Nrf2基因沉默的人支气管上皮细胞模型的建立
发布时间：2018-11-11 09:54
【摘要】：目的构建沉默Nrf2(Nuclear Factor-erythroid 2-related Factor 2,Nrf2)基因表达的RNA干扰质粒,建立稳定转染Nrf2干扰质粒的人支气管上皮细胞株BEAS-2B(Human Bronchial Cells),并对其在该细胞中的沉默效果进行鉴定。方法根据Genbank上公布的人Nrf2核苷酸序列设计4条RNA抑制靶序列。化学合成后载入PGU6/RFP/Neo质粒载体构建成短发夹RNA(shRNA)表达载体,分别转染至BEAS-2B细胞,G418筛选稳定转染细胞株,RT-PCR(Real time-RCR)检测目标基因Nrf2的表达水平。结果成功构建shRNA质粒表达载体,G418浓度为400mg/L时能够筛选到稳定转染的细胞株,构建的稳定细胞系均能特异性的抑制Nrf2基因的表达。结论通过RNA干扰(RNA Interfering,RNAi)技术能够成功构建BEAS-2B细胞Nrf2下调表达的细胞模型,为研究Nrf2在肺癌抗氧化通路中的作用机制奠定了基础。
[Abstract]:Objective to construct a RNA interference plasmid that silenced the expression of Nrf2 (Nuclear Factor-erythroid 2-related Factor 2nrf2) gene and to establish a stable transfected Nrf2 interference plasmid BEAS-2B (Human Bronchial Cells), cell line of human bronchial epithelium. And its silencing effect in the cell was identified. Methods four RNA suppression target sequences were designed according to the human Nrf2 nucleotide sequence published on Genbank. The short hairpin RNA (shRNA) expression vector was constructed by chemical synthesis and loaded into PGU6/RFP/Neo plasmid vector, and then transfected into BEAS-2B cells. G418 was used to screen the stable transfection cell line. RT-PCR (Real time-RCR) was used to detect the expression level of the target gene Nrf2. Results the expression vector of shRNA plasmid was successfully constructed. The stable transfected cell lines could be screened when G418 concentration was 400mg/L, and the stable cell lines could specifically inhibit the expression of Nrf2 gene. Conclusion the down-regulated expression of Nrf2 in BEAS-2B cells can be successfully constructed by RNA interference (RNA Interfering,RNAi), which lays a foundation for the study of the mechanism of Nrf2 in the antioxidant pathway of lung cancer.
【作者单位】： 泰山医学院公共卫生学院;郑州大学公共卫生学院;
【基金】：国家自然科学基金(81573203)
【分类号】：R734.2
本文编号：2324487
[Abstract]:Objective to construct a RNA interference plasmid that silenced the expression of Nrf2 (Nuclear Factor-erythroid 2-related Factor 2nrf2) gene and to establish a stable transfected Nrf2 interference plasmid BEAS-2B (Human Bronchial Cells), cell line of human bronchial epithelium. And its silencing effect in the cell was identified. Methods four RNA suppression target sequences were designed according to the human Nrf2 nucleotide sequence published on Genbank. The short hairpin RNA (shRNA) expression vector was constructed by chemical synthesis and loaded into PGU6/RFP/Neo plasmid vector, and then transfected into BEAS-2B cells. G418 was used to screen the stable transfection cell line. RT-PCR (Real time-RCR) was used to detect the expression level of the target gene Nrf2. Results the expression vector of shRNA plasmid was successfully constructed. The stable transfected cell lines could be screened when G418 concentration was 400mg/L, and the stable cell lines could specifically inhibit the expression of Nrf2 gene. Conclusion the down-regulated expression of Nrf2 in BEAS-2B cells can be successfully constructed by RNA interference (RNA Interfering,RNAi), which lays a foundation for the study of the mechanism of Nrf2 in the antioxidant pathway of lung cancer.
【作者单位】： 泰山医学院公共卫生学院;郑州大学公共卫生学院;
【基金】：国家自然科学基金(81573203)
【分类号】：R734.2
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