口腔扁平苔鲜中树突状细胞亚群及其Toll样受体表达谱的研究

Oral lichen planus (OLP) is commonly accepted as an autoimmunal disease because of its autoimmunity T lymphocytes infiltration in lamina propia and increased apoptosis of epithelium cell as the target. But its specific autoantigen keeps unclear, and thus makes it short of specific therapy..As the most important antigen-presentation cells and their antigen-specific receptors, dendritic cells (DC) and Toll-like receptors (TLRs) were applied as the strategy for finding the autoantigen in similar autoimmunal diseases. And some impressive presedents had been acquired, while analogous researches in OLP kept no launching..In preliminary experiments, we have found that DC’s amount and percentage increased and TLR2/4/9 mRNA highly expressed in 3 cases of OLP, compared with the healthy control. As a result, a study hypothesis is made that functional DC phenotypes infiltrate in OLP lesions and their TLRs recognize OLP-autoantigens so as to induce the following immune responses..With the techiques of Flow-cytometry, immunohistology, reverse-transcription PCR, and laser scanning confocal microscope, the authors hope to (1)systematicly evaluate DCs’ phenotypes and distribution; uncover the expression profile of DCs’ chemokine receptors/ligands and cytokines so as to find DCs’ immune function in OLP; (2) find out DC's TLRs expression profile and distribution in cell and tissue. The results are expected of contribution to the future studies on OLP's autoantigen and so that some specific therapies could be established for advantaging and accelerating the final conquer on oral lichen planus.

口腔扁平苔藓（OLP）是一种T淋巴细胞介导的自身免疫病，但是其自身抗原不明，且缺乏特异性治疗方法。树突状细胞（DC）及其Toll样受体（TLRs）是最重要的抗原提呈细胞和抗原识别受体，应用其作为研究策略发现自身抗原，在相关疾病领域已有成功先例，本项目的预初实验结果发现3例斑纹型OLP中，DC的数量增加、比例上升且TLR2/4/9mRNA高表达。因此本项目提出研究假设：OLP中存在DC的功能亚群，并通过其TLRs识别结合自身抗原，介导免疫应答。通过流式细胞术、免疫组化、免疫荧光、RT-PCR等方法，本项目将：1.阐明DC在OLP中的亚群和分布，通过对其趋化因子受体/配体和细胞因子的研究，证实DC在OLP中具有免疫应答的功能；2.分析OLP组织中DC识别抗原的TLRs特征性表达谱，为分析OLP自身抗原的类型和来源，开发OLP的特异性免疫干预新疗法提供依据。

背景：口腔扁平苔藓(oral lichen planus, OLP)是最常见的与T细胞免疫相关的慢性炎症性口腔黏膜病，其致病抗原依然不明。树突状细胞(dendritic cells, DCs)是最主要的抗原提呈细胞，其Toll样受体(Toll-like receptors, TLRs)是识别抗原、诱导适应性免疫的重要分子，研究DC在OLP中的优势亚群及其相关TLRs，有助于揭示OLP致病抗原来源和类型。.目的：分析评价与正常人群相比，OLP人群损害组织和外周血中的树突状细胞亚群的数量差异；分析其相关TLRs的表达差异；分析差异表达的TLRs信号通路分子的转录和表达差异，为进一步的固有免疫和病因学研究提供基础。.方法：收集OLP病例和对照样本，并记录病例信息。.（1）流式细胞术检测两组人群外周血和组织中的CD11c+ mDC1、CD141+ mDC2、CD123+ pDC、CD207+ LC共4类DC亚群数量比，评价其差异；IHC染色观察DC亚群在OLP损害和健康对照组织中的数量和分布；.（2）检测相关趋化因子及其受体和细胞因子mRNA在组织中的转录情况；.（3）检测OLP损害组织中优势表达的TLRs，及其相关信号通路活化情况。.结果：（1）两组人群外周血中4类DC亚群数量均无统计学差异；组织中，OLP组mDC1和pDC的浸润数量明显增加（p<0.01），mDC2和LC亚群无统计学差异。组化染色证实mDC1和pDC亚群在OLP损害中数量增加，浸润部位与T细胞浸润带密切相关。LC主要浸润在上皮，其数量无明显增加；组化染色无法分析mDC2亚群。（2）DC相关趋化因子（CCL2/21、CXCL9）、趋化因子受体（CCR1/2/5/6/7、CXCR3/4）以及IFN-γ的mRNA均在OLP损害中升高（p<0.05），OLP损害中IFN-α的mRNA转录升高但无统计学差异。（3）TLR1/2/4/7/8/9/10在OLP损害组织中高表达，以TLR7/8/9为著（p<0.001），其相关的MyD88-IRF7-IFN-α信号通路在OLP损害组织中活化。.结论：mDC1和pDC亚群是OLP损害中主要的抗原提呈细胞，OLP更倾向于是一种局部的炎症；DC相关趋化因子及其受体在OLP损害中高表达；TLR7/8/9及下游IRF7-IFN-α通路活化可能是OLP固有免疫阶段的重要事件。