细粒棘球蚴GLUT摄取宿主葡萄糖的调控机制及其抗包虫药物靶点研究

Cystic echinococcosis (CE) is one of the neglected and life-threatening zoonoses, which is caused by the larval stage of Echinococcus granulosus (Eg). It’s urgent to develop novel drug targets for the helminth. Block of glucose uptake from the intermediate host is a way to develop the anti-echinococcosis drug target. However, its mechanism is still unknown. We found that E. granulosus insulin receptor signaling pathway could influence the quantity of glucose in the larvae of E. granulosus and is an important anti-echinococcosis drug target. And glucose transport protein (GLUT) inhibitor could reduce the viability of larvae of E. granulosus. As glucose in the E. granulosus was transported from the environment of intermediate host. It’s suggested that EgGLUT might control the glucose uptake from the intermediate host and is an important anti-echinococcosis drug target. In this work, we plan to clone the gene sequence of EgGLUT, prepare the antibody of EgGLUT, and detect the expression, location, glucose uptake function and translocation of EgGLUT by the confocal laser scanning microscope and other molecular biology technology. Then we would identify the effects of Eg insulin receptor signaling pathway and EgAMPK signaling pathway on the glucose uptake of EgGLUT, viability of larvae and pathogenicity, by treated with insulin or AICAR(AMPK agonist) and inhibitors (EgIR signaling pathway, EgAMPK signaling pathway and EgGLUT), in vitro and in vivo. The curative effect and safety of EgGLUT inhibitor would be evaluated, in vitro and in vivo. Furthermore, the relationship between the EgGLUT function and different type of CE (active and inactive) was analyzed, in the specimen of CE patient. This study would lay a basis for further research on the mechanism of glucose uptake in E. granulosus, and provides a theoretical proof for clinical treatment of hydatid disease.

细粒棘球蚴（Eg）感染所致囊型包虫病（CE），危害严重，亟需新药物。阻抑Eg摄取中间宿主体内葡萄糖，是研发抗包虫药物的思路之一，然而Eg摄取宿主葡萄糖的机制未知。我们前期在Eg中发现了葡萄糖转运蛋白（GLUT）和胰岛素受体（IR）信号通路，及其抑制剂能高效抑制Eg幼虫的活性。为阐明EgGLUT摄取宿主葡萄糖的作用及其调控机制，对致病性的影响和鉴定药物靶点。本课题拟通过激光共聚焦等实验，鉴定EgGLUT的表达、定位、葡萄糖摄取和转位等；体内外实验，分别通过正反向干预EgIR、EgAMPK和EgGLUT等，确定EgGLUT的调控机制及其对葡萄糖摄取、Eg幼虫活性和致病性的影响；GLUT抑制剂的体内外抗包虫药效实验，鉴定药靶；CE患者标本中分析EgGLUT功能与CE（活跃、非活跃）的相关性。本课题是阐明Eg幼虫摄取葡萄糖的调控机制，鉴定抗包虫药物靶点的重要组成部分，为研发新型抗包虫药物奠定基础。

囊型包虫病（Cystic echinococcosis，CE），危害严重，亟需研发新型药物。Echinococcus granulosus糖代谢通路是研发治疗CE药物的重要靶点。为了阐明E. granulosus幼虫从中间宿主体内摄取葡萄糖的分子机制，本项目通过基因克隆、抗体制备、siRNA干预、葡萄糖摄取检测和体内外药效评估等实验，研究了E. granulosus的葡萄糖转运蛋白（EgGLUT），及其上游信号通路调控E. granulosus葡萄糖摄取的机制，初步评估了其抑制剂治疗CE的药效。本项目发现：①E. granulosus幼虫中表达具有葡萄糖摄取功能的EgGLUT1和EgGLUT3等蛋白；②EgGLUT1和EgGLUT3等蛋白与人的同源基因序列差异较大，可作为药物靶点用于开发特异性针对E. granulosus的药物；③EgIGF-1R和EgAMPK信号通路分别通过调控EgGLUT的蛋白表达，调控E. granulosus幼虫的葡萄糖摄取功能；④干预EgGLUT1、EgIGF-1R和EgAMPK的表达或功能，能高效降低E. granulosus幼虫的活性，其抑制剂（WZB117、BMS-536924和Compound C等）是治疗CE的重要候选化合物。因此，本项目证明，EgIGF-1R和EgAMPK信号通路分别通过调控EgGLUT的表达，干预E. granulosus幼虫的葡萄糖摄取功能，从而影响E. granulosus幼虫的糖代谢通路。本研究初步阐明了E. granulosus幼虫葡萄糖摄取的分子机制，为临床CE的治疗提供新的药物靶点。