Apak稳定敲除细胞系的建立及其p53活性和凋亡水平的变化

摘要

目的：利用CRISPR／Cas9慢病毒系统在人结肠癌细胞（ HCT116细胞）中建立稳定及永久性Apak敲除细胞系，检测Apak敲除后细胞部分生物学活性、p53活性和凋亡水平的变化。方法构建lentiCRISPR v2－sgRNA Apak敲除质粒，进行慢病毒包装，感染HCT116细胞，嘌罗霉素筛选出阳性克隆。通过蛋白质免疫印迹检测Apak蛋白水平，筛选得到Apak稳定敲除细胞系。分别通过报告基因实验、流式细胞术和克隆形成实验测定Apak稳定敲除细胞系中p53活性、细胞凋亡率和克隆形成能力。结果通过测序证明lentiCRISPR v2－sgRNA Apak敲除质粒正确，蛋白质免疫印迹证实Apak敲除细胞系中Apak表达缺失。在Apak敲除细胞系中p53的转录活性增强，Apak敲除细胞凋亡增加、克隆形成能力降低。结论获得Apak稳定敲除细胞系，便于后期Apak功能的研究。%Objective To establish an Apak gene stable and permanent knockout cell line using CRISPR/Cas9 system in human colon cancer cells ( HCT116 cells), and study the effect of Apak knock-out on p53 activity and apoptosis. Methods The lentiCRISPR v2-sgRNA Apak expression plasmid was co-transfected with lentivirus coated plasmids pSPAX2 and pMD2.G.The supernatant was collected, filtered, and used to infect HCT116 cells.The positive clones were screened out by puromycin culture and Western blot was used to detect Apak knockout cell lines.Luciferase reporter gene assay, flow cytometry analysis and colony formation assay were used to examine p53 activity and apoptosis of Apak knockout cells, respectively.Results Apak knockout HCT116 cell lines were generated in which p53 activity and apoptosis were increased,but the colony formation was decreased.Conclusion The Apak stable knockout cell lines of HCT116 are successfully generated by CRISPR/Cas9 system for further functional study.