幽门螺杆菌东亚株引起胃癌高发的分子机制研究

The epidemiological investigation showed that the incidence of gastric cancer in countries with prevalence of East-Asia Helicobacter pylori (H .pylori) strains was even higher than that in countries with prevalence of Western H.pylori strains, and East-Asia H. pylori had stronger carcinogenicity than Western type. But the pathogenic mechanism was unclear. We hypothesized that the structure of the virulence factor CagA was changed after transmission of Western H. pylori to East-Aisa region, which endowed East-Asia H. pylori with stronger carcinogenicity, resulting in high incidence of gastric carcinoma in East Asian countries as the different mechanism from Western type. In the study, East-Asia H. pylori strains are isolated from gastric biopsy specimens from the patients with chronic atrophic gastritis, peptic ulcer and gastric cancer. The animal models are established by H. pylori isolates and their oncogenic potential is observed. Then human gastric cancer cell line AGS cells are infected with the isolates and control Western strains, and the differently expressed proteins are obtained by two-dimensional electrophoresis (2-DE) and identified by LC-MS/MS to explore the cause of high incidence of gastric carcinoma. Further, the eukaryotic expression vector, carrying the full-length East-Asia cagA or Western cagA gene, are constructed and used to transfect AGS cells to study the effector proteins binding to East-Asia CagA and their phosphorylation modification by the co-immunoprecipitation and proteomics technology. Finally, the differential proteins and effector proteins are verified in gastric cancer tissues. Our results shall reveal the molecular mechanism of East-Asia H. pylori leading to high incidence of gastric carcinoma and elucidate the development and progression of gastric cancer.

流行病学资料显示幽门螺杆菌（H.pylori）东亚株流行的国家胃癌发病率远高于流行西方株的国家，H.pylori东亚株比西方株有更强的致癌性，但是机制并不清楚。我们推测西方株在传播到东亚国家后，其毒力因子CagA结构发生改变，赋予东亚株有更强的致癌性，以不完全同于西方株的致癌机制，引起东亚国家胃癌高发。本研究将从慢性萎缩性胃炎、消化性溃疡及胃癌标本中分离H.pylori东亚株，以西方株为对照，建立胃癌的动物模型，观察东亚株的致癌能力；将H.pylori东亚株感染胃癌细胞，用蛋白质组学技术，寻找东亚株感染细胞后的差异表达蛋白，探索胃癌高发的原因；构建含东亚型cagA或西方型cagA的表达载体，转染胃癌细胞，联用免疫共沉淀和蛋白质组学技术，研究东亚型CagA在胃癌细胞中的效应蛋白及其磷酸化修饰，并在胃癌组织中加以验证。揭示H.pylori东亚株引起胃癌高发的分子机制，阐明胃癌的发生发展。

幽门螺杆菌（H.pylori）东亚株比西方株有更强的致癌性，但机制不清楚。本项目从临床胃疾病组织中分离H.pylori，PCR扩增H.pylori毒毒蛋白基因cagA，构建载体并测序后转变成氨基酸序列；根据CagA蛋白序列，分析临床分离株中东、西方株的感染情况，获取东亚株CagA的特征性序列和结构；选取典型H.pylori东、西方株，感染蒙古沙鼠，研究H.pylori东亚株体内诱导胃癌形成的能力；用H.pylori东亚株感染胃癌细胞，通过蛋白质组学技术，获得H.pylori东亚株感染细胞后特异的差异表达蛋白，探索胃癌高发的原因；构建东、西方株cagA真核载体，转染胃癌细胞后研究其对细胞生长、增殖和凋亡的影响; 最后从临床胃癌组织中分离癌相关纤维细胞（Cancer associated fibroblasts，CAFs）、癌周正常纤维细胞（Normal fibroblasts, NFs）及胃癌细胞，用H.pylori东亚株感染CAFs及NFs，探讨肿瘤微循环在胃癌发生发展中作用。用RNA-seq技术获取CAFs与NFs之间的差异表达基因。. 取得的主要成果有：（1）成功分离到H.pylori 临床株26株，获得H.pylori东亚株毒素蛋白CagA的特征性序列、结构及聚类，发现东、西方株CagA在氨基酸组成和结构上有显著差异。获得东亚株CagA的特征性空间结构。（2）用H.pylori感染蒙古沙鼠24月，成功诱导出与人胃相关疾病相似的发展进程：急性胃炎－急性糜烂性胃炎－萎缩性胃炎－肠型上皮化生－胃癌。（3）通过蛋白质组学技术，获得H.pylori东亚株感染胃癌细胞后特异的差异表达蛋白。（4）首次发现H.pylori感染细胞后，能活化胃黏膜上皮细胞中NLRP3炎症小体通路，但在胃癌细胞中却抑制了NLRP3炎症小体的反应链，并且活性氧（ROS）途径参与了H.pylori对 NLRP3 炎症小体的活化作用。（5）成功从胃癌组织中分离到癌相关纤维细胞（CAFs）、癌周正常纤维细胞（NFs）及胃癌细胞，在体外均可以被H.pylori吸附。H.pylori和CAFs共同作用，促进了原代胃癌细胞的运动能力。（6）通过RNA-seq技术获得了CAFs与NFs之间的差异表达基因及其参与的生物学过程和信号通路。这些研究成果将有助于探索H.pylori东亚株引起胃癌高发的分子机制。