人支气管上皮细胞中苯并芘诱导TNF-α表达的分子机制

摘要

Objective: To study B[a]P on TNF-α mRNA expression regulation mechanism in Beas-2B cells through constructing TNF-α promoter luciferase reporter gene vector.Methods: The expression of TNF-α mRNA was detected by Real time-PCR with in 24 hours in Beas-2B cells exposed to B[a]P. We constructed TNF-α pro-moter luciferase reporter gene vector by molecular cloning technique and detected the promoter activity. Also we detected the change trend of TNF-α promoter activity in Beas-2B cells and 293T cells exposed to B[a]P.Results:Upon B[a]P exposure, the expression of TNF-α mRNA increased gradually with in 24 hours in Beas-2B cells and B[a]P can induce the TNF-α promoter activity. The TNF-α promoter activity also enhanced in 293Tcells exposed to B[a]P environment.Conclusion: We successfully construct the TNF-α promoter luciferase reporter gene plasmids and explained that B[a]P can induce the expression of TNF-α mRNA at transcriptional level without cell speciifcity.%目的：构建肿瘤坏死因子-α（TNF-α）启动子双荧光素酶报告基因载体，研究苯并芘（B[a]P）暴露对TNF-αmRNA表达的调控机制。方法：采用Realtime-PCR技术检测B[a]P暴露下，人支气管上皮细胞（Beas-2B）中TNF-αmRNA随时间的变化趋势。通过分子克隆技术构建TNF-α启动子双荧光素酶报告基因载体并检测其活性。进一步检测Beas-2B细胞和人肾上皮细胞293T细胞暴露于B[a]P环境下，TNF-α启动子活性的变化趋势。结果：Realtime-PCR检测B[a]P暴露下，24h内，Beas-2B细胞中TNF-αmRNA表达量随时间延长升高。且B[a]P刺激Beas-2B细胞24h内，TNF-α启动子活性也呈升高趋势。B[a]P刺激293T细胞， TNF-α启动子活性也会升高。结论：成功构建了TNF-α启动子双荧光素酶报告基因载体。而且，B[a]P能够促进TNF-αmRNA的转录从而促进TNF-αmRNA的表达，且promoter1要比promoter2活性强，没有细胞特异性。