【作者】 张宝祥；

【导师】 涂彩霞；

【作者基本信息】 大连医科大学 ， 中西医结合皮肤病与性病学， 2013， 博士

【摘要】 白癜风是较常见的色素脱失性疾病，其中非节段型白癜风的发病机制与自身免疫功能异常有关。近来，CD8~+T细胞在白癜风发病机制中的作用受到重视，而T细胞从外周循环归巢至皮肤发挥功能并不是一个随机过程，皮肤淋巴细胞归巢抗原（Cutaneous Lymphocyte-associated Antigen, CLA）是效应性T细胞表面重要的皮肤归巢分子，可介导效应性T细胞从外周血向皮肤归巢。新近的研究提示皮肤归巢的CD8~+T细胞（CLA~+CD8~+T细胞）可能是白癜风致病的自身反应性细胞。调节性T细胞（regulatory T cell, Treg）是近年发现的具有免疫抑制功能T细胞亚群，对于维持机体免疫动态平衡具有至关重要的作用，越来越多的证据显示Treg的抑制功能下降，是自身反应性细胞激活的重要因素，我们推测非节段型白癜风患者外周血Treg对自身反应性细胞抑制功能下降参与了白癜风的发病机制。对于白癜风的治疗尚无特效疗法，中药治疗白癜风的历史悠久，但治疗机理有待研究，在有免疫调节作用的中药有效成分中找到对白癜风自身反应性CLA~+CD8~+T细胞具有抑制作用中药单体，旨在为临床上应用和推广提供实验依据，是目前研究的一个新方向。目的：1.检测非节段型白癜风患者外周血CD8~+T细胞、CLA~+CD8~+T细胞的数量，并分析其杀伤功能相关蛋白穿孔素、颗粒酶B、FasL以及归巢相关趋化因子受体CCR4（C-C chemokine receptor4）、CCR10的表达2.分析白癜风患者外周血Treg对自身皮肤归巢的CD8~+T细胞抑制功能是否下降及Treg功能性调节分子的表达。3.探究具有免疫调节作用的中药单体对白癜风患者皮肤归巢的CD8~+T细胞是否具有抑制作用，并初步探讨其作用机制。方法：1.对非节段型白癜风患者进展期15例、稳定期12例，正常人15例，采用流式细胞术，检测外周血CD8~+T细胞、CLA~+CD8~+T细胞的数量，并分析其杀伤功能相关蛋白穿孔素、颗粒酶B、FasL以及归巢相关趋化因子受体CCR4、CCR10的表达。2.对非节段型白癜风患者进展期7例、稳定期6例，正常人6例，采用流式细胞术检测外周血Treg功能性调节分子的表达。通过免疫磁珠法分选外周血CD8~+T细胞和Treg，根据CFSE（carboxyfluorescein diacetate succinimidyl ester）标记技术，采用流式细胞术检测外周血CLA~+CD8~+T细胞的增殖能力，并分析Treg对自身CLA~+CD8~+T细胞增殖的抑制能力。3.以9例非节段型进展期白癜风患者为研究对象，对文献报道能抑制T细胞功能且所属中药在古中医方剂中被用于治疗白癜风的中药单体，包括芹黄素、汉黄芩素、山奈酚、芍药苷和京尼平苷，通过免疫磁珠法分选外周血CLA~+CD8~+T细胞，采用CCK-8法检测这些中药单体对于外周血CLA~+CD8~+T细胞无杀伤毒性浓度范围，进而在此浓度范围内研究这些中药单体对外周血CLA~+CD8~+T细胞的抑制功能，并初步探讨其作用机制。结果：1.进展期白癜风外周血CLA~+CD8~+T细胞数量及功能检测1）外周血CD8~+T细胞的数量在白癜风进展期、稳定期和对照组之间无明显差异；CD8~+T细胞穿孔素、颗粒酶B和FasL的表达在三组间均无明显差异。2）外周血CLA~+CD8~+T细胞的数量在进展期明显高于稳定期和对照组，且在进展期的数量与白癜风面积及严重程度评分（Vitiligo Area Scoring Index, VASI）正相关；CCR4在CD8~+T细胞的表达在进展期明显高于稳定期和对照组，而CCR10的表达在三组间无明显差异。3）外周血CLA~+CD8~+T细胞穿孔素、颗粒酶B的表达在白癜风进展期明显高于稳定期和对照组，FasL的表达在三组间无明显差异；CCR4和CCR10在CLA~+CD8~+T细胞的表达在三组间均无明显差异。2.进展期白癜风患者外周血Treg对自身CLA~+CD8~+T细胞抑制功能检测1）外周血Treg表达功能性调节分子细胞毒T淋巴细胞相关抗原4（cytotoxicT lymphocyte-associated antigen-4, CTLA-4）水平在进展期明显高于稳定期和对照组，而表达淋巴细胞活化基因-3分子（lymphocyte activation gene-3, LAG-3）、CD39和CD73水平在三组间无明显差异。2）外周血CLA~+CD8~+T细胞的增殖能力在三组间无明显差异。3）外周血Treg对于自身CLA~+CD8~+T细胞的抑制能力在白癜风进展期明显低于稳定期和对照组。3.具有免疫调节作用的中药单体对CLA~+CD8~+T细胞抑制功能检测1）芹黄素可抑制进展期白癜风患者外周血CLA~+CD8~+T细胞增殖，而汉黄芩素、山奈酚、芍药苷和京尼平苷无明显抑制作用。2）芹黄素可抑制白癜风CLA~+CD8~+T细胞分泌IL-2，但对IFN-γ和TNF-α无明显抑制作用。3）芹黄素能够诱导白癜风CLA~+CD8~+T细胞周期停滞在G0/G1期。结论：1.非节段型进展期白癜风患者外周血CLA~+CD8~+T细胞数量增加，杀伤功能相关蛋白穿孔素、颗粒酶B的表达增强，提示CLA~+CD8~+T细胞是白癜风的自身反应性细胞并参与了白癜风的发病机制。2.非节段型进展期白癜风患者外周血Treg对自身CLA~+CD8~+T细胞抑制功能下降，可能与CLA~+CD8~+T细胞数量增加有关。3.芹黄素对非节段型进展期白癜风患者外周血激活的CLA~+CD8~+T细胞具有抑制作用，从免疫学发病机制方面为芹黄素用于白癜风的治疗提供了实验依据。更多还原

【Abstract】 Vitiligo is a depigmentation disorder affecting around1%to2%of the generalpopulation. It can be separated into segmental and non-segmental types. The latter is themost common, and generally described as having an autoimmune etiology. Recentworks suggest that CD8~+T cells play an important role in the pathogenesis ofvitiligo.The migration of circulating T lymphocytes to the skin is not a random process,and the cutaneous lymphocyte-associated antigen (CLA) antigen is considered a homingreceptor for effector memory T cells with tropism for the skin. Recent studies suggestthat the CLA~+CD8~+T cells may be the autoreactive cells in vitiligo, and the studies onthe characterization of peripheral CLA~+CD8~+T cells will be helpful to reveal theimmune abnormalities of vitiligo.Regulatory T cells (Tregs) play a crucial role in the maintenance of peripheraltolerance via suppression of self-reactive T cells. More and more studies suggested thatan attenuated suppressive function of peripheral Tregs on self-reactive T cells may leadto self-reactive immune responses. We hypothesized that there may be a decreasedinhibition function of peripheral Tregs on autologous CLA~+CD8~+T cells in thepathogenesis of vitiligo.Traditional Chinese medicines (TCMs) were used for the treatment of vitiligo witha long history, but the therapeutic mechanism remains to be studied. Recently, someingredients of Chinese medicines which were used to treat vitiligo in TCM prescriptionswere reported for their inhibition to T cell. Thus, studies on ingredients of TCMs withsuppressive function to self-reactive CLA~+CD8~+T cells may provide experimental basisfor the application of these ingredients in the treatment of vitiligo. Objective:1. To investigate the frequencies of peripheral CD8~+T cells and CLA~+CD8~+T cells,and their expression of cytotoxic molecules (perforin、granzyme-B and FasL), andmigration-related molecules (CCR4and CCR10) in non-segmental vitiligo patients.2. To investigate the inhibition effects of peripheral Tregs on proliferation ofautologous CLA~+CD8~+T cells in non-segmental vitiligo patients and the expressionlevels of functional molecules on Tregs.3. To seek for ingredients of TCMs with suppressive effects on self-reactiveCLA~+CD8~+T cells, and investigate the mechanisms.Methods:1. Twenty seven patients (15with active and12with stable non-segmental vitiligo)and15healthy control volunteers were enrolled in the study. The frequencies of CD8~+T cells and CLA~+CD8~+T cells in peripheral blood of participants were investigated byflow cytometry, and their expression levels of cytotoxic molecules (perforin,granzyme-B and FasL) and chemokine receptors (CCR4, CCR10) were evaluated also.2. In7active non-segmental vitiligo patients,6stable patients and6controls, theexpression levels of functional molecules on Tregs in peripheral blood of participantswere investigated by flow cytometry. The proliferative responses of peripheralCLA~+CD8~+T cells were assessed in the absence or presence of autologous Tregs usingCFSE.3. Nine patients with active non-segmental vitiligo patients were enrolled in thispart. Five ingredients of TCMs, including apigenin, wogonin, kaempferol, peoniflorinand geniposide, were tested. To test the cytotoxicity of the ingredients, the viability ofcells was evaluated by the CCK-8assay. And the inhibition effects and relatedmechanisms of these ingredients on CLA~+CD8~+T cells were investigated.Results:1. Detection of frequency and hyper-activated cytotoxic functions of peripheralCD8+CLA~+T cells from active vitiligo patients1) There was no significant difference in the percentages of peripheral CD8~+T cellsamong the active vitiligo, stable vitiligo and control group. The expression levels ofcytotoxic molecules (perforin、granzyme-B and FasL) were of no significant differenceamong the three groups.2) The frequencies of peripheral CD8+CLA~+T cells were significantly higher in theactive vitiligo compared with the stable vitiligo and control group, and a positive correlation was found between Vitiligo Area Scoring Index (VASI) and the frequenciesof CLA~+CD8~+T cells. The expression levels of CCR4on CD8~+T cells were significantlyhigher in the active than in the stable vitiligo or control group, but the expression levelsof CCR10on CD8~+T cells were of no significant difference among the three groups.3) Both the expression levels of perforin and granzyme-B on peripheralCD8+CLA~+T cells were significantly higher in the active vitiligo compared with thestable vitiligo and control group, but the expression levels of FasL on CLA~+CD8~+T cellswere of no significant difference among the three groups. And the expression levels ofCCR4and CCR10on CLA~+CD8~+T cells were of no significant difference among thethree groups.2. Detection of suppressive function of Tregs on the proliferation of autologousCLA~+CD8~+T cells1) The expression levels of cytotoxic T lymphocyte-associated antigen-4(CTLA-4)on peripheral Tregs were significantly higher in the active vitiligo compared with thestable vitiligo and control group, but the expression levels of lymphocyte activationgene-3(LAG-3)、CD39and CD73were of no significant difference among the threegroups.2) The percentages of proliferating peripheral CLA~+CD8~+T cells stimulated withanti-CD3/CD28mAbs were of no significant difference among the three groups.However, after cocultured with autologous Tregs, the percentages of proliferatingperipheral CLA~+CD8~+T cells were significantly higher in the active vitiligo comparedwith the stable vitiligo and control group.3) Tregs showed an attenuated suppressive function on the proliferation ofautologous CLA~+CD8~+T cells in the active vitiligo compared with the stable vitiligo andcontrol group.3. Analysis of inhibition effects of ingredients of TCMs with functions ofimmuno-regulation on peripheral CD8+CLA~+T cells from active vitiligo patients1) The proliferative responses of anti-CD3/CD28mAbs stimulated peripheralCLA~+CD8~+T cells from active non-segmental vitiligo patients were inhibited obviouslyby apigenin in a dose-dependent manner, while Wogonin, kaempferol, peoniflorin andgeniposide showed no inhibition effects.2) The secretion of IL-2of peripheral CLA~+CD8~+T cells was inhibited obviously.However, the production of IFN-γ and TNF-α was not effected by apigenin.3) Apigenin induces peripheral CLA~+CD8~+T cell cycle arrest at G0/G1phase. Conclusion:1. Patients with active non-segmental vitiligo have a higher frequency of peripheralCD8+CLA~+T cells with hyper-activated cytotoxic functions (perforin and granzyme-B),which may be involved in the pathogenesis of non-segmental vitiligo.2. Tregs showed an attenuated suppressive function on the proliferation ofautologous CLA~+CD8~+T cells, which may be responsible for the increased population ofperipheral CLA~+CD8~+T cells in the active non-segmental vitiligo.3. Apigenin showed an obvious inhibition on the proliferation of activatedperipheral CD8+CLA~+T cells, which provides experimental basis for the application ofapigenin in the treatment of vitiligo.更多还原