阴道毛滴虫黏附宿主细胞关键分子及其功能研究

Trichomonas vaginalis (T. vaginalis) is a widespread and serious sexually transmitted pathogen. The adherence to the surface of the host cell is the precondition for its parasitism and pathogenicity. The adhesion protein 65 (AP65) plays a key role in the process of adhesion. The study on AP65 and its receptor on the surface of host cell contributes to reveal the molecular mechanism of T. vaginalis adhesion to the host cell. In this project, the interaction between AP65 and its receptor molecules mediating the adhesion of T. vaginalis to host cells is studied on T. vaginalis and human vaginal epithelial cells. The function of AP65 in the process of T. vaginalis adhesion to host cell is explored by T. vaginalis AP65 gene deletion strain or T. vaginalis wild strain blocked by AP65 antibody infecting human vaginal epithelial cells in vitro and mice in vivo, respectively. Then, the receptors of AP65 are screened and identified by yeast two hybrid, pull down and co-immunoprecipitation. Finally, human vaginal epithelial cells AP65 receptors deletion strain and AP65 receptors blocked wild strain human vaginal epithelial cells by the antibody of are used to analyze the role of AP65 receptors in the process of T. vaginalis adhesion to host cell. The purpose of this study is to elucidate that the interaction between AP65 and its receptor molecules mediates the adhesion of T. vaginalis to host cells, provide basis for searching the drug targets of anti-T. vaginalis, and afford new ideas for prevention and treatment of trichomoniasis.

阴道毛滴虫黏附宿主细胞是其寄生和致病的前提，在黏附过程中虫体表面蛋白AP65发挥重要作用。研究AP65蛋白及其宿主细胞上的受体分子有助于揭示阴道毛滴虫黏附宿主细胞的分子机制。本项目拟采取阴道毛滴虫和人阴道上皮细胞，①通过阴道毛滴虫AP65基因缺失株或AP65抗体封闭、阻断的野生株分别体外感染人阴道上皮细胞和体内感染小鼠，探究AP65蛋白在阴道毛滴虫黏附宿主细胞中的功能；②采用酵母双杂交、拉下实验和免疫共沉淀筛选、鉴定人阴道上皮细胞上与AP65蛋白相互作用的受体分子；③阴道毛滴虫分别感染人阴道上皮细胞AP65受体分子缺失株和抗体封闭、阻断后的野生株，分析AP65受体分子在阴道毛滴虫黏附宿主细胞中的作用。以期阐明AP65与其受体分子相互作用介导阴道毛滴虫黏附宿主细胞的分子机制，为寻找抗阴道毛滴虫的药物靶点提供依据，并为阴道毛滴虫病的防治提供新思路。

阴道毛滴虫（Trichomonas vaginalis, T. vaginalis）是一种常见的以性传播为主的寄生原虫，常寄生于女性的阴道和男性的尿道、前列腺、睾丸、附睾及包皮下组织。阴道毛滴虫作为一种非病毒性性传播病原体，主要是通过黏附于宿主细胞表面感染、寄生及致病。所以，黏附于宿主细胞表面是阴道毛滴虫寄生和致病的关键性环节，而阴道毛滴虫黏附蛋白65（TvAP65）在阴道毛滴虫黏附宿主细胞过程中发挥重要作用。因此，研究TvAP65蛋白及其宿主细胞上的受体分子有助于揭示阴道毛滴虫黏附宿主细胞的分子机制。本项目通过RNA干扰技术降低了TvAP65蛋白在阴道毛滴虫滋养体中的表达，并发现TvAP65蛋白低表达虫株黏附宿主细胞的能力降低、抑制宿主细胞增殖减弱、导致宿主细胞死亡下降；动物实验结果表明，TvAP65蛋白主动免疫、TvAP65多克隆抗体被动免疫及封闭能够显著地延长腹腔感染阴道毛滴虫滋养体的小鼠的存活时间。免疫荧光指出TvAP65蛋白分子可以结合到阴道上皮细胞（VK2/E6E7）表面；以TvAP65蛋白分子为诱饵，采用酵母双杂交筛选、鉴定到阴道上皮细胞上的13个蛋白分子与TvAP65相互作用；采用免疫共沉淀进一步验证，BNIP3作为阴道上皮细胞上的受体分子与TvAP65蛋白相互作用。借助RNA干扰技术降低BNIP3在阴道上皮细胞中的表达，分析TvAP65是通过与BNIP3相互作用在阴道毛滴虫黏附宿主细胞中发挥作用，结果表明TvAP65与阴道上皮细胞上的BNIP3以“配体-受体”的方式介导阴道毛滴虫黏附宿主细胞。本研究阐明了TvAP65蛋白及其受体分子BNIP3在阴道毛滴虫黏附宿主细胞中的作用机制，为寻找抗阴道毛滴虫的药物靶点和阴道毛滴虫病的诊断分子提供依据，为研究抗阴道毛滴虫的新型药物和阴道毛滴虫病的分子诊断奠定基础，为阴道毛滴虫病的防治及减小其危害提供思路。