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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。
OBJECTIVE: To establish a stable H.pylori-infected human gastric epithelial cell model, screen and identify the expression of microRNAs (miRNAs) associated with H.pylori infection, and lay a foundation for further study on the regulatory mechanism of miRNAs related to infection. Methods: Human gastric epithelial cells were infected with H.pylori strain at MOI = 100: 1, and the infection model was evaluated by detecting the expression of inflammatory cytokines and key enzymes of inflammatory reaction. The cell infection was analyzed by miRNA V3.0 microarray Before and after the miRNAs expression profile changes, the use of real-time quantitative PCR technology and Northern hybridization expression of miRNAs were significantly different analysis identified. Results: After 24 h infection of H.pylori cells, the secretion of proinflammatory cytokine IL-8 was significantly increased (P <0.01). The expression of COX-2, a key enzyme in initiating inflammatory response, was significantly increased. Chromosome data showed that the miRNAs that were significantly differentially expressed more than 2-fold with H.pylori infection included PREDICTED-MIR191, miR-155, miR-92b, miR-30b, miR-146a and miR- Of miR-181b, miR-324. The results of real-time PCR and Northern blot showed that the expression of miR-155 and miR-146a were significantly increased in H.pylori-infected cell model (P <0.01). Conclusion: The increased expression of miR-155 and miR-146a in infected cell models suggests that both may be involved in the immune regulation of H. pylori infection.