【作者】 杨连娟；

【导师】 温海；

【作者基本信息】 第二军医大学 ， 皮肤病与性病学（专业学位）， 2013， 博士

【摘要】 口腔念珠菌病（Oral candidiasis）是由念珠菌属真菌感染所引起的急性、亚急性或慢性口腔黏膜疾病。近年来，由于抗生素和免疫抑制剂在临床上的广泛应用，导致菌群失调或免疫力降低，从而使内脏、皮肤及黏膜真菌感染者日益增多，口腔黏膜念珠菌病的发生率也相应增高。口腔念珠菌病可按病损特征及病变部位等分为假膜型、萎缩型、增殖型等口腔念珠菌病，常见的口腔念珠菌病有念珠菌性唇炎及念珠菌性口角炎。长期慢性口腔念珠菌病还有恶变的可能，口腔念珠菌病中最主要的病原菌是白念珠菌，此菌为卵圆形芽生酵母样菌，革兰阳性，有芽孢，延长的芽生细胞极似菌丝，故名假丝白念珠菌。念珠菌对热的抵抗力不强，但对干燥、日光、紫外线及化学制剂等抵抗力较强。白念珠菌是典型的条件致病菌，平时寄生在体内的酵母型念珠菌并无致病性，而当它发展为菌丝型时，才有致病性。白念珠菌的感染及致病机理主要包括四个步骤：附着、芽管和菌丝的形成、分泌蛋白酶、菌落相转换。目前口腔念珠菌病以局部治疗为主，但严重病例及慢性念珠菌感染常需辅以全身抗真菌药物治疗才能奏效。常用的抗真菌药物主要有制霉菌素、咪康唑和氟康唑等。T细胞通过其组合分泌的细胞因子来行使其对靶细胞的所有作用。新近被阐述的辅助T细胞Th22亚群以不能产生IFN-γ，IL-4和IL-17，但可以分泌TNF-α和IL-22为特征。Th22细胞属于一类新型白细胞，与其它免疫细胞很少或没有直接作用，其选择性地作用于上皮细胞。最新研究结果显示，IL-22和TNF-α在某些皮肤疾病（如银屑病、特应性皮炎等）中，可以协同激活角质形成细胞的固有免疫反应。本文主要致力于研究IL-22和TNF-α是否在白念珠菌感染的口腔黏膜上皮细胞中也存在类似的固有免疫反应激活机制，从而产生抗真菌作用。为此，第一部分实验中我们首先建立了小鼠白念珠菌口腔粘膜感染模型，用ELISA方法检测了模型组体内TNF-α、IL-6、IL-22、IFN-γ、IL-17和TGF-β的水平和局部粘膜组织内抗菌趋化因子（CXCL9、CXCL10）的水平，用免疫沉淀法检测局部粘膜组织中IL-22BP和TNF-αBP的表达水平，在此部分实验基础上我们证明了IL-22和TNF-α是与小鼠口腔黏膜上皮细胞固有免疫反应相关的细胞因子。随后我们在第二部分实验中用IL-22和TNF-α体外刺激人原代口腔黏膜上皮细胞，用实时荧光定量PCR检测固有免疫相关因子CXCL‐9、CXCL‐10、CXCL‐11、C1s、C1r、S100A7、HBD2的mRNA表达水平，我们观察到IL-22和TNF-α对其固有免疫反应的激活具有协同作用，IL-22可以增加数种依赖TNF-α的免疫调节因子，诸如初始补体因子C1r及C1s，抗菌肽S100A7及HBD-2和抗菌趋化因子CXCL-9/-10/-11等的诱导和分泌。在此基础上我们进一步研究其胞内信号通路，结果显示这种IL-22和TNF-α的协同机制是经由胞内的MAP激酶及其下游的转录因子AP-1家族介导的。在第三部分实验中我们通过给口腔感染了白念珠菌的小鼠腹腔注射IL-22和TNF-α后，可以观察到黏膜损伤症状明显减轻，存活率明显增加。另外，在人原代口腔黏膜上皮细胞的念珠菌感染体外模型中同样也观察到了IL-22和TNF-α对黏膜细胞有显著的协同保护作用，显著抑制了念珠菌的生长，降低了白念珠菌对上皮细胞的毒性作用。综上所述，我们通过体内外模型实验证实了IL-22和TNF-α在口腔黏膜免疫中是一个强有力的细胞因子组合。这为开发用于治疗口腔黏膜念珠菌感染性疾病的抗真菌药物提供了一个潜在的靶点。更多还原

【Abstract】 Oral candidiasis is an acute, subacute or chronic oral mucosal diseases caused bycandida species. In recent years, due to the extensive application of antibiotics andimmunosuppressive agents in clinical which causes dysbacteriosis or Decreasedimmunity, the number of internal organs, skin and mucosal fungal infections is increasing,so as the incidence of oral mucosal candidiasis. Oral candidiasis can be divided intopseudomembranous type, atrophic type and proliferative type according to lesioncharacteristics and location. Common oral candidiasis includes Candida albicans cheilitisand angular cheilitis. There is the possibility of malignant transformation of chronic oralcandidiasis. The most important pathogens of oral candidiasis is candida albicans. Thisbacterium is oval budding yeast like fungi, gram-positive, rod-shaped, extending buddingcells resembling pseudohyphal Candida albicans hyphal, hence the name. Candida’sresistance is not strong to heat, but strong to dry, sunlight, ultraviolet radiation andchemical agents. Candida albicans is a typical opportunistic pathogen, usually notpathogenic when parasitic in vivo, and when it develops into the mycelia type, have thepathogenicity. The infection and pathogenic mechanism of Candida albicans mainlyincludes four steps: adhesion, germ tube and hyphal formation, secretion of protease,colony conversion. At present, the treatment of Candida albicans is mainly by localtherapy, but severe cases of chronic Candida infections often need to be supplementedwith systemic antifungal therapy to be effective. The common antifungal agents includenystatin, miconazole and fluconazole.T cell exercises its full impact on target cell through the combinatorial secretion ofcytokines. The newly elaborated Th22subsets of helper t cells cannot produce IFN-γ，IL-4and IL-17，but is characterized by the secretion of TNF-α and IL-22. Th22cellsbelong to a new class of leukocytes, with little or no direct effect on other immune cells,but selective effect on epithelial cells. The latest research results show that IL-22andTNF-α can synergisticly activate the innate immune response of keratinocytes in certain skin disorders (such as psoriasis, allergic eczema, etc.). This article mainly focuses onstudying whether TNF-α and IL-22have the similar activation mechanism in the Candidaalbicans infected oral mucosal epithelial cells, resulting in anti-fungal activity. To thisend, we first established a mouse Candida albicans oral infection model and on this basisproved that IL-22and TNF-α are the cytokines associated with innate immune responsein mice oral mucosal epithelial cells. Subsequently, in the primary human oral epithelialcells, we observed that IL-22and TNF-α have a synergistic effect on activation of theinnate immune response. IL-22could increase the number of several TNF-α dependentimmune regulatory factors, such as initial complement factor C1r and C1s, theantibacterial peptide S100A7and HBD-2(human βdefensin2) and induction andsecretion of chemotactic factor CXCL-9/-10/-11in human primary keratinocytes. Furtherstudy of intracellular signaling pathways reveal that such a coordination mechanism ofIL-22and TNF-α is mediated via the MAP kinase and its downstream intracellulartranscription factor AP-1family. By intraperitoneal injection of IL-22and TNF-α inmouse with oral infection of Candida albicans, mucosal injury can be significantlyreduced. In addition, the protective effect of IL-22and TNF-α on mucosal cells was alsoobserved in primary human oral mucosal epithelial cells infected with Candida albicans.In conclusion, we demonstrated that IL-22and TNF-α is a powerful combination ofcytokines in the mucosal immune. This provides a potential target for the development oforal mucosa antifungal drugs: IL-22.更多还原