人支气管上皮细胞中苯并芘诱导TNF-α表达的分子机制

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摘要目的：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-α mRNA表达的调控机制。方法：采用Real time-PCR技术检测B[a]P暴露下，人支气管上皮细胞（Beas-2B）中TNF-α mRNA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：Real time-PCR检测B[a]P暴露下，24 h内，Beas-2B细胞中TNF-α mRNA表达量随时间延长升高。且B[a]P刺激Beas-2B细胞24 h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-α mRNA的转录从而促进TNF-α mRNA的表达，且promoter1要比promoter2活性强，没有细胞特异性。

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	代佳
	吕建祎
	张敏
	蔡秦真
	高基民

关键词 ：肿瘤坏死因子, 启动子荧光素酶报告基因, Beas-2B细胞, 苯并芘, 肺肿瘤

Abstract：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 promoter 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 specificity.

Key words： TNF-α the promoter luciferase reporter gene Beas-2Bcells B[a]P lung tumor

收稿日期: 2014-07-15

基金资助:国家自然科学基金重大研究计划培育项目（91029 706）；浙江省自然科学基金重点项目（LY12C070001）；温州医科大学校人才启动经费项目（89211004）。

通讯作者: 高基民，教授，Email：jimingao64@163.com。

作者简介: 代佳（1985-），女，陕西咸阳人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2014/V44/I11/781

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