幽门螺杆菌感染细胞模型的建立及感染相关microRNAs的筛选鉴定

目的：建立稳定的幽门螺杆菌（H.pylori）感染人胃上皮细胞模型;筛选并鉴定H.pylori感染相关microRNAs（miRNAs）的表达,为深入研究感染相关miRNAs的调控作用机制奠定基础。方法：将H.pylori标准株按MOI=100：1感染人胃上皮细胞,通过检测炎性细胞因子及炎症反应关键酶的表达综合评价感染模型;采用博奥公司miRNAs V3.0芯片分析细胞感染前后miRNAs表达谱变化,运用实时定量PCR技术和Northern杂交对表达显著差异的miRNAs进行分析鉴定。结果：H.pylori感染细胞24 h后,细胞分泌促炎细胞因子IL-8显著升高（P〈0.01）;启动炎症反应的关键酶COX-2的表达明显增加。芯片数据显示：H.pylori感染引起超过2倍显著差异表达的miRNAs包括：表达上调的PREDICTED-MIR191、miR-155、miR-92b、miR-30b、miR-146a、miR-16等,和表达降低的miR-181b、miR-324。实时定量PCR和Northern杂交结果显示感染相关miR-155和miR-146a在H.pylori感染细胞模型中表达均显著增加（P〈0.01）。结论：miR-155和miR-146a在感染细胞模型中的表达增加提示二者可能参与H.pylori感染的免疫调控过程。

Establishment of helicobacter pylori infection cell model and identification of infection-associated microRNAs

Objective： To establish stable gastric epithelium cells model infected by Helicobacter pylori（H.pylori）,and to screen and identify H.pylori infection-related microRNAs（miRNAs） so as to investigate the regulatory mechanism of miRNAs.Methods： H.pylori were added to cells at a multiplicity of infection of 100：1.The infection model was monitored by the release of interleukin-8（IL-8） and the expression of COX-2.The expression profile of cellular miRNAs during H.pylori infection were analyzed by microarray（Capi-talBio,Beijing）.The significantly altered miRNAs were identified by the quantitative RT-PCR and Northern blot assays.Results： After the incubation of H.pylori with the gastric epithelium cells for 24h,IL-8 release and COX-2 protein expression in cells were detected.The expression of miRNAs could be significantly altered during H.pylori infection by microarray,including the up-regulation of PRE-DICTED-MIR191,miR-155,miR-92b,miR-30b,miR-146a,miR-16 and the down-regulation of miR-181b and miR-324.In consensus with the findings from microarray,miR-155 and miR-146a in infection models increased significantly（P0.01）.Conclusion： The upregu-lation of miR-155 and miR-146a in infection models implied that they may be involved in the immune regulation of H.pylori infection.