轮状病毒感染对细胞内Ca2+介导的脂筏依赖性NHE3转运的调控作用

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.

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。

背景：轮状病毒（rotavirus，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+升高可调控钠-氢交换蛋白3（Na+/H+ exchanger 3，NHE3）的转运。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，据此本研究提出假说，RV感染可调控细胞内Ca2+水平，通过脂筏依赖性NHE3转运机制降低细胞表面NHE3蛋白的量及其生物活性，减少钠水吸收，引起腹泻症状。.内容：构建RV感染表达3HA-NHE3单层极化Caco-2/BBe细胞模型，新生鼠离体回肠和在体模型，分别利用利用胆固醇破坏剂MβCD、细胞内钙离子络合剂BAPTA-AM、Cdc42阻断剂Pril-1和Clathrin阻断剂CPZ等不同阻断剂进行干预实验，通过BCECF-AM法测定Na+-H+交换活性，细胞表面NHE3生物素化法，免疫荧光法等技术研究细胞内Ca2+介导的脂筏依赖性转运机制在RV感染对NHE3蛋白调控机制中发挥的作用。.结果：RV感染能够明显抑制细胞表面NHE3蛋白的表达水平和生物活性，此抑制作用可被MβCD、BAPTA-AM和Pril-1所完全拮抗，而不能被 CPZ完全拮抗。在体模型研究结果发现，RV感染可升高新生鼠回肠含水量，这一作用可被MβCD、BAPTA-AM和Pril-1明显拮抗，而CPZ的拮抗作用不明显。.结论：RV感染抑制NHE3的作用可能是经由细胞内Ca2+介导的脂筏依赖性转运途径发挥的，例如Cdc42转运途径。而RV感染抑制NHE3的作用与Clathrin转运途径有一定的关系，但不是Clathrin依赖性途径的，还有其他的转运途径的参与。脂筏、细胞内钙离子水平和Cdc42可能是RV感染性腹泻的潜在治疗靶点。.意义：本课题深入探索RV感染性腹泻的发生机制，为RV感染性腹泻的预防和治疗寻找新的治疗靶点提供线索，为开发新的RV感染性腹泻治疗药物提供研究基础。