氧化应激敏感的TRPM2活化NLRP3炎症小体参与白癜风异常免疫应答的机制研究
发布时间：2018-07-16 07:31
【摘要】：背景:白癜风是一种常见的自身免疫性皮肤病,其黑素细胞破坏主要系CD8~+T细胞介导的免疫损伤所致。文献及我们以往的研究表明,氧化应激是导致白癜风发病的重要原因,并证实角质形成细胞在氧化应激下,对白癜风局部免疫微环境发挥重要调控作用,可诱导大量趋化因子如CXCL10、CXCL16分泌,通过与受体CXCR3、CXCR6结合介导CD8~+T细胞向皮肤迁移,最终导致黑素细胞特异损伤。然而,角质形成细胞在氧化应激下如何调控趋化信号介导CD8~+T细胞迁移,以及对CD8~+T细胞其他免疫功能的影响,仍有待进一步阐明。NLRP3炎症小体是由NLRP3、ASC及pro-caspase-1组成的复合物,是天然免疫系统的重要组成部分,活化后诱导促炎因子IL-1β、IL-18的成熟与释放。越来越多的研究证实NLRP3炎症小体活化可诱导趋化因子分泌从而介导免疫细胞组织定向迁移,还可调控免疫细胞增殖、活化等过程,在多种自身免疫性疾病的启动阶段发挥重要作用。最近有学者提出NLRP3炎症炎症小体参与白癜风发病,但其在白癜风中的具体功能作用及激活机制,尚不清楚。研究证实,线粒体氧化应激是促进NLRP3向线粒体转位并激活NLRP3炎症小体的重要和必要因素。进一步发现,NLRP3炎症小体组装依赖胞内钙信号,细胞膜上钙通道开放介导胞内钙超载,促进Ca~(2+)内流至线粒体,是导致线粒体损伤、NLRP3炎症小体活化的关键机制。最新文献发现,氧化应激敏感的TRPM2通道,介导Ca~(2+)内流,是线粒体氧化应激产生的重要机制,也参与调控氧化应激诱导的趋化因子表达、免疫细胞迁移等免疫过程。由此我们提出假说:白癜风患者角质形成细胞在氧化应激下,TRPM2钙通道开放介导Ca~(2+)内流,诱导线粒体损伤,很可能是活化NLRP3炎症小体的重要机制,NLRP3炎症小体活化可能参与调控角质形成细胞趋化因子表达、免疫细胞皮肤迁移等异常免疫反应,最终导致黑素细胞破坏及白斑形成。目的:研究白癜风患者表皮氧化应激活化角质形成细胞NLRP3炎症小体的机制,并阐明NLRP3炎症小体活化对白癜风CD8~+T细胞皮肤迁移及免疫效应的调控作用。方法:1.采用免疫组化鉴定白癜风皮损区NLRP3等炎症小体表达情况,ELISA检测白癜风患者外周血IL-1β的表达及治疗前后变化,并采用real-time PCR检测皮损区IL-1β表达并分析其与H_2O_2蓄积、趋化因子CXCL10、CXCL16表达的关系。2.在原代角质形成细胞中,模拟体内氧化应激环境,采用流式检测线粒体ROS生成、线粒体膜电位变化;采用real-time PCR、Western Blot、细胞免疫荧光、ELISA等鉴定NLRP3表达、定位以及活化情况,明确氧化应激对角质形成细胞中NLRP3炎症小体的激活作用。3.在原代角质形成细胞中,模拟体内氧化应激环境,Western Blot检测TRPM2表达,采用Fluo-4探针检测Ca~(2+)内流情况;在体外Ha Ca T细胞中转染TRPM2 Si RNA干涉TRPM2表达,检测Ca~(2+)内流、线粒体损伤及NLRP3炎症小体活化的各项指标,明确TRPM2介导的Ca~(2+)内流是否为线粒体氧化应激活化NLRP3炎症小体所必需。4.在Ha Ca T细胞中,采用Si RNA干涉Ha Ca T细胞中TRPM2、NLRP3表达,结合IL-1β中和抗体及IL-1R受体阻滞剂,ELISA检测Ha Ca T细胞中趋化因子CXCL10及CXCL16的分泌,阐明角质形成细胞中TRPM2活化的NLRP3炎症小体对趋化因子表达的影响及机制。5.分选进展期白癜风患者外周血CD8~+T细胞,给予干涉TRPM2、NLRP3或中和IL-1β的Ha Ca T上清刺激处理,或阻断CD8~+T细胞表面IL-1R,采用流式细胞数检测CD8~+T细胞表面趋化因子受体CXCR3、CXCR6表达,明确角质形成细胞中TRPM2活化的NLRP3炎症小体对CD8~+T细胞表面趋化因子受体表达的影响。6.分选进展期白癜风患者外周血CD8~+T细胞,利用Transwell实验,检测干涉TRPM2、NLRP3的Ha Ca T上清对白癜风患者CD8~+T细胞的迁移作用;并结合人重组趋化因子CXCL10、CXCL16试剂进行回复实验,或流式分选消除CD8~+T细胞上CXCR3及CXCR6表达,进行趋化实验,明确TRPM2活化的NLRP3炎症小体是否通过调控CXCL10-CXCR3及CXCL16-CXCR6信号影响白癜风CD8~+T细胞迁移。7.分选进展期白癜风患者外周血CD8~+T细胞,给予干涉TRPM2、NLRP3的Ha Ca T上清刺激处理,流式检测CD8~+T细胞分泌效应分子IFN-γ的能力,分析TRPM2活化的NLRP3炎症小体对白癜风CD8~+T细胞免疫效应功能的调控作用。结果:1.免疫组化检测白癜风皮损周组织中NLRP1、NLRP3、NLRC4以及AIM2的表达,发现以NLRP3上调最明显,且主要表达于角质形成细胞。ELISA结果显示,进展期白癜风患者外周血IL-1β(5.617±0.174 pg/m L,n=30)较健康对照(6.821±0.367 pg/m L,n=18)显著下调(P0.01);采用标准疗法治疗2月后,外周IL-1β水平较治疗前无明显变化(n=30,P=0.7154)。但在白癜风皮损周,IL-1βm RNA水平显著上调2.04±0.28倍(P0.01),并与皮损局部高浓度的H_2O_2(r=0.6588,P=0.0055)、以及上调的CXCL10(r=0.6118,P=0.0118)、CXCL16(r=0.5853,P=0.0172)m RNA水平均呈正相关。2.在原代角质形成细胞中,H_2O_2可呈剂量依赖方式诱导线粒体膜电位下降,促进线粒体ROS蓄积,导致线粒体损伤。此外,H_2O_2可显著上调NLRP3 m RNA及蛋白水平,促进NLRP3及其接头分子ASC向线粒体转位,诱导NLRP3炎症小体中Caspase-1及IL-1β的剪切成熟,同时效应分子IL-1β表达显著增加。表明H_2O_2可激活角质形成细胞中NLRP3炎症小体。3.在原代角质形成细胞中,H_2O_2可诱导TRPM2表达上调,显著促进Ca~(2+)内流;而转染TRPM2 Si RNA后,可显著抑制H_2O_2诱导的Ca~(2+)内流、线粒体膜电位下降及ROS生成;同时H_2O_2诱导的NLRP3、ASC线粒体转位及Caspase-1、IL-1β的剪切也被阻断。表明TRPM2介导的Ca~(2+)内流是氧化应激活化NLRP3炎症小体所必需。4.干涉Ha Ca T细胞中TRPM2、NLRP3表达显著降低Ha Ca T细胞中氧化应激诱导的趋化因子CXCL10、CXCL16分泌,以干涉TRPM2最为显著;此外,利用IL-1β中和抗体及IL-1R受体阻滞剂阻断IL-1β/IL-R信号,也可抑制氧化应激诱导的CXCL10、CXCL16表达。表明角质形成细胞中TRPM2活化的NLRP3炎症小体参与调控其自身分泌趋化因子,且至少部分是通过IL-1β/IL-R信号调控的。5.分选进展期白癜风患者外周CD8~+T细胞,流式检测发现Ha Ca T细胞氧化应激模型上清可以显著诱导CD8~+T细胞表面趋化标志CXCR3、CXCR6表达,而予以干涉了TRPM2、NLRP3的Ha Ca T细胞氧化应激模型上清处理后,相比未干涉组CXCR3、CXCR6表达均下调;此外,中和Ha Ca T细胞氧化应激模型中IL-1β或阻断CD8~+T细胞表面IL-1R也有类似效果。表明角质形成细胞中TRPM2活化的NLRP3炎症小体也可调控CD8~+T细胞表面趋化因子受体表达,并且是依赖IL-1β/IL-R信号的。6.利用Transwell模型研究Ha Ca T细胞上清对白癜风CD8~+T细胞的迁移,发现干涉TRPM2、NLRP3的Ha Ca T氧化应激模型上清对进展期白癜风患者CD8~+T细胞的迁移能力显著下降,而加入人重组趋化因子rh CXCL10、rh CXCL16进行回复实验,发现CD8~+T细胞趋化能力显著上调。此外,消除白癜风患者CD8~+T细胞上CXCR3、CXCR6表达均会影响其迁移。该部分研究证实角质形成细胞中TRPM2活化的NLRP3炎症小体通过CXCL10-CXCR3、CXCL16-CXCR6信号调控白癜风CD8~+T细胞迁移。7.分选进展期白癜风患者外周CD8~+T细胞,流式检测发现Ha Ca T细胞氧化应激模型可以显著诱导CD8~+T细胞表达IFN-γ,而干涉了TRPM2、NLRP3的Ha Ca T细胞上清处理CD8~+T细胞后,相比未干涉组其IFN-γ表达显著减少。表明TRPM2活化的NLRP3炎症小体对白癜风CD8~+T细胞表达IFN-γ、发挥免疫效应起关键调控作用。结论:通过本课题研究,我们首次明确氧化应激条件下,角质形成细胞中TRPM2介导Ca~(2+)内流,诱导线粒体损伤,从而活化NLRP3炎症小体的具体机制;并率先揭示了TRPM2活化的NLRP3炎症小体通过调控趋化因子CXCL10、CXCL16分泌、以及CD8~+T细胞表面CXCR3、CXCR6的表达影响白癜风CD8~+T细胞皮肤迁移;此外还发现角质形成细胞中TRPM2活化的NLRP3炎症小体可以促进白癜风CD8~+T细胞分泌效应分子IFN-γ,参与白癜风发生及进展。本研究首次证实NLRP3炎症小体在氧化应激诱导的CD8~+T细胞特异免疫应答中发挥关键桥梁作用,为氧化应激启动自身免疫反应的机制提供了新证据,也为临床治疗提供了新思路和靶点。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R758.41
，
本文编号：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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R758.41
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本文编号：2125681
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