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目的探究靶向人端粒酶逆转录酶(hTERT)的核糖核酸干扰(RNAi)对白血病HL-60细胞生长及hTERT基因的作用。方法用已经构建好的表达针对pSilencer1.0-U6/hTERT siRNA质粒载体,经RNAi-Mate转染试剂转染HL-60细胞。将转染细胞分为对照组(3亚组)和实验组(3亚组),即pSilencer1.0-U6空质粒组、RNAi-Mate转染试剂组及生理盐水空白对照组为3个对照组,pSilencer1.0-U6/hTERT质粒转染后24 h组、pSilencer1.0-U6/hTERT质粒转染后72 h组、pSilencer1.0-U6/hTERT质粒转染后120 h组为3个实验组。用反转录聚合酶链反应(RT-PCR)法检测细胞hTERT基因mRNA的表达,Western blot法检测细胞hTERT蛋白的表达,Annexin V-FITC/PI流式细胞仪双染法检测细胞凋亡情况,用四甲基偶氮唑盐(MTT)比色法检测细胞增殖抑制率。结果实验组(3亚组)HL-60细胞hTERT蛋白表达水平(0.47±0.09、0.32±0.07、0.51±0.08)、mRNA(0.31±0.08、0.28±0.05、0.32±0.06)低于对照组(3亚组)(0.73±0.14、0.76±0.15、0.75±0.11,0.55±0.13、0.49±0.08、0.58±0.19);实验组(3亚组)细胞增殖抑制率[(23.7±4.0)%、(35.1±5.9)%、(29.6±3.8)%]及凋亡率[(11.95±3.61)%、(14.61±2.97)%、(12.82±3.01)%]均高于对照组(3亚组)(P<0.05),但实验组各亚组间、对照组各亚组间差异无统计学意义(P>0.05)。结论载体介导的靶向hTERT基因的RNAi技术在体外能抑制HL-60细胞hTERT蛋白和基因的表达,能抑制细胞增殖,增加细胞凋亡。
Objective To investigate the effect of RNAi targeting human telomerase reverse transcriptase (hTERT) on the growth of HL-60 leukemia cells and hTERT gene. Methods HL-60 cells were transfected with pSilencer1.0-U6 / hTERT siRNA plasmid vector by RNAi-Mate transfection reagent. The transfected cells were divided into control group (3 subgroups) and experimental group (3 subgroups), namely, pSilencer1.0-U6 empty plasmid group, RNAi-Mate transfection reagent group and normal saline control group were 3 control groups , pSilencer1.0-U6 / hTERT plasmid transfected 24 h group, pSilencer1.0-U6 / hTERT plasmid transfected 72 h group, pSilencer1.0-U6 / hTERT plasmid 120 h after transfection group was 3 experimental groups . The expression of hTERT mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), the expression of hTERT protein was detected by Western blot, and the apoptosis was detected by flow cytometry with Annexin V-FITC / PI The inhibitory rate of cell proliferation was detected by MTT assay. Results The expression of hTERT protein in HL-60 cells in the experimental group (3 subgroups) was significantly lower than that in the control group (0.47 ± 0.09,0.32 ± 0.07,0.51 ± 0.08) and (3 ± 0.08,0.28 ± 0.05,0.32 ± 0.06) (23.7 ± 4.0)%, (35.1%) in the experimental group (subgroup) (0.73 ± 0.14,0.76 ± 0.15,0.75 ± 0.11,0.55 ± 0.13,0.49 ± 0.08,0.58 ± 0.19) (P <0.05). The apoptotic rate was significantly higher than that in control group (subgroup 3) (P <0.05) <0.05). However, there was no significant difference between the subgroups of the experimental group and the control group (P> 0.05). Conclusion The vector-mediated RNAi technology targeting hTERT gene can inhibit the expression of hTERT protein and gene in HL-60 cells in vitro and inhibit cell proliferation and apoptosis.