轮状病毒（RV）感染性腹泻的发生与其NSP4蛋白引起细胞内Ca2+升高有关。前期工作中我们发现细胞内Ca2+升高可调控NHE3转运。预实验中发现RV感染表达3HA-NHE3单层极化Caco-2/BBe细胞1h后，NHE3活性抑制，而NHE3蛋白总量未发生明显变化。细胞表面NHE3约有50%位于脂筏，据此提出假说，RV感染可调控细胞内Ca2+水平，影响脂筏依赖性NHE3转运，降低细胞表面NHE3蛋白的量及其生物活性，减少钠水吸收，引起腹泻症状。为验证假说，本课题构建RV感染表达3HA-NHE3单层极化Caco-2/BBe细胞模型，乳鼠离体和在体回肠模型，利用MβCD和BAPTA-AM进行干预实验，通过BCECF-AM法测定Na+-H+交换活性，细胞表面NHE3生物素化法，激光共聚焦等技术研究RV感染对Ca2+介导的脂筏依赖性NHE3转运的调控，探讨RV感染性腹泻的发生机制，寻找新的治疗靶点。

Rotavirus (RV) infectious diarrhea is associated with elevation of intracellular Ca2+ induced by non structural protein 4 (NSP4) of RV. Our previous studies showed that NHE3 trafficking could be regulated by elevation of intracellular Ca2+. In preliminary experiments, we found that activity of NHE3 was significantly inhibited by infection with RV for 1 hour in monolayer polarized Caco-2/BBe cells expressing 3HA-NHE3, but total amount of NHE3 protein had no obvious change. It is well known that 50% of surface NHE3 was located in lipid raft. We proposed the hypothesis that RV infection could decrease surface express and biological activity of NHE3 by intracellular Ca2+ mediated NHE3 trafficking through a lipid raft-dependent mechanism and diarrhea was induced by decreasing sodium and water absorption. In this study, we will construct cell model of RV infectious monolayer polarized Caco-2/BBe cells expressing 3HA-NHE3, in vivo and in vitro ileum models of neonatal mice to verify the hypothesis. The regulation of RV on NHE3 trafficking through lipid raft-dependent mechanism mediated by intracellular Ca2+ was determined by using technologies such as measurement of Na+-H+ exchange activity using BCECF-AM, NHE3 surface biotinylation and confocal microscope in the presence or absence of MβCD or BAPTA-AM. In this study, we will investigate the mechanism of RV infectious diarrhea and provide clues of new targets for RV therapy.