VacA蛋白介导TRAF1及IL-8在萎缩性胃炎中的免疫调控机制研究

Our group previously selected hyper-virulent cagAvacAs1m1 H.pylori, the atrophic gastritis-caused strain through H.pylori infected cell and animal models, and the enrichment of TNF receptor-associated factor-1 (TRAF1) via NF-κB is found in VacA+H.pylori infected gastric mucosa. Moreover, the IL-8 secreting is directly dependent on the TRAF1 upregulation. This study therefore aims to further investigate as follows: 1) Construct recombinant VacA protein to explore the subsequent TRAF1 and IL-8 expression in vacA+H.pylori infected gastric epithelial cell models；and potential downstream effectors of TRAF1 by Mass Spectrometry (MS), Chromatin Immunoprecipitation sequencing (ChIP-seq), bioinformatics techniques and Luciferase Reporter Assay; 2) Verify the TRAF1-dependent NF-κB signaling in IL-8 expression, immune regulation as well as the varying biological properties in TRAF1 overexpressing and silencing condition. 3) The TRAF1, IL-8, and other cytokines profiling in peripheral blood and gastric mucosa in animal models under VacA+H.pylori both infected and eradicated circumstances; TRAF1 knockout animals are then utilized to test the above indexes as well as immunoregulating mechanisms by means of T lymphocytes activities. These studies will highlight the fine immunoregulations of vacA-TRAF1-IL-8 axis, further elucidate its functions in the pathological process and prognosis of H.pylori associated atrophic gastritis.

项目组前期经细胞感染及动物感染模型筛选出致萎缩性胃炎的cagAvacAs1m1型Hp强毒力菌株，并发现vacA可能通过NF-κB途径使胃黏膜TRAF1表达升高，且IL-8分泌直接依赖TRAF1上调。本项目将进一步阐明VacA-TRAF1-IL-8轴免疫调控机制及其在Hp相关萎缩性胃炎病理转归中的作用:1、构建VacA重组蛋白，利用胃上皮细胞模型，了解VacA蛋白对TRAF1、IL-8表达的影响；采用质谱及ChIP-seq技术、生物信息学及荧光素酶实验筛选TRAF1下游靶基因;2、利用TRAF1过表达和沉默表达手段,观察依赖TRAF1的NF-κB途径对IL-8表达、免疫调控及生物学功能的影响;3、利用动物模型，了解vacA+Hp感染及根除对外周血和胃黏膜TRAF1、IL-8表达和细胞因子的影响；构建TRAF1基因敲除的Hp动物感染模型，观察上述指标的变化及其对T淋巴细胞活性的免疫调控机制。

项目组前期经细胞感染及动物感染模型筛选出致萎缩性胃炎的cagAvacAs1m1型Hp强毒力菌株，并发现vacA可能通过NF-κB途径使胃黏膜TRAF1表达升高，且IL-8分泌直接依赖TRAF1上调。本项目在前期基础上，成功构建了TRAF1稳定过表达及沉默表达细胞系。过表达TRAF1，促进细胞的增殖及迁移，上调磷酸化p65、4-1BB及下游趋化因子IL-8的表达，激活NF-κB通路；干扰TRAF1，抑制细胞的增殖及迁移，下调磷酸化p65、4-1BB及下游趋化因子IL-8的表达，抑制NF-κB通路的活化。课题组进一步构建了VacA重组蛋白，发现VacA重组蛋白在体外实验中具有促进细胞凋亡，抑制细胞增殖的作用，且呈浓度依赖性，使促凋亡蛋白Bax表达升高，抗凋亡蛋白Bcl-xl表达降低，上调4-1BB的表达，在TRAF1调控下激活NF-κB通路，促进炎症因子IL-8的表达。本研究结果探索了TRAF1对胃上皮细胞生物学特性及NF-κB通路活化的影响，进一步验证了“vacA→TRAF1→炎症NF-κB通路相关基因→IL-8”调控轴的存在,为后续筛选TRAF1的下游基因及其他潜在信号通路提供了一定实验基础和数据支持,为vacA+Hp萎缩性胃炎发展进程中找到相关性分子标志物提供了更多的线索。