| 摘要: |
| 目的:利用RNA干扰的方法,构建针对人核转录因子κB抑制子(IκB)α、β和ε的共同干扰载体,转染人肝细胞癌HepG2细胞,建立稳定、共同下调IκBα、IκBβ和IκBε的HepG2细胞系,为后续研究奠定基础。 方法:构建3个针对人IκBα基因的干扰载体pcDNATM6.2-miR-IκBα(1,2,3),转染HepG2细胞,在mRNA和蛋白水平检测3个干扰载体对IκBα表达的抑制作用,筛选出抑制效果最好的干扰载体。以同样的方法构建并筛选出分别针对人IκBβ和IκBε基因的最佳干扰载体,通过酶切连接反应将分别针对IκBα、IκBβ和IκBε基因的3个最佳干扰片段串联连入pcDNATM6.2-miR干扰载体中,构建可同时干扰IκBα、IκBβ和IκBε的共同干扰载体pcDNATM6.2-miR-IκBα-IκBβ-IκBε。将共同干扰载体转染HepG2细胞,通过G418筛选,建立IκBα、IκBβ和IκBε的表达共同稳定下调的HepG2细胞系,并以蛋白质印迹法检测IκBα、IκBβ和IκBε的蛋白表达。 结果:成功构建共同干扰载体pcDNATM6.2-miR-IκBα-IκBβ-IκBε；Western印迹结果显示,与正常及对照载体pcDNATM6.2-miR-Neg转染的HepG2细胞相比,在稳定转染pcDNATM6.2-miR-IκBα-IκBβ-IκBε的HepG2细胞系中,IκBα、IκBβ和IκBε的表达均稳定下调。 结论:成功建立了IκBα、IκBβ和IκBε共同稳定下调的人肝细胞癌HepG2细胞系,为后续研究奠定了基础。 |
| 关键词: 核转录因子κB 抑制子 RNA干扰 HepG2细胞 干扰载体 |
| DOI：10.3724/SP.J.1008.2009.0998 |
| 投稿时间：2009-08-10修订日期：2009-08-10 |
| 基金项目:上海市自然科学基金(08ZR1405500). |
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| Establishment of a stable HepG2 cell line with simultaneous knockdown of nuclear factor-kappaB inhibitors IκBα, IκBβ and IκBε genes |
| FU Jing,CHEN Yao,REN Yi-bin,QIAN You-wen,WANG Hong-yang* |
| (International Cooperation Laboratory on Signal Transduction,Eastern Hepatobiliary Hospital,Second Military Medical University,Shanghai 200438,China) |
| Abstract: |
| Objective:To establish a human hepatic cancer cell line HepG2 with stable,simultaneous knockdown of three nuclear factor-kappaB inhibitors (IκBα,IκBβ,and IκBε)by RNA interference using pcDNATM6.2-miR-IκBα-IκBβ-IκBε co-targeting IκBα,IκBβ,and IκBε genes,so as to lay a foundation for future study.Methods: We designed and constructed three interfering plasmids pcDNATM6.2-miR-IκBα(1,2,3)targeting human IκBα gene; the three vectors were transfected into HepG2 cells separately and the most effective interfering fragment was identified by RT-PCR and Western blotting analysis. Similarly,we also constructed and selected the most effective interfering vectors targeting IκBβ and IκBε. Then we chained the three most effective interfering fragments (miR-IκBα,miR-IκBβ,and miR-IκBε) into pcDNATM6.2-miR vector to construct pcDNATM6.2-miR-IκBα-IκBβ-IκBε vector,which was used to transfect HepG2 cells and the transfectants were selected by G418.The expression of IκBα,IκBβ,and IκBε in the transfectants was examined by Western blotting analysis.Results: We successfully constructed the pcDNATM6.2-miR-IκBα-IκBβ-IκBε vector. Western blotting analysis showed that,compared with the normal HepG2 cells and those transfected with pcDNATM6.2-miR-Neg vector,the protein expression of IκBα,IκBβ,and IκBε was stably down-regulated in cells transfected with pcDNATM6.2-miR-IκBα-IκBβ-IκBε vector.Conclusion: We have successfully established a HepG2 cell line with simultaneous knockdown of IκBα,IκBβ,and IκBε,paving a way for future study. |
| Key words: inhibitor of nuclear factor-kappaB RNA interference HepG2 cells interfering vector |
