摘要
目的探讨核呼吸因子-1(NRF-1)对氯化钴(cobalt chloride,CoCl2)诱导细胞凋亡的影响。方法采用CRISPR在线工具构建NRF-1-sgRNA,CRISPR/Cas9质粒酶切及T4连接酶的连接构建NRF-1基因敲除的慢病毒重组质粒lentiCRISPR-NRF-1-sgRNA,慢病毒包装后转染至NRK-52E细胞,通过嘌呤霉素筛选、流式细胞仪单选、T7E1酶切分析、Sanger测序及Western blot法检测NRF-1基因的表达水平;用含CoCl2(终浓度为600μmol·L-1)的完全培养液将NRF-1敲除组(mutant)、空载体组(vector)和正常对照组(control)细胞培养24 h后,用流式细胞仪检测细胞的凋亡率,用Western blot法检测HIF-1a,Cleaved-caspase-3,Caspase-3,Bax,Bcl-2蛋白表达水平。结果测序结果显示NRF-1-sgRNA正确插入到lentiCRISPR质粒中,T7E1酶切分析显示,sgRNA2为NRF-1基因的有效靶点;Sanger测序结果证明NRF-1基因的9个碱基被敲除;与vector组比较,NRF-1敲除组(mutant1、mutant2、mutant3)组细胞株中NRF-1蛋白表达水平降低(P<0.01);CoCl2诱导缺氧时,mutant组细胞凋亡率高于vector组和contorl组(P均<0.01);当mutant组细胞与vector组比较时,HIF-1a、Cleaved-caspase-3、Bax蛋白表达水平增高,Bcl-2蛋白表达水平降低(P均<0.01)。结论 NRF-1基因的敲除可以增加CoCl2诱导的大鼠肾细胞NRK-52E的凋亡水平。
Objective To investigate the effect of nuclear respiratory factor-1(NRF-1)on apoptosis induced by cobalt chloride(CoCl2).Methods NRF-1-sgRNA was constructed by CRISPR on-line tools,and the NRF-1 knock-out lentivirus recombinant plasmid lenti CRISPR-NRF-1-sgRNA was constructed by enzyme digestion of CRISPR/Cas9 plasmid and T4 ligase.The lentivirus was packaged and transferred to NRK-52E cells.The expression level of NRF-1 gene was detected by puromycin screening,flow cytometry single selection,T7E1 digestion,Sanger sequencing and Western blot.The cells of NRF-1 knockout group(mutant),vector group and control group were cultured in complete culture medium containing CoCl2(final concentration of 600μmol·L-1)for 24 h.The apoptosis rate of cells was detected by flow cytometry,and the expression levels of HIF-1a,Cleaved-caspase-3,Caspas-3,Bax,Bcl-2 were detected by Western blot.Results Sequencing results showed that the NRF-1-sgRNA was correctly inserted into the lenti CRISPR plasmid.T7E1 restriction analysis revealed that sgRNA2 was a valid target for the NRF-1 gene.Sanger sequencing results showed that 9 bases of the NRF-1 gene were knocked out.Compared with the empty vector group,the expression level of NRF-1 protein in the cell line of the NRF-1 knockout group(mutant1,mutant2,mutant3)were reduced(P<0.01).When CoCl2 induced hypoxia,the apoptosis rate of the mutant group was higher than that of the empty vector group and the normal control group(P all<0.01).When the mutant group cells were compared with the empty vector group and the normal control group,the expression levels of HIF-1a,Cleaved-caspase-3 and Bax increased,and the expression levels of Bcl-2 protein decreased(P all<0.01).Conclusion Knockout of NRF-1 gene can increase the apoptosis level of NRK-52E induced by CoCl2.
作者
董飞
孙红丽
牛楠
李卉
赵瑞
王婵
赵巍
DONG Fei;SUN Hongli;NIU Nan;LI Hui;ZHAO Rui;WANG Chan;ZHAO Wei(Ningxia Medical University,Yinchuan750004,China;Research Center for Medical Science and Technology,Ningxia Medical University,Yinchuan750004,China)
出处
《宁夏医科大学学报》
2020年第4期351-357,共7页
Journal of Ningxia Medical University
