CD44介导ROS/Akt信号通路调控H.pylori诱导胃上皮间质转化的机制研究

Our previous project funded by the National Natural Science Fund showed that Hp infection could induce the generation of ROS, causing DNA injury, promoting the apotisis of gastric epithelial cells. At the same time, Hp infection could promote cell proliferation via activating Akt pathway. ROS and Akt pathway interacted, playing a key role in the modulation of H.pylori( Hp) induced apotisis and proliferation of gastric epithelial cells. Meanwhile, different from traditional opinions that ROS/Akt pathway has the cancer-promoting activity, recently researchers found that the activation of ROS/Akt pathway could inhibit tumor growth, indicating that ROS/Akt pathway is a double-edged sword for tumor development. Therefore, during the process of Hp inducing epithelial-mesenchymal transition of gastric epithelial cells, what affects the bidirectional regulationof ROS/Akt pathway? With a character as tumor stem cell marker, CD44(CD44V9) could regulate the redox state of gastric epithelial cells and overcome the apotisis caused by ROS. Thereby, we speculate that it could be the key point for promoting ROS/Akt pathway, inducing epithelial-mesenchymal transition of gastric epithelial cells. Combined with the influence of macrophage microenvironment,our project will study CD44(CD44V9) mediating ROS/Akt pathway on regulating epithelial-mesenchymal transition of gastric epithelial cells induced by Hp, for a better understanding of the mechanisms of Hp related diseases and providing a new thought for blocking epithelial-mesenchymal transition of gastric epithelial cells induced by Hp infection.

我们前期研究表明，Hp感染可诱导ROS生成，促进胃上皮细胞凋亡，同时也可激活Akt信号通路促细胞增殖，ROS和Akt信号通路相互影响，在Hp诱导胃上皮细胞凋亡增殖的调控中起关键作用。新近研究提示激活的ROS/Akt信号可抑制肿瘤生长，表明ROS/Akt信号通路是肿瘤进展的双刃剑。那么在Hp诱导胃上皮间质转化过程中，ROS/Akt信号通路是否有双向调控作用？CD44（CD44V9）分子可以调节胃上皮细胞氧化还原状态，可克服ROS积聚导致的细胞凋亡，因此我们推测其可能是促进ROS/Akt信号通路调控Hp诱导胃胃上皮间质转化的关键因素。本课题结合巨噬细胞微环境的影响，从分子、细胞、动物和临床不同层面研究CD44（CD44V9）分子介导ROS/Akt信号通路调控Hp诱导胃上皮细胞间质转化的作用，以期更好理解Hp致病机制，为阻断Hp感染诱导胃上皮细胞恶性转化提供新思路。

背景和目的：胃癌是全球第五大常见癌症，有研究表明幽门螺杆菌（Helicobacter pylori, HP）感染可影响胃上皮间充质转化（epithelial-mesenchymal transition，EMT）。CD44v9与肿瘤的预后和侵袭等有关，且胃黏膜中CD44v9阳性细胞的出现与HP感染密切相关。HP感染可使活性氧（reactive oxygen species，ROS）水平升高和缺氧诱导因子1α（hypoxia inducible factor-1α，HIF-1α）表达上调，ROS和HIF-1α在胃癌的侵袭转移过程也发挥重要作用。因此，本研究拟从人体组织标本、细胞及动物水平，阐述ROS/CD44v9/HIF-1α轴在HP感染致胃癌发生发展中的作用及其机制。方法：将HP菌株与胃癌细胞株共培养，检测HP感染对CD44v9、HIF-1α和ROS表达以及对胃EMT过程的影响；分别过表达和敲减CD44v9检测其对HIF-1α表达和对HP诱导的胃EMT的影响；分别抑制HIF-1α和ROS水平，探索其对胃EMT的影响；通过免疫共沉淀技术检测CD44v9蛋白与HIF-1α蛋白的相互作用；利用人胃癌组织芯片和人体胃黏膜内镜标本，分析CD44v9和HIF-1α的表达及其与胃粘膜病变和胃癌临床病理特征的相关性；构建AGS胃癌细胞裸鼠移植瘤模型，检测CD44v9对移植瘤生长和侵袭的作用；结果：HP感染可上调胃癌细胞CD44v9和HIF-1α的表达，使ROS水平增高，促进胃EMT过程；抑制CD44v9表达则显著抑制HP诱导的胃EMT，同时HIF-1α的表达也被显著抑制；抑制HIF-1α的表达可显著抑制HP诱导的胃癌细胞的侵袭迁移；CD44v9通过去泛素化稳定HIF-1α的表达；CD44v9的高表达与胃癌的临床病理特征显著相关；随着胃黏膜病变进展，CD44v9的表达逐渐增加，且与HP感染相关；沉默CD44v9可显著抑制裸鼠移植瘤的生长和侵袭，抑制HIF-1α的表达；ROS可上调CD44v9和HIF-1α表达，NAC可抑制HP诱导的胃EMT过程。结论：HP感染可诱导胃EMT过程，上调胃癌细胞ROS、CD44v9和HIF-1α的表达；HP感染可能通过ROS/CD44v9/HIF-1α轴调控胃EMT过程，从而促进胃癌的进展。