慢病毒介导的CAV1过表达对HL-60细胞增殖与凋亡的影响

摘要

Abstract This study was purposed to explore the effect of lentivirus-mediated CAV1 overexpression on proliferation and apoptosis in HL-60 cells.Recombinant lentiviral expression vector pcDNA-EF1-CAV1 was constructed,and cotransfected the 293TN cells with a mixture of pPACK packaging plasmids.Then collecting virus suspension infects the HL-60 cells,which make CAV1 gene stable transfection and high expression in the cells.The CAV1 protein expression status in HL-60 cells transfected was elvaluated through Western blot method.Proliferative activity and apoptosis of HL-60 cells before and after transfection were detected by CCK-8 method and flow cytometry,respectively.The results showed that the PCR-positive clone screening and results of nucleotide sequencing confirmed that the CAV1 gene inserted into the expression vector pcDNA-EF1-GFP correctly,recombinant lentiviral particles Lv-CAV1 transfected HL-60 cells successfully and with transfection rate up to 90％.The result of Western blot showed that CAV1 protein expression in HL-60 cells significantly increased at 48 hours after transfection.CCK-8 result indicated that cell proliferation activity increased 48 h after transfection (P ＜ 0.05),flow cytometry testing results displayed that apoptosis rate of HL-60 cells obvirously decreased after transfection (P ＜ 0.01).It is concluded that the overexpression of CAV1 in HL-60 cells can inhibit cell proliferation activity and promote cell apoptosis.%本研究旨在探讨慢病毒介导的CAV1过表达对HL-60细胞增殖与凋亡的影响.构建慢病毒重组表达载体pcDNA-EF1-CAV1,并与pPACK包装质粒混合物共转染至293TN细胞后,收集病毒液感染HL-60细胞,使CAV1基因在细胞中稳定转染并高表达.采用Western blot方法评价转染后HL-60 CAV1蛋白表达情况.采用CCK-8法、流式细胞术分别检测转染前后HL_60细胞的增殖活性和凋亡情况.结果表明:PCR阳性克隆筛选及核苷酸测序结果证实CAV1基因正确插入表达载体pcDNA-EF1-GFP中.重组慢病毒颗粒Lv-CAV1成功转染HL-60细胞,转染率达90％.Western blot检测结果显示,转染后48 h HL-60细胞中CAV1蛋白表达明显增高.CCK-8检测结果显示转染后48 h HL-60细胞增殖活性降低(P＜0.05),流式细胞仪检测结果显示转染后HL-60细胞凋亡率明显增加(P＜0.01).结论:CAV1过表达对HL-60细胞具有抑制细胞的增殖活性、促进细胞凋亡的作用.