EgIR通过PI3K/AKT调控细粒棘球蚴存活的机制研究

Echinococcosis is one of the high infected and life-threatening zoonoses in the 7 provinces of western China including Xinjiang, of which 95% is caused by the metacestode larval stage of Echinococcus granulosus (Eg). The EgIR/PI3K/AKT signaling pathway relating Eg growth, survival and pathogenicity is rarely reported. In our previous study, we present evidence for the presence of EgIR，which could interact with human insulin and infuluence the survival of Eg metacestode.However, the interference of EgIR/ERK signaling pathway has little impact on the survival of metacestode of Eg.Therefore, we propose a scientific hypothesis that EgIR control the survival of Eg metacestode through PI3K/AKT signaling pathway. In this work, we focus on EgPI3K/AKT/mTOR expression, location and biological function by immunohistochemistry and yeast two-hybrid, then we identify the effects of insulin and EgIR/PI3K/AKT inhibitors on the in-vitro cultivated protoscolex and pathogenicity in Eg infected mice model in vivo. This study laid a basis for further research on drug and vaccine against cystic echinococcosis, which choose EgIR/PI3K/AKT signaling pathway and its members as promising targets, and provide a theoretical proof for clinical treatment of hydatid disease.

95%以上包虫病由细粒棘球蚴（Eg）感染所致囊型包虫病（CE），新疆是高发区，Eg中EgIR/PI3K/AKT信号通路研究尚属空白。前期研究发现EgIR能够与人胰岛素发生交互作用，其功能与Eg蚴虫的活性相关，而EgIR/ERK信号通路的干预对Eg蚴虫活性的影响较弱，提示EgIR通过PI3K/AKT分子调控Eg蚴虫的活性。本课题拟克隆EgIR信号通路关键成员（EgPI3K/AKT/mTOR），检测表达并定位；酵母双杂交等实验鉴定EgIR/EgPI3K/AKT/mTOR等基因的生物学功能及交互作用；Eg体外培养和体内接种感染动物方式，通过正向激活和反向干预实验，确定EgIR/PI3K/AKT信号通路对细粒棘球蚴存活和致病性的影响；探索其用于治疗CE的可行性和有效性。本课题第一次将EgIR/PI3K/AKT信号通路引入CE研究领域，将极大丰富信号网络通路在包虫病致病机制中所起的作用。

本项目通过体内外实验，克隆获得细粒棘球绦虫EgPI3K P110、EgPI3K P85和EgAKT基因序列，成功制备获得EgPI3K P110抗体，完成性质鉴定等实验。应用AZD8055（mTOR）、BEZ235（mTOR/PI3K）、GSK690693（AKT）、everolimus（mTOR）、tacrolimus（mTOR）等PI3K/AKT通路抑制剂体外分别干预原头蚴和囊泡，并探索干预对细粒棘球绦虫蚴虫活性的影响。利用体内动物模型检测了tacrolimus治疗包虫病的药效和安全性及其机制。在此基础上，我们探索了PI3K/AKT通路上游EgIR的抑制剂PQ401和阿苯达唑联用治疗包虫病的药效，及从EgIR抑制剂PQ401、BMS536924和OSI-906 中筛选出具有较好药效的BMS536924，并检测了BMS536924对细粒棘球绦虫原头蚴的基因表达谱的影响。另外，我们针对PI3K/AKT通路下游糖代谢通路进行探索，克隆获得葡萄糖转运蛋白GLUT，并发现其抑制剂WZB117在体外能高效的抑制Eg原头蚴活性，为进一步深入探索细粒棘球绦虫EgIR信号通路作为抗包虫药物靶点和作用机制，奠定了重要的工作基础。