隐孢子虫入侵介导的宿主细胞自噬相关分子鉴定及功能研究

隐孢子虫病是由隐孢子虫引起的一种重要人畜共患寄生虫病。研究认为自噬是细胞维持稳态的一种机制，在细胞与病原体相互作用的过程中发挥着重要作用。但目前尚无宿主细胞自噬与隐孢子虫感染之间的相互作用，以及哪些自噬分子与虫体入侵介导相关的研究报道。本申请项目拟围绕隐孢子虫入侵宿主细胞这一环节，应用透射电镜、亚细胞共定位技术研究宿主细胞自噬小体的形成及分布，建立子孢子入侵率计算方法（qRT-PCR⊿⊿CT），综合分析自噬水平上调、下调对隐孢子虫感染的影响。进而通过解析隐孢子虫入侵后Atg5、Atg7、Atg8、Atg12等自噬相关分子在转录和翻译水平上的表达差异来鉴定虫体入侵介导的自噬关键作用分子，并利用细胞转染、RNAi技术构建靶Atg分子过表达和缺失突变株研究其在入侵过程中的生物学功能。预期成果将阐明与隐孢子虫入侵介导相关的自噬关键作用分子，为下一步寻找与之互作的隐孢子虫受体蛋白奠定基础。

Cryptosporidiosis caused by Cryptosporidium spp. has been considered as a zoonotic disease with public health significance in the world. Accumulating evidence supports the pivotal role of autophagy in host defense against intracellular pathogens based on its homeostatic mechanism. However, it is still unclear about the effect of autophagy in C. parvum infection and which kind of autophagy related genes (Atg) main take part in such invasion process. This project propose to focus on Cryptosporidium invasion to host cell. Together with qRT-PCR ⊿⊿CT calculate method of sporozoite invasion rate, transmission electron microscope and subcellular localization technology will be used to observe the autophagosome formation and location, in order to comprehensively analyze the effect of cell autophagy in C. parvum infection. Then we will identify the major autophagy related molecule according to analyze the expression of Atg5, Atg7, Atg8 and Atg12 in transcription and translation level, and functionally verify such target Atg proteins through RNA-mediated gene silencing, over expression. Therefore, target Atg proteins which play a critical role in autophagy could be identified and its interplay between host cell autophagy and C. parvum infection will be clarified. These findings may provide strong data support for seeking possible regulatory pathways with its interacting receptor protein of C. parvum in future.