【摘要】：目的構(gòu)建沉默Nrf2(Nuclear Factor-erythroid 2-related Factor 2,Nrf2)基因表達(dá)的RNA干擾質(zhì)粒,建立穩(wěn)定轉(zhuǎn)染Nrf2干擾質(zhì)粒的人支氣管上皮細(xì)胞株BEAS-2B(Human Bronchial Cells),并對(duì)其在該細(xì)胞中的沉默效果進(jìn)行鑒定。方法根據(jù)Genbank上公布的人Nrf2核苷酸序列設(shè)計(jì)4條RNA抑制靶序列�；瘜W(xué)合成后載入PGU6/RFP/Neo質(zhì)粒載體構(gòu)建成短發(fā)夾RNA(shRNA)表達(dá)載體,分別轉(zhuǎn)染至BEAS-2B細(xì)胞,G418篩選穩(wěn)定轉(zhuǎn)染細(xì)胞株,RT-PCR(Real time-RCR)檢測(cè)目標(biāo)基因Nrf2的表達(dá)水平。結(jié)果成功構(gòu)建shRNA質(zhì)粒表達(dá)載體,G418濃度為400mg/L時(shí)能夠篩選到穩(wěn)定轉(zhuǎn)染的細(xì)胞株,構(gòu)建的穩(wěn)定細(xì)胞系均能特異性的抑制Nrf2基因的表達(dá)。結(jié)論通過(guò)RNA干擾(RNA Interfering,RNAi)技術(shù)能夠成功構(gòu)建BEAS-2B細(xì)胞Nrf2下調(diào)表達(dá)的細(xì)胞模型,為研究Nrf2在肺癌抗氧化通路中的作用機(jī)制奠定了基礎(chǔ)。
[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.
【作者單位】： 泰山醫(yī)學(xué)院公共衛(wèi)生學(xué)院;鄭州大學(xué)公共衛(wèi)生學(xué)院;
【基金】：國(guó)家自然科學(xué)基金(81573203)
【分類號(hào)】：R734.2

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