外泌体介导IL-17信号途径在吸烟与白念珠菌协同促进口腔白斑发生发展中的作用机制研究

Smoking and Candida albicans infection are crucial risk factors of oral leukoplakia. Smoking can impair the local immune of oral mucosa. Smoking and Candida albicans can promote occurrence and development of oral leukoplakia synergistically, while the mechanism is unclear. Recent studies have shown that the epithelial cells invaded by pathogenic microorganism can activate the receptor cell to secrete IL-17 through exosome-mediated intercellular information communication. Most recently, oral mucosal epithelial cells have been confirmed to orchestrate the defense response against Candida albicans via IL-17 signaling pathway. Our recent data indicated that cigarette smoke extract could reduce the expression of exosome marker CD63, synergistically inhibiting IL-17 level with Candida albicans. Herein, we presume that smoking and Candida albicans could negatively regulate the exosome-mediated IL-17 signal pathway synergistically, suppress the production of downstream human beta defensins and other key effectors, inhibit the defense function of oral mucosal epithelial cells, aggravate the invasion of pathogenic microorganism into oral mucosa and promote the occurrence and development of oral leukoplakia. We plan to set up the in vitro cell culture model exposed to cigarette smoke, cell-Candida albicans co-culture model, animal models of active smoking and occurrence and development of oral mucosal precancerous lesion. We also plan to conduct clinical specimens assay, isolation and purification of salivary exosomes in combination with the in vitro verification of the exosomes function. By using some experimental methods, we will confirm our hypothesis in the ways of clinical specimens, animal models, cellular and molecular mechanism. This study provides some new ideas and new ways to improve the prevention, treatment and prognosis of oral leukoplakia.

吸烟与白念珠菌感染是口腔白斑的关键危险因素。吸烟抑制口腔黏膜防御功能，与白念珠菌协同促进口腔白斑发生发展，但机制不明。新近研究表明，受微生物侵袭的上皮细胞通过外泌体介导的细胞间信息交流激活受体细胞分泌IL-17，而最近研究证实口腔黏膜上皮细胞通过IL-17信号途径主导了针对白念珠菌的防御反应。我们前期发现，香烟烟雾提取物可抑制口腔黏膜上皮细胞内外泌体标志物的表达，并与白念珠菌协同降低了IL-17的水平。因此，我们推测吸烟与白念珠菌协同作用可负向调控外泌体介导IL-17信号途径，降低其下游人β防御素等效应分子的产生，抑制口腔黏膜上皮细胞的防御功能，导致白念珠菌等微生物侵袭加剧，促进口腔白斑的发生发展。课题组拟构建体外吸烟细胞模型、细胞-白念珠菌共培养模型、主动吸烟及口腔黏膜癌前病变发生发展动物模型，通过临床样本检测、唾液外泌体体外效应验证，采用多种研究手段验证假说，为口腔白斑的防治提新思路。

吸烟和白念珠菌感染是口腔白斑的重要危险因素之一，不但增加口腔白斑的患病风险，而且还与上皮异常增生的严重程度密切相关，促进口腔白斑的发展，导致口腔鳞癌的发生。外泌体是一种具有高度特异性的纳米级囊泡，介导细胞间信息交流，调控信号通路，行使固有免疫效应，在细胞微环境中发挥重要作用。推断口腔黏膜上皮细胞分泌的外泌体也极可能发挥防御功能，抵御病原微生物的侵袭，在吸烟抑制口腔黏膜上皮细胞的防御功能及口腔黏膜癌变过程起作用。IL-17作为重要的细胞因子，在针对各种病原微生物的炎症反应和防御功能中起到不可替代的作用，以往研究证实口腔黏膜上皮细胞通过IL-17信号途径主导了针对白念珠菌的防御反应，IL-17在口腔白念珠菌感染的防御反应中发挥关键性的“指挥”角色。课题组构建体外吸烟细胞模型、细胞-白念珠菌共培养模型、主动吸烟动物模型、口腔癌前病变发生发展动物模型、口腔念珠菌病动物模型、口腔移植瘤动物模型，通过临床样本检测、血浆唾液外泌体分离纯化结合体外细胞验证，采用多种研究手段和方法，从临床样本-动物模型-细胞验证-分子机制等多层次验证假说。本研究发现外泌体在口腔白斑发生发展过程中发挥重要作用；体外实验确认吸烟和白念珠菌影响口腔黏膜上皮细胞防御分子释放水平，香烟烟雾可抑制细胞外泌体水平，而白念珠菌可协同促进该抑制作用；口腔黏膜上皮细胞来源外泌体和健康人唾液来源外泌体对白念珠菌有抑菌作用；健康大鼠血浆外源性外泌体在大鼠癌变模型中发挥抑瘤作用；主动吸烟加重小鼠口腔白斑病的异常增生程度，吸烟可进一步促进口腔黏膜的癌变进程；通过体内外实验，成功验证外泌体介导IL-17 信号途径协同促进口腔白斑的发生发展。本课题从外泌体这一全新角度对口腔黏膜上皮细胞与白念珠菌之间的相互作用进行深入的研究，探究外泌体介导的IL-17信号途径在口腔白斑发生发展中的潜在作用及其机制，探寻口腔白斑病、口腔念珠菌病等黏膜病防治的新靶点。