产黑普氏菌通过TLR4引发局部免疫失衡促进口腔扁平苔藓发病的作用和机制研究

Recent studies have shown that the oral commensal bacteria may trigger the immune imbalance during the pathogenesis of OLP. The Toll like receptors (TLRs) are important molecules that recognize the microorganism and participate in the innate immunity and inducing the adaptive immunity . Our previous research, which was funded by the Natural Science Foundation of China, revealed that the bacterial structure of OLP buccal surface was significantly different from healthy control. In addition, the TLR4 expression was increased and the bacteria content was significantly elevated in OLP lesion and positively correlated with the expression of inflammatory cytokines. Thus we hypothesized that Prevotella melaninogenica may trigger the immune imbalance of OLP through TLR4. However the specific mechanism is still unclear. Therefore, this project would further study the correlation between the bacteria and the occurrence of OLP; to study whether oral epithelial cells and dendritic cells would produce immune recognition to Prevotella melaninogenica through TLR4 and the subsequent immune response and mechanism. RNAseq combined with bioinformatics research would utilized to find the different roles of oral epithelial cells and dendritic cells during the local immune imbalance. This project explores the role and the mechanism of Prevotella melaninogenica in OLP from the molecular, cellular and animal levels and provides theoretical and experimental basis for the development of new diagnostic and treatment strategies.

最新研究表明口腔共生菌可能是促进OLP免疫失调的动因。Toll样受体（TLRs）是机体识别微生物，引发固有免疫并促进适应性免疫的关键分子。本课题组前期在国家自然科学基金资助下，通过宏基因组学研究发现产黑普氏菌在OLP病损黏膜表面微生物群落中构成比升高，且OLP病损组织中TLR4表达升高，该菌含量显著增加并与炎症因子表达水平呈正相关，推测该菌可能通过TLR4引发局部免疫失衡促进OLP发病。为验证此假说，本项目将进一步研究该菌与OLP发生的相关性，从组织学水平明确产黑普氏菌入侵OLP组织的数量、定位及其与OLP局部炎症应答的相关性；研究口腔上皮角质细胞和树突状细胞对产黑普氏菌的免疫识别和应答机制，口腔上皮角质细胞与树突状细胞是否通过TLR4对产黑普氏菌产生免疫识别，研究口腔上皮角质细胞与树突状细胞在产黑普氏菌作用下的免疫效应和信号转导机制，采用RNAseq技术结合生物信息学研究上皮角质细胞与树突状细胞在免疫失衡中的不同作用及产黑普氏菌与细胞相互作用的可能机制。本课题深入探索产黑普氏菌引发OLP的分子机制，为研发新型诊疗策略提供理论依据和实验基础。

最新研究表明口腔共生菌可能是促进OLP免疫失调的重要因素。本课题组前期通过宏基因组学研究发现产黑普氏菌在OLP病损黏膜表面微生物群落中构成比升高，而唾液链球菌的构成比降低，推测共生菌可能引发局部免疫失衡而促进OLP发病。为验证此假说，本项目进一步研究产黑普氏菌与OLP发生的相关性，采用qPCR方法检测OLP活检组织中产黑普氏菌的含量以及局部炎症细胞因子的表达情况，结果显示OLP病损组织中该菌含量显著增加且与IL-6表达水平呈正相关；通过荧光原位杂交实验发现在61.54%的OLP病损组织中检测到产黑普氏菌信号，且在上皮与固有层连接的上皮乳突处富集，而正常黏膜组织中未发现该菌；将产黑普氏菌与巨噬细胞共培养，发现该菌能黏附并侵入巨噬细胞，激活巨噬细胞NF-κB通路，以上研究提示该菌可能是促进OLP免疫失调的重要因素。进一步将产黑普氏菌与人原代口腔上皮细胞共培养，采用RNAseq技术检测该菌感染4h及24h后上皮细胞mRNA表达谱的变化，结果发现Ca2+通道相关基因表达异常；差异基因除在经典炎症通路富集外，还在血管生成、角质细胞分化与上皮形成、荷尔蒙代谢等生物过程中富集。此外，本课题还研究了唾液链球菌治疗OLP的疗效及其抑炎机制。研究结果发现，唾液链球菌K12含片治疗OLP能有效改善充血糜烂体征、缓解疼痛，治疗后病损黏膜局部的唾液链球菌定植增加。唾液链球菌可能通过增加紧密连接蛋白表达而促进上皮屏障功能以及抑制NF-κB炎症通路而发挥抗炎作用。综上所述，本课题深入探索了共生菌在OLP发生发展中的作用机制，为研发新型诊疗策略提供理论依据和实验基础，唾液链球菌K12含片作为一种新型的治疗策略，可在临床中推广应用，尤其在治疗OLP的长期用药与疗效维持方面可能具有一定的临床意义。