Integrin介导的宿主细胞F-actin聚集在鸡球虫入侵过程中的作用研究

Filamentous actin (F-actin) aggregation cause cytoskeletal rearrangement and play an important role in the course of pathogens invasion the host cell. While some singaling pathways regulate F-actin polymerization . Integrin was considered an important signal transduction recertor and can active F-actin polymerization,Which played a leading role in foreign microbial invasion of host cell. Chicken coccidiosis currently popular in the world, the damage is very serious, resistance problem is outstanding, and the invasion of mechanism is still not clear, so no effective measures to control it in clinic now. Therefore we carry out the research On the basis of that Eimeria tenella infected HCT - 8 cell and established cell culture model. Regard as integrin mediateing the host cell F-actin polymerization that impact on sporozoites invasion process as the main line. Analysis the relationship among sporozoites invasion and F-actin aggregation using subcellular localization. Study the effect of sporozoites invasion, through antibody blocking, gene knock out and overexpression of ITGA2 change the activity of the integrin, furter analyze the effect by integrin mediates F-actin aggregation cause cytoskeleton rearrangement in the invasion of sporozoites, Our study is very great significance in the aspects of replenish or announce Eimeria tenella invasion mechanism and control chicken coccidiosis.

病原入侵宿主细胞过程中，由F-actin聚集引起的宿主细胞骨架重排起着至关重要的作用。而宿主细胞F-actin聚集受众多信号通路的调节，位于信号通路的众多受体中，integrin被认为是激活宿主细胞F-actin聚集重排信号转导的重要受体，在外源微生物入侵宿主细胞过程中起主导作用。而在鸡球虫研究方面尚未有相关工作的报道，原因是在鸡球虫入侵过程中对宿主细胞本身的变化研究较少，导致对鸡球虫的入侵机制不清楚。因此，本项目组在柔嫩艾美耳球虫子孢子感染HCT-8细胞培养模型的基础上，利用亚细胞定位的方法和抑制试验分析子孢子入侵与F-actin聚集的关系，通过分子生物学技术改变integrin的活性，分析子孢子入侵率的变化，阐明由integrin介导的宿主细胞F-actin聚集引起的细胞骨架重排对鸡球虫入侵的作用，为补充或者解析鸡球虫的入侵机制提供理论基础。

E. tenella子孢子入侵 HCT-8 细胞并发育至第二代裂殖体，以此建立并优化鸡球虫体外细胞培养模型，当子孢子接入 HCT-8 细胞6h，入侵率最高。亚细胞定位显示，当子孢子入侵HCT-8 细胞时，宿主细胞F-actin在入侵与粘附部位聚集。将抑制宿主细胞F-actin聚集的细胞松弛素D与HCT-8细胞孵育后感染子孢子，结果子孢子的入侵率明显降低，同样亚细胞定位显示，抑制剂作用后，宿主细胞F-actin聚集明显减少。将ITGA2单克隆抗体与HCT-8细胞孵育阻断ITGA2的功能，感染子孢子，结果子孢子的入侵率明显降低。亚细胞定位显示，抗体阻断后宿主细胞F-actin聚集明显减少；将子孢子感染ITGA2基因敲除系细胞，结果子孢子的入侵率明显降低。亚细胞定位显示，基因敲除细胞中F-actin聚集明显减少；将子孢子感染ITGA2基因过表达系细胞，结果子孢子的入侵率明显增加。亚细胞定位显示，基因过表达细胞中F-actin聚集明显增加。综上，宿主细胞F-actin聚集与鸡球虫入侵密切相关；当integrin活性发生变化时，直接导致宿主细胞F-actin聚集及子孢子的入侵率发生相应的变化；因此， integrin介导的宿主细胞F-actin聚集引起的细胞骨架重排对鸡球虫入侵起重要的调控作用。