载体介导的RNA i技术对HL-60细胞端粒酶逆转录酶基因表达和细胞增殖的影响

Effect of hTERT gene expression and activity of proliferation on HL-60 cell line by vector-based RNAi technology

目的探讨由载体介导的靶向端粒酶逆转录酶(hTERT)基因的RNA i技术对人类髓系白血病HL-60细胞hTERT基因表达和细胞增殖的影响。方法用表达针对hTERT基因siRNA的质粒载体转染HL-60细胞,以转染后24、72、120 h细胞组作为3个实验组,以空质粒组、转染试剂组及空白对照组作为3个对照组,RT-PCR法检测HL-60细胞hTERT基因mRNA的表达,MTT法检测HL-60细胞增殖活性。结果转染后24、72、120 h组HL-60细胞hTERT基因mRNA相对表达水平分别为0.31±0.08、0.28±0.05、0.32±0.06,质粒组、转染试剂组、空白对照组分别为0.55±0.13、0.49±0.08、0.58±0.19,3个实验组HL-60细胞hTERT mRNA表达水平低于3个对照组,差异有显著性(P<0.05),3个实验组组间比较差异无显著性(P>0.05),3个对照组间表达水平比较差异无显著性(P>0.05);24、72、120 h实验组细胞增殖活性抑制率分别为(23.7±4.0)%、(35.1±5.9)%、(29.6±3.8)%,质粒组分别为(3.7±0.8)%、(2.1±0.5)%、(2.7±0.9)%,转染试剂组分别为(4.0±1.8)%、(2.6±1.3)%、(2.2±1.1)%,空白对照组均为0,3个实验组HL-60细胞增殖活性抑制率明显高于各对照组,差异有显著性(P<0.05),3个实验组及组间比较差异无显著性(P>0.05),3个对照组组间比较差异无显著性(P>0.05)。结论载体介导的靶向hTERT基因的RNA i技术能在体外抑制HL-60细胞hTERT基因的表达,细胞增殖活性受抑制。

Objective To explore the effect of RNAi technology targeting hTERT by vector on hTERT mRNA expression and the antiproliferative effects of HL-60 cells.Methods hTERT-siRNA plasmid was transfected into HL-60 cells by RNAi-Mate.The empty plasmid,transfected reagent and blank groups were used as controls.Cells were collected at 24,72,120 h,respectively.After transfection,the expression of hTERT mRNA and the antiproliferative effects of cells were detected by RT-PCR and MTT assay.Results hTERT mRNA expression of test groups at 24,72,120 h were 0.31±0.08,0.28±0.05 and 0.32±0.06 respectively,while the expression level of empty plasmid,transfected reagent and blank groups were 0.55±0.13,0.49±0.08,0.58±0.19 respectively.Compared with control groups,hTERT mRNA expression in test groups was significantly lower（P0.05）;there were no significant differences among three test groups（P0.05）;compared with blank group,hTERT mRNA expressions in the empty plasmid and transfected reagent groups had no significant differences（P0.05）.The antiproliferative effects of RNAi in HL-60 cells in 24 h,72 h,120 h test groups were（23.7±4.0）%,（35.1±5.9）%,（29.6±3.8）%,respectively;they were（3.7±0.8）%,（2.1±0.5）%,（2.7±0.9）% in the empty plasmid group and（4.0±1.8）%,（2.6±1.3）%,（2.2±1.1）% in transfected reagent group.The antiproliferative effects of cells in test groups were higher than that in control groups（P0.05）;there were no distinguished differences among three test groups（P0.05）;compared with blank group,the antiproliferative effects in the empty plasmid and transfected reagent groups had no distinguished differences（P0.05） in 24 h,72 h,120 h.Conclusion RNAi technology targeting hTERT by vector could inhibit the expression of hTERT gene and the activity of HL-60 cells proliferation in vitro.