(fā)病的重要原因,并證實角質形成細胞在氧化應激下,對白癜風局部免疫微環(huán)境發(fā)揮重要調控作用,可誘導大量趨化因子如CXCL10、CXCL16分泌,通過與受體CXCR3、CXCR6結合介導CD8~+T細胞向皮膚遷移,最終導致黑素細胞特異損傷。然而,角質形成細胞在氧化應激下如何調控趨化信號介導CD8~+T細胞遷移,以及對CD8~+T細胞其他免疫功能的影響,仍有待進一步闡明。NLRP3炎癥小體是由NLRP3、ASC及pro-caspase-1組成的復合物,是天然免疫系統的重要組成部分,活化后誘導促炎因子IL-1β、IL-18的成熟與釋放。越來越多的研究證實NLRP3炎癥小體活化可誘導趨化因子分泌從而介導免疫細胞組織定向遷移,還可調控免疫細胞增殖、活化等過程,在多種自身免疫性疾病的啟動階段發(fā)揮重要作用。最近有學者提出NLRP3炎癥炎癥小體參與白癜風發(fā)病,但其在白癜風中的具體功能作用及激活機制,尚不清楚。研究證實,線粒體氧化應激是促進NLRP3向線粒體轉位并激活NLRP3炎癥小體的重要和必要因素。進一步發(fā)現,NLRP3炎癥小體組裝依賴胞內鈣信號,細胞膜上鈣通道開放介導胞內鈣超載,促進Ca~(2+)內流至線粒體,是導致線粒體損傷、NLRP3炎癥小體活化的關鍵機制。最新文獻發(fā)現,氧化應激敏感的TRPM2通道,介導Ca~(2+)內流,是線粒體氧化應激產生的重要機制,也參與調控氧化應激誘導的趨化因子表達、免疫細胞遷移等免疫過程。由此我們提出假說:白癜風患者角質形成細胞在氧化應激下,TRPM2鈣通道開放介導Ca~(2+)內流,誘導線粒體損傷,很可能是活化NLRP3炎癥小體的重要機制,NLRP3炎癥小體活化可能參與調控角質形成細胞趨化因子表達、免疫細胞皮膚遷移等異常免疫反應,最終導致黑素細胞破壞及白斑形成。目的:研究白癜風患者表皮氧化應激活化角質形成細胞NLRP3炎癥小體的機制,并闡明NLRP3炎癥小體活化對白癜風CD8~+T細胞皮膚遷移及免疫效應的調控作用。方法:1.采用免疫組化鑒定白癜風皮損區(qū)NLRP3等炎癥小體表達情況,ELISA檢測白癜風患者外周血IL-1β的表達及治療前后變化,并采用real-time PCR檢測皮損區(qū)IL-1β表達并分析其與H_2O_2蓄積、趨化因子CXCL10、CXCL16表達的關系。2.在原代角質形成細胞中,模擬體內氧化應激環(huán)境,采用流式檢測線粒體ROS生成、線粒體膜電位變化;采用real-time PCR、Western Blot、細胞免疫熒光、ELISA等鑒定NLRP3表達、定位以及活化情況,明確氧化應激對角質形成細胞中NLRP3炎癥小體的激活作用。3.在原代角質形成細胞中,模擬體內氧化應激環(huán)境,Western Blot檢測TRPM2表達,采用Fluo-4探針檢測Ca~(2+)內流情況;在體外Ha Ca T細胞中轉染TRPM2 Si RNA干涉TRPM2表達,檢測Ca~(2+)內流、線粒體損傷及NLRP3炎癥小體活化的各項指標,明確TRPM2介導的Ca~(2+)內流是否為線粒體氧化應激活化NLRP3炎癥小體所必需。4.在Ha Ca T細胞中,采用Si RNA干涉Ha Ca T細胞中TRPM2、NLRP3表達,結合IL-1β中和抗體及IL-1R受體阻滯劑,ELISA檢測Ha Ca T細胞中趨化因子CXCL10及CXCL16的分泌,闡明角質形成細胞中TRPM2活化的NLRP3炎癥小體對趨化因子表達的影響及機制。5.分選進展期白癜風患者外周血CD8~+T細胞,給予干涉TRPM2、NLRP3或中和IL-1β的Ha Ca T上清刺激處理,或阻斷CD8~+T細胞表面IL-1R,采用流式細胞數檢測CD8~+T細胞表面趨化因子受體CXCR3、CXCR6表達,明確角質形成細胞中TRPM2活化的NLRP3炎癥小體對CD8~+T細胞表面趨化因子受體表達的影響。6.分選進展期白癜風患者外周血CD8~+T細胞,利用Transwell實驗,檢測干涉TRPM2、NLRP3的Ha Ca T上清對白癜風患者CD8~+T細胞的遷移作用;并結合人重組趨化因子CXCL10、CXCL16試劑進行回復實驗,或流式分選消除CD8~+T細胞上CXCR3及CXCR6表達,進行趨化實驗,明確TRPM2活化的NLRP3炎癥小體是否通過調控CXCL10-CXCR3及CXCL16-CXCR6信號影響白癜風CD8~+T細胞遷移。7.分選進展期白癜風患者外周血CD8~+T細胞,給予干涉TRPM2、NLRP3的Ha Ca T上清刺激處理,流式檢測CD8~+T細胞分泌效應分子IFN-γ的能力,分析TRPM2活化的NLRP3炎癥小體對白癜風CD8~+T細胞免疫效應功能的調控作用。結果:1.免疫組化檢測白癜風皮損周組織中NLRP1、NLRP3、NLRC4以及AIM2的表達,發(fā)現以NLRP3上調最明顯,且主要表達于角質形成細胞。ELISA結果顯示,進展期白癜風患者外周血IL-1β(5.617±0.174 pg/m L,n=30)較健康對照(6.821±0.367 pg/m L,n=18)顯著下調(P0.01);采用標準療法治療2月后,外周IL-1β水平較治療前無明顯變化(n=30,P=0.7154)。但在白癜風皮損周,IL-1βm RNA水平顯著上調2.04±0.28倍(P0.01),并與皮損局部高濃度的H_2O_2(r=0.6588,P=0.0055)、以及上調的CXCL10(r=0.6118,P=0.0118)、CXCL16(r=0.5853,P=0.0172)m RNA水平均呈正相關。2.在原代角質形成細胞中,H_2O_2可呈劑量依賴方式誘導線粒體膜電位下降,促進線粒體ROS蓄積,導致線粒體損傷。此外,H_2O_2可顯著上調NLRP3 m RNA及蛋白水平,促進NLRP3及其接頭分子ASC向線粒體轉位,誘導NLRP3炎癥小體中Caspase-1及IL-1β的剪切成熟,同時效應分子IL-1β表達顯著增加。表明H_2O_2可激活角質形成細胞中NLRP3炎癥小體。3.在原代角質形成細胞中,H_2O_2可誘導TRPM2表達上調,顯著促進Ca~(2+)內流;而轉染TRPM2 Si RNA后,可顯著抑制H_2O_2誘導的Ca~(2+)內流、線粒體膜電位下降及ROS生成;同時H_2O_2誘導的NLRP3、ASC線粒體轉位及Caspase-1、IL-1β的剪切也被阻斷。表明TRPM2介導的Ca~(2+)內流是氧化應激活化NLRP3炎癥小體所必需。4.干涉Ha Ca T細胞中TRPM2、NLRP3表達顯著降低Ha Ca T細胞中氧化應激誘導的趨化因子CXCL10、CXCL16分泌,以干涉TRPM2最為顯著;此外,利用IL-1β中和抗體及IL-1R受體阻滯劑阻斷IL-1β/IL-R信號,也可抑制氧化應激誘導的CXCL10、CXCL16表達。表明角質形成細胞中TRPM2活化的NLRP3炎癥小體參與調控其自身分泌趨化因子,且至少部分是通過IL-1β/IL-R信號調控的。5.分選進展期白癜風患者外周CD8~+T細胞,流式檢測發(fā)現Ha Ca T細胞氧化應激模型上清可以顯著誘導CD8~+T細胞表面趨化標志CXCR3、CXCR6表達,而予以干涉了TRPM2、NLRP3的Ha Ca T細胞氧化應激模型上清處理后,相比未干涉組CXCR3、CXCR6表達均下調;此外,中和Ha Ca T細胞氧化應激模型中IL-1β或阻斷CD8~+T細胞表面IL-1R也有類似效果。表明角質形成細胞中TRPM2活化的NLRP3炎癥小體也可調控CD8~+T細胞表面趨化因子受體表達,并且是依賴IL-1β/IL-R信號的。6.利用Transwell模型研究Ha Ca T細胞上清對白癜風CD8~+T細胞的遷移,發(fā)現干涉TRPM2、NLRP3的Ha Ca T氧化應激模型上清對進展期白癜風患者CD8~+T細胞的遷移能力顯著下降,而加入人重組趨化因子rh CXCL10、rh CXCL16進行回復實驗,發(fā)現CD8~+T細胞趨化能力顯著上調。此外,消除白癜風患者CD8~+T細胞上CXCR3、CXCR6表達均會影響其遷移。該部分研究證實角質形成細胞中TRPM2活化的NLRP3炎癥小體通過CXCL10-CXCR3、CXCL16-CXCR6信號調控白癜風CD8~+T細胞遷移。7.分選進展期白癜風患者外周CD8~+T細胞,流式檢測發(fā)現Ha Ca T細胞氧化應激模型可以顯著誘導CD8~+T細胞表達IFN-γ,而干涉了TRPM2、NLRP3的Ha Ca T細胞上清處理CD8~+T細胞后,相比未干涉組其IFN-γ表達顯著減少。表明TRPM2活化的NLRP3炎癥小體對白癜風CD8~+T細胞表達IFN-γ、發(fā)揮免疫效應起關鍵調控作用。結論:通過本課題研究,我們首次明確氧化應激條件下,角質形成細胞中TRPM2介導Ca~(2+)內流,誘導線粒體損傷,從而活化NLRP3炎癥小體的具體機制;并率先揭示了TRPM2活化的NLRP3炎癥小體通過調控趨化因子CXCL10、CXCL16分泌、以及CD8~+T細胞表面CXCR3、CXCR6的表達影響白癜風CD8~+T細胞皮膚遷移;此外還發(fā)現角質形成細胞中TRPM2活化的NLRP3炎癥小體可以促進白癜風CD8~+T細胞分泌效應分子IFN-γ,參與白癜風發(fā)生及進展。本研究首次證實NLRP3炎癥小體在氧化應激誘導的CD8~+T細胞特異免疫應答中發(fā)揮關鍵橋梁作用,為氧化應激啟動自身免疫反應的機制提供了新證據,也為臨床治療提供了新思路和靶點。
[Abstract]:Background: vitiligo is a common autoimmune dermatosis in which melanocytes are damaged mainly by CD8~+T cell mediated immune damage. The literature and our previous studies have shown that oxidative stress is an important cause of the pathogenesis of vitiligo, and that keratinocytes are partly immune to vitiligo under oxidative stress. Play an important role in inducing a large number of chemokines, such as CXCL10, CXCL16 secretion, through binding with the receptor CXCR3, CXCR6 to mediate the migration of CD8~+T cells to the skin, and eventually lead to the specific damage to melanocytes. However, how to regulate the migration of CD8~+T cells mediated by chemotaxis and other CD8~+T cells under oxidative stress. The effect of immune function remains to be further clarified that the.NLRP3 inflammatory body is a complex composed of NLRP3, ASC and pro-caspase-1. It is an important part of the natural immune system. After activation, it induces the maturation and release of IL-1 beta, IL-18, and more and more studies have confirmed that the activation of NLRP3 inflammatory corpuscles can induce chemokine secretion and thus lead to the secretion of chemokine. Mediating the directed migration of immune cells and regulating the proliferation and activation of immune cells, it plays an important role in the initiation of a variety of autoimmune diseases. Recently, some scholars have suggested that NLRP3 inflammatory corpuscles are involved in the pathogenesis of vitiligo, but the specific function and activation mechanism of the inflammatory cells in vitiligo are not clear. Oxidative stress in mitochondria is an important and necessary factor to promote the transposition of NLRP3 to mitochondria and activate NLRP3 inflammatory bodies. It is further found that the assembly of NLRP3 inflammatory corpuscles depends on intracellular calcium signal, calcium channel on the cell membrane mediates intracellular calcium overload and promotes the flow of Ca~ (2+) to mitochondria, which leads to mitochondrial damage and NLRP3 inflammatory corpuscle activation. Key mechanism. The latest literature found that oxidative stress sensitive TRPM2 channel, mediating Ca~ (2+) internal flow, is an important mechanism of mitochondrial oxidative stress, and also involved in the regulation of oxidative stress induced chemokine expression, immune cell migration and other immune processes. Therefore, we put forward a false saying that keratinocytes in vitiligo patients are under oxidative stress, TR PM2 calcium channel is open to mediate Ca~ (2+) internal flow and induce mitochondrial damage. It is very likely to be an important mechanism for activating NLRP3 inflammatory corpuscles. The activation of NLRP3 inflammatory corpuscle may participate in the regulation of the expression of keratinocyte chemoattractant factor, immune cell skin migration and other abnormal immune responses, and eventually lead to melanocyte destruction and leukoplakia formation. The epidermal oxidation of the patients with purpura should activate the mechanism of the keratinocyte NLRP3 inflammatory body, and clarify the regulation of the activation of NLRP3 inflammatory corpuscle on the skin migration and immune effect of vitiligo CD8~+T cells. Methods: 1. the expression of NLRP3 and other inflammatory bodies in the skin lesion of vitiligo was identified by immunohistochemistry. ELISA was used to detect the peripheral blood of vitiligo patients. The expression of IL-1 beta and the changes before and after treatment, and using real-time PCR to detect the expression of IL-1 beta in the lesion area and analyze the relationship with H_2O_2 accumulation, chemokine CXCL10, CXCL16 expression,.2. in the primary keratinocytes, simulated oxidative stress environment in the body, and the change of mitochondrial membrane potential by flow cytometry, and real-time PC. R, Western Blot, cell immunofluorescence, ELISA and other identification of NLRP3 expression, location and activation, the activation of oxidative stress to NLRP3 inflammatory bodies in keratinocytes,.3. in the primary keratinocytes, the oxidative stress environment in the body was simulated, Western Blot was used to detect TRPM2 expression, and Fluo-4 probe was used to detect the flow of Ca~. TRPM2 Si RNA interfered TRPM2 expression in Ha Ca T cells in vitro to detect Ca~ (2+) internal flow, mitochondrial damage and NLRP3 inflammatory corpuscle activation. P3 expression, combined with IL-1 beta neutralization antibody and IL-1R receptor blocker, ELISA detection of chemokine CXCL10 and CXCL16 in Ha Ca T cells, clarifying the effect of NLRP3 inflammatory cells activated by TRPM2 in keratinocytes on the expression of chemokine and mechanism.5. to separate the peripheral blood cells of patients with vitiligo. Neutralizing the Ha Ca T supernatant of IL-1 beta, or blocking the IL-1R on the surface of CD8~+T cells, using flow cytometry to detect the chemokine receptor CXCR3 on the surface of CD8~+T cells, CXCR6 expression, and the influence of NLRP3 inflammatory bodies activated by TRPM2 in the keratinocytes on the expression of chemokine receptors on the surface of the CD8~+T cells CD8~+T cells in peripheral blood were used to detect the migration of TRPM2, Ha Ca T supernatant on the CD8~+T cells of patients with vitiligo and NLRP3 Ha Ca T, combined with recombinant chemokine CXCL10, CXCL16 reagent for recovery experiments, or flow sorting to eliminate CXCR3 and expression on CD8~+T cells. 3 whether or not the inflammatory corpuscle affects the peripheral blood CD8~+T cells of vitiligo patients through the regulation of CXCL10-CXCR3 and CXCL16-CXCR6 signals in the.7. separation of vitiligo CD8~+T cells, giving interference TRPM2, NLRP3 Ha Ca T supernatant, and the ability of flow detection of CD8~+T cells to secrete the molecule IFN- gamma. The effect on the immune effect of vitiligo CD8~+T cells. Results: 1. immunohistochemistry was used to detect the expression of NLRP1, NLRP3, NLRC4 and AIM2 in the peripheral tissue of vitiligo. It was found that the up-regulation of NLRP3 was most obvious, and the main expression in the keratinocyte.ELISA results showed that the peripheral blood IL-1 beta (5.617 + 0.174 pg/m L, n=30) of the patients with vitiligo. Compared with healthy controls (6.821 + 0.367 pg/m L, n=18) significantly down (P0.01), after February, the level of IL-1 beta was not significantly changed (n=30, P=0.7154) after February, but the level of IL-1 beta m RNA was up to 2.04 + 0.28 times (P0.01) in the skin lesion of vitiligo. CXCL10 (r=0.6118, P=0.0118), CXCL16 (r=0.5853, P=0.0172) m RNA levels are all positive correlation.2. in the primary keratinocytes. H_2O_2 can induce mitochondrial membrane potential decrease in a dose-dependent manner, promote mitochondrial ROS accumulation and lead to mitochondrial damage. The head molecule ASC transposition to mitochondria induces the shear maturation of Caspase-1 and IL-1 beta in the NLRP3 inflammatory body, and the expression of IL-1 beta of the effector molecule increases significantly. It indicates that H_2O_2 can activate the NLRP3 inflammatory corpuscle.3. in the keratinocytes of the keratinocytes, and H_2O_2 can induce the up regulation of TRPM2 expression and promote Ca~ (2+) internal flow. After 2 Si RNA, the flow of H_2O_2 induced Ca~ (2+), mitochondrial membrane potential and ROS formation were inhibited, while H_2O_2 induced NLRP3, ASC mitochondrial translocation and Caspase-1, IL-1 beta were also blocked. The oxidative stress induced chemotactic factor CXCL10, CXCL16 secretion in Ha Ca T cells, was most significant in interfering TRPM2. Moreover, IL-1 beta neutralization antibody and IL-1R receptor blocker blocking IL-1 beta /IL-R signal could also inhibit CXCL10 and CXCL16 expression induced by oxidative stress, indicating that the activation of inflammation in keratinocytes is small. The body participates in the regulation of its autotactic chemokine, and at least partly through the.5. separation of the peripheral CD8~+T cells of patients with vitiligo through the IL-1 beta /IL-R signal. Flow detection found that the supernatant of Ha Ca T cell oxidative stress model can significantly induce the surface chemotaxis of CD8~+T cells CXCR3, CXCR6 expression, while interfering TRPM2, NLRP3 After the a Ca T cell oxidative stress model supernatant, the expression of CXCR6 was downregulated compared to the uninterfered group CXCR3, and the IL-1 beta or blocking CD8~+T cell surface IL-1R in the oxidative stress model of Ha Ca T cells also had similar effects. It is expressed in body, and is dependent on the.6. of IL-1 beta /IL-R signal to use Transwell model to study the migration of Ha Ca T cell supernatant to vitiligo CD8~+T cells, and to find interference TRPM2. NLRP3 Ha Ca oxidative stress model supernatant significantly reduced the migration ability of vitiligo cells in progressive vitiligo patients. 16 the chemotactic ability of CD8~+T cells was significantly up-regulated. In addition, the removal of CXCR3 and CXCR6 expression on CD8~+T cells in patients with vitiligo could affect their migration. This part of the study confirmed that the NLRP3 inflammatory bodies activated by TRPM2 in keratinocytes through CXCL10-CXCR3, CXCL16-CXCR6 signals regulate the CD8~+T cell migration.7. separation of vitiligo. The flow cytometry found that the oxidative stress model of Ha Ca T cells could significantly induce the expression of IFN- gamma in CD8~+T cells, but interfered with TRPM2, and the Ha Ca T cell of NLRP3's Ha Ca was treated with the CD8~+T cells. The expression of IFN- gamma in 8~+T cells plays a key role in regulating the immune effect. Conclusion: through this study, we have first identified the specific mechanism of TRPM2 mediated Ca~ (2+) inflow in keratinocytes, inducing mitochondrial damage, and activating the specific mechanism of NLRP3 inflammatory corpuscles in the keratinocytes, and first revealing the TRPM2 activated NLRP3 inflammation corpuscles. The regulation of chemokine CXCL10, CXCL16 secretion, and the expression of CXCR3, CXCR6 on the surface of CD8~+T cells affects the skin migration of vitiligo CD8~+T cells. Furthermore, the NLRP3 inflammatory corpuscle activated by TRPM2 in keratinocytes can promote the CD8~+T cell secretory effect molecule IFN- gamma of vitiligo and participate in the occurrence and progress of vitiligo. This study is the first evidence of this study. The solid NLRP3 inflammatory body plays a key role in the specific immune response of CD8~+T cells induced by oxidative stress, which provides new evidence for the mechanism of oxidative stress to start the autoimmune reaction and provides new ideas and targets for clinical treatment.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R758.41
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本文編號：2125681
[Abstract]:Background: vitiligo is a common autoimmune dermatosis in which melanocytes are damaged mainly by CD8~+T cell mediated immune damage. The literature and our previous studies have shown that oxidative stress is an important cause of the pathogenesis of vitiligo, and that keratinocytes are partly immune to vitiligo under oxidative stress. Play an important role in inducing a large number of chemokines, such as CXCL10, CXCL16 secretion, through binding with the receptor CXCR3, CXCR6 to mediate the migration of CD8~+T cells to the skin, and eventually lead to the specific damage to melanocytes. However, how to regulate the migration of CD8~+T cells mediated by chemotaxis and other CD8~+T cells under oxidative stress. The effect of immune function remains to be further clarified that the.NLRP3 inflammatory body is a complex composed of NLRP3, ASC and pro-caspase-1. It is an important part of the natural immune system. After activation, it induces the maturation and release of IL-1 beta, IL-18, and more and more studies have confirmed that the activation of NLRP3 inflammatory corpuscles can induce chemokine secretion and thus lead to the secretion of chemokine. Mediating the directed migration of immune cells and regulating the proliferation and activation of immune cells, it plays an important role in the initiation of a variety of autoimmune diseases. Recently, some scholars have suggested that NLRP3 inflammatory corpuscles are involved in the pathogenesis of vitiligo, but the specific function and activation mechanism of the inflammatory cells in vitiligo are not clear. Oxidative stress in mitochondria is an important and necessary factor to promote the transposition of NLRP3 to mitochondria and activate NLRP3 inflammatory bodies. It is further found that the assembly of NLRP3 inflammatory corpuscles depends on intracellular calcium signal, calcium channel on the cell membrane mediates intracellular calcium overload and promotes the flow of Ca~ (2+) to mitochondria, which leads to mitochondrial damage and NLRP3 inflammatory corpuscle activation. Key mechanism. The latest literature found that oxidative stress sensitive TRPM2 channel, mediating Ca~ (2+) internal flow, is an important mechanism of mitochondrial oxidative stress, and also involved in the regulation of oxidative stress induced chemokine expression, immune cell migration and other immune processes. Therefore, we put forward a false saying that keratinocytes in vitiligo patients are under oxidative stress, TR PM2 calcium channel is open to mediate Ca~ (2+) internal flow and induce mitochondrial damage. It is very likely to be an important mechanism for activating NLRP3 inflammatory corpuscles. The activation of NLRP3 inflammatory corpuscle may participate in the regulation of the expression of keratinocyte chemoattractant factor, immune cell skin migration and other abnormal immune responses, and eventually lead to melanocyte destruction and leukoplakia formation. The epidermal oxidation of the patients with purpura should activate the mechanism of the keratinocyte NLRP3 inflammatory body, and clarify the regulation of the activation of NLRP3 inflammatory corpuscle on the skin migration and immune effect of vitiligo CD8~+T cells. Methods: 1. the expression of NLRP3 and other inflammatory bodies in the skin lesion of vitiligo was identified by immunohistochemistry. ELISA was used to detect the peripheral blood of vitiligo patients. The expression of IL-1 beta and the changes before and after treatment, and using real-time PCR to detect the expression of IL-1 beta in the lesion area and analyze the relationship with H_2O_2 accumulation, chemokine CXCL10, CXCL16 expression,.2. in the primary keratinocytes, simulated oxidative stress environment in the body, and the change of mitochondrial membrane potential by flow cytometry, and real-time PC. R, Western Blot, cell immunofluorescence, ELISA and other identification of NLRP3 expression, location and activation, the activation of oxidative stress to NLRP3 inflammatory bodies in keratinocytes,.3. in the primary keratinocytes, the oxidative stress environment in the body was simulated, Western Blot was used to detect TRPM2 expression, and Fluo-4 probe was used to detect the flow of Ca~. TRPM2 Si RNA interfered TRPM2 expression in Ha Ca T cells in vitro to detect Ca~ (2+) internal flow, mitochondrial damage and NLRP3 inflammatory corpuscle activation. P3 expression, combined with IL-1 beta neutralization antibody and IL-1R receptor blocker, ELISA detection of chemokine CXCL10 and CXCL16 in Ha Ca T cells, clarifying the effect of NLRP3 inflammatory cells activated by TRPM2 in keratinocytes on the expression of chemokine and mechanism.5. to separate the peripheral blood cells of patients with vitiligo. Neutralizing the Ha Ca T supernatant of IL-1 beta, or blocking the IL-1R on the surface of CD8~+T cells, using flow cytometry to detect the chemokine receptor CXCR3 on the surface of CD8~+T cells, CXCR6 expression, and the influence of NLRP3 inflammatory bodies activated by TRPM2 in the keratinocytes on the expression of chemokine receptors on the surface of the CD8~+T cells CD8~+T cells in peripheral blood were used to detect the migration of TRPM2, Ha Ca T supernatant on the CD8~+T cells of patients with vitiligo and NLRP3 Ha Ca T, combined with recombinant chemokine CXCL10, CXCL16 reagent for recovery experiments, or flow sorting to eliminate CXCR3 and expression on CD8~+T cells. 3 whether or not the inflammatory corpuscle affects the peripheral blood CD8~+T cells of vitiligo patients through the regulation of CXCL10-CXCR3 and CXCL16-CXCR6 signals in the.7. separation of vitiligo CD8~+T cells, giving interference TRPM2, NLRP3 Ha Ca T supernatant, and the ability of flow detection of CD8~+T cells to secrete the molecule IFN- gamma. The effect on the immune effect of vitiligo CD8~+T cells. Results: 1. immunohistochemistry was used to detect the expression of NLRP1, NLRP3, NLRC4 and AIM2 in the peripheral tissue of vitiligo. It was found that the up-regulation of NLRP3 was most obvious, and the main expression in the keratinocyte.ELISA results showed that the peripheral blood IL-1 beta (5.617 + 0.174 pg/m L, n=30) of the patients with vitiligo. Compared with healthy controls (6.821 + 0.367 pg/m L, n=18) significantly down (P0.01), after February, the level of IL-1 beta was not significantly changed (n=30, P=0.7154) after February, but the level of IL-1 beta m RNA was up to 2.04 + 0.28 times (P0.01) in the skin lesion of vitiligo. CXCL10 (r=0.6118, P=0.0118), CXCL16 (r=0.5853, P=0.0172) m RNA levels are all positive correlation.2. in the primary keratinocytes. H_2O_2 can induce mitochondrial membrane potential decrease in a dose-dependent manner, promote mitochondrial ROS accumulation and lead to mitochondrial damage. The head molecule ASC transposition to mitochondria induces the shear maturation of Caspase-1 and IL-1 beta in the NLRP3 inflammatory body, and the expression of IL-1 beta of the effector molecule increases significantly. It indicates that H_2O_2 can activate the NLRP3 inflammatory corpuscle.3. in the keratinocytes of the keratinocytes, and H_2O_2 can induce the up regulation of TRPM2 expression and promote Ca~ (2+) internal flow. After 2 Si RNA, the flow of H_2O_2 induced Ca~ (2+), mitochondrial membrane potential and ROS formation were inhibited, while H_2O_2 induced NLRP3, ASC mitochondrial translocation and Caspase-1, IL-1 beta were also blocked. The oxidative stress induced chemotactic factor CXCL10, CXCL16 secretion in Ha Ca T cells, was most significant in interfering TRPM2. Moreover, IL-1 beta neutralization antibody and IL-1R receptor blocker blocking IL-1 beta /IL-R signal could also inhibit CXCL10 and CXCL16 expression induced by oxidative stress, indicating that the activation of inflammation in keratinocytes is small. The body participates in the regulation of its autotactic chemokine, and at least partly through the.5. separation of the peripheral CD8~+T cells of patients with vitiligo through the IL-1 beta /IL-R signal. Flow detection found that the supernatant of Ha Ca T cell oxidative stress model can significantly induce the surface chemotaxis of CD8~+T cells CXCR3, CXCR6 expression, while interfering TRPM2, NLRP3 After the a Ca T cell oxidative stress model supernatant, the expression of CXCR6 was downregulated compared to the uninterfered group CXCR3, and the IL-1 beta or blocking CD8~+T cell surface IL-1R in the oxidative stress model of Ha Ca T cells also had similar effects. It is expressed in body, and is dependent on the.6. of IL-1 beta /IL-R signal to use Transwell model to study the migration of Ha Ca T cell supernatant to vitiligo CD8~+T cells, and to find interference TRPM2. NLRP3 Ha Ca oxidative stress model supernatant significantly reduced the migration ability of vitiligo cells in progressive vitiligo patients. 16 the chemotactic ability of CD8~+T cells was significantly up-regulated. In addition, the removal of CXCR3 and CXCR6 expression on CD8~+T cells in patients with vitiligo could affect their migration. This part of the study confirmed that the NLRP3 inflammatory bodies activated by TRPM2 in keratinocytes through CXCL10-CXCR3, CXCL16-CXCR6 signals regulate the CD8~+T cell migration.7. separation of vitiligo. The flow cytometry found that the oxidative stress model of Ha Ca T cells could significantly induce the expression of IFN- gamma in CD8~+T cells, but interfered with TRPM2, and the Ha Ca T cell of NLRP3's Ha Ca was treated with the CD8~+T cells. The expression of IFN- gamma in 8~+T cells plays a key role in regulating the immune effect. Conclusion: through this study, we have first identified the specific mechanism of TRPM2 mediated Ca~ (2+) inflow in keratinocytes, inducing mitochondrial damage, and activating the specific mechanism of NLRP3 inflammatory corpuscles in the keratinocytes, and first revealing the TRPM2 activated NLRP3 inflammation corpuscles. The regulation of chemokine CXCL10, CXCL16 secretion, and the expression of CXCR3, CXCR6 on the surface of CD8~+T cells affects the skin migration of vitiligo CD8~+T cells. Furthermore, the NLRP3 inflammatory corpuscle activated by TRPM2 in keratinocytes can promote the CD8~+T cell secretory effect molecule IFN- gamma of vitiligo and participate in the occurrence and progress of vitiligo. This study is the first evidence of this study. The solid NLRP3 inflammatory body plays a key role in the specific immune response of CD8~+T cells induced by oxidative stress, which provides new evidence for the mechanism of oxidative stress to start the autoimmune reaction and provides new ideas and targets for clinical treatment.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R758.41
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本文編號：2125681
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