NRF-1基因敲除对NRK-52E细胞凋亡的影响

摘要

目的 探讨NRF-1基因对NRK-52E细胞凋亡的影响.方法 构建敲除NRF-1基因的慢病毒重组质粒lentiCRISPR-NRF-1-sgRNA,包装慢病毒,转染NRK-52E细胞,实验设control组、vector组和mutant组;通过嘌磷霉素筛选、T7E1酶切分析、Sanger测序及Western blot法筛选NRF-1基因稳定敲除的细胞系;用含H2O2的完全培养液(终浓度为500μmol·L-1)将control组、vector组和mutant组细胞培养4h建立NRK-52E细胞氧化应激损伤模型.Western blot法检测氧化应激条件下各组细胞中cyt-c、Cleaved-caspase3、Bcl-2、Bax蛋白表达水平;采用流式细胞术检测各组细胞的凋亡率.结果 T7E1酶切分析显示,sgRNA2为NRF-1基因的有效靶点;Sanger测序结果获得了3个基因缺失的突变体;Western blot结果显示,与control组及vector组比较,mutant组细胞中NRF-1蛋白表达水平降低(P＜0.05或＜0.01);NRF-1基因敲除后,与control组和vector组相比,氧化应激损伤时,mutant组细胞中的cyt-c、Cleaved-caspase3、Bax表达水平增高,Bcl-2表达水平降低(P＜0.05或＜0.01);Annexin V-FITC/PI检测细胞凋亡结果显示,与control组和vector组比较,氧化应激条件下mutant组细胞凋亡率增高[(11.45±0.64)％vs(3.45±0.14)％,(11.45±0.64)％vs(3.60±0.28)％)(P均＜0.01).结论 NRF-1基因敲除增强了氧化应激条件下NRK-52E细胞凋亡水平.%Objective To explore the effect of NRF-1 on apoptosis of NRK-52 E cells. Methods Lentivirus recombinant plasmid lentiCRISPR-NRF-1-sgRNA was constructed to knock down the NRF-1 gene. Lentivirus was packaged and transfected into NRK-52 E cells. Cells were divided into control group, vector group and mutant group. NRF-1 stable knockout cell lines were screened by purinomycin screening, T7 E1 enzyme digestion analysis, Sanger sequencing and Western blot assay.The oxidative stress injury model of NRK-52 E cells was established by culturing the cells of three groups for 4 hours with a complete culture medium containing H2 O2(the final concentration is 500μmol·L-1). Western blot was used to detect the expression of cyt-c, cleaved-caspase 3, Bcl-2 and Bax proteins in the cells under oxidative stress, and flow cytometry was used to detect the apoptosis rate of each group. Results T7 E1 enzyme digestion analysis showed that sg RNA2 was an effective target of NRF-1 gene. Sanger sequencing showed that three mutants with gene deletion were obtained. Western blot results showed that the expression of NRF-1 protein in mutant group was lower than that in control group and vector group (P ＜0.05 or ＜0.01). Compared with control group and vector group, the expression of cyt-c, cleaved-caspase 3 and Bax in mutant group increased and the expression of Bcl-2 in mutant group decreased when oxidative stress damage occurred (P＜0.05 or ＜0.01). The results of Annexin V-FITC/PI staining showed that compared with control and vector group, the apoptosis rate of mutant group was significantly higher[ (11.45±0.64) ％ vs (3.45±0.14) ％, (11.45±0.64) ％ vs (3.60±0.28) ％, P＜0.01) ]. Conclusion NRF-1 knockout enhances the apoptosis of NRK-52 E cells under oxidative stress.