沙眼衣原体毒力基因的鉴定及其对输卵管病变致病机制的研究

Genital chlamydia trachomatis infection is the leading cause of sexually transmitted disease in the world, which often causes consistent infection and female tubal infertility. The pathogenic mechanism remains unclear due to the lack of genetic manipulation tools. Our preliminary study has shown that the virulence genes for tubal pathology were located in chlamydia plasmid, however, the exact virulence genes have not been identified yet. This project plans to use chlamydia muridarum-mouse as an animal model, which is often used for studying chlamydia trachomatis pathogenic mechanism, knocking out the plasmid encoded 8 open reading frames separately, constructing recombinant plasmids with a certain open reading frame knock out and transforming the plasmid to a plasmid cured chlamydia strain. The virulence genes will be identified according to the different pathogenicity of the transformed strains. The project will try to explore the pathogenic mechanism of the virulence genes by comparing glycogen synthase gene transcription and expression , Toll receptor 2 signaling pathways stimulation between virulence strains and virulence gene knock out strains, as well as the neutralization ability of virulence gene encoded protein against the anti-chlamydia activity of human anti-microbial peptides. Completion of the project can not only lay a foundation for illustrate the pathogenic mechanism of chlamydia trachomatis but also provide a new way for clinical treatment of chlamydia trachomatis, eventually reducing the tubal infertility rate in female patients.

沙眼衣原体（C.t）泌尿生殖道感染是目前国内外最常见的性传播疾病，临床迁延难愈，是女性患者发生输卵管性不孕的重要原因之一。由于缺乏基因操纵工具，C.t对输卵管病变的致病机制尚不明确。前期研究发现决定输卵管病变的毒力基因位于衣原体质粒上，但迄今尚未鉴定出确切的毒力基因。本课题计划以常用的研究C.t致病机制的鼠肺炎沙眼衣原体-小鼠为动物模型，通过基因重组技术，将鼠肺炎沙眼衣原体质粒所编码的8个开放阅读框架分别进行敲除，构建基因敲除的重组质粒并将其转化到缺失质粒的衣原体中，根据转化菌株对小鼠输卵管致病力的不同鉴定出毒力基因；通过比较毒力基因携带株和缺失株糖原合成酶基因的转录及表达、对TLR2信号通路的激活以及毒力基因编码蛋白抵抗人类抗菌多肽杀伤衣原体的能力来探索毒力基因的致病机制。本项目的完成可为阐明C.t的致病机制奠定基础，为临床治疗C.t感染、降低C.t所致输卵管性不孕的发生率提供新思路

课题研究结果显示衣原体质粒编码蛋白pgp3在体外对衣原体在细胞间的播散以及糖原合成能力均无影响，在体内pgp3则是促进CM繁殖、上行感染及对输卵管致病的重要因子。因此pgp3被鉴定为衣原体质粒编码的重要毒力因子之一。研究发现抗菌多肽LL-37、CRAMP、HNP2、HBD2、HBD3可以杀伤多种血清型衣原体 ，其中以LL-37和CRAMP作用最强，pgp3可以有效抑制抗菌多肽对衣原体的杀伤作用。进一步研究发现pgp3可以通过其中间段M（67-132）结合LL-37并抑制其杀伤功能；另外，pgp3还可以结合其他的抗菌多肽如CRAMP、HNP2、HBD2、HBD3并抑制这些抗菌多肽对衣原体的杀伤作用。LL-37是中性粒细胞的趋化因子，pgp3可以有效阻断LL-37对中性粒细胞的趋化作用，这有助于衣原体在下生殖道的繁殖及播散。pgp3还可以直接刺激小鼠巨噬细胞及树突状细胞产生促炎症因子IL-6及MIP-2，这可能有助于输卵管部位炎症的形成。. Pgp3与LL-37结合后形成稳定的复合体并且可以阻断LL-37的部分功能，考虑到LL-37在银屑病的发病机制中发挥了重要的作用，本课题运用咪喹莫特诱导小鼠银屑病样皮炎的模型，探索了pgp3对银屑病皮损形成的影响，结果发现pgp3外用或局部皮下注射均可有效抑制小鼠银屑病样皮炎的发展，该发现在国际上属首次发现，目前正在进行详细机制研究并计划申报专利。