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目的探讨由载体介导的靶向端粒酶反转录酶(hTERT)基因的RNA干扰(RNAi)技术对白血病HL-60细胞hTERT基因、蛋白表达及细胞凋亡的影响。方法用已经构建好的表达针对hTERT基因siRNA的质粒载体经转染试剂RNAi-Mate转染HL-60细胞;以转染后24 h、72 h、120 h细胞作为实验组,以空质粒、转染试剂及空白组作对照,采用反转录-PCR(RT-PCR)检测细胞hTERT基因mRNA表达,Western blot检测细胞hTERT蛋白表达,膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/PI双染法流式细胞仪检测细胞凋亡。结果转染后24 h、72 h、120 h实验组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;24 h、72 h、120 h实验组蛋白相对表达水平分别为0.47±0.09、0.32±0.07、0.51±0.08,质粒组、转染试剂组、空白对照组分别为0.75±0.11、0.76±0.15、0.73±0.14;24 h、72 h、120 h实验组,质粒组,转染试剂组,空白对照组凋亡率分别为(11.95±3.61)%、(14.61±2.97)%、(12.82±3.01)%、(4.25±2.16)%、(5.09±1.97)%、(3.76±1.84)%。24 h、72 h、120 h实验组hTERT mRNA、蛋白表达水平均低于各对照组(Pa<0.05),细胞凋亡率高于各对照组(Pa<0.05),hTERT mRNA、蛋白表达水平、细胞凋亡率实验组组间比较差异均无统计学意义(Pa>0.05);质粒组、转染试剂组hTERT mRNA、蛋白表达水平、细胞凋亡率与空白对照组比较差异均无统计学意义(Pa>0.05)。结论载体介导的靶向hTERT基因的RNAi技术能在体外抑制HL-60细胞hTERT基因和蛋白的表达,增加细胞凋亡。
Objective To investigate the effects of RNA interference (RNAi) targeting hTERT gene on hTERT gene, protein expression and apoptosis in HL-60 leukemia cells. Methods The transfected HL-60 cells were transfected with the plasmid vector of siRNA targeting hTERT gene by RNAi-Mate. The transfected cells were transfected with HL-60 cells at 24 h, 72 h and 120 h after transfection. The expression of hTERT mRNA was detected by reverse transcription-PCR (RT-PCR). The expression of hTERT protein was detected by Western blot. Annexin V-FITC ) / PI double staining flow cytometry apoptosis. Results The relative expression levels of hTERT mRNA in HL-60 cells at 24 h, 72 h and 120 h after transfection were 0.31 ± 0.08, 0.28 ± 0.05 and 0.32 ± 0.06, respectively. The plasmid group, transfection reagent group and blank control group 0.55 ± 0.13,0.49 ± 0.08,0.58 ± 0.19; The relative expression levels of protein in 24 h, 72 h and 120 h groups were 0.47 ± 0.09, 0.32 ± 0.07 and 0.51 ± 0.08, respectively. The plasmid group, transfection reagent group, blank The apoptosis rates in the control group were 0.75 ± 0.11,0.76 ± 0.15 and 0.73 ± 0.14, respectively; the apoptosis rates in the experimental group, the plasmid group, the transfection reagent group and the blank control group at 24 h, 72 h and 120 h were (11.95 ± 3.61)%, (14.61 ± 2.97)%, (12.82 ± 3.01)%, (4.25 ± 2.16)%, (5.09 ± 1.97)% and (3.76 ± 1.84)%, respectively. The mRNA and protein expression of hTERT in 24 h, 72 h and 120 h experimental groups were lower than those in control group (Pa <0.05) There was no significant difference between the experimental group and the control group (P> 0.05). There was no significant difference in the expression of hTERT mRNA, protein and apoptosis between the plasmid group and transfection reagent group and the blank control group (Pa> 0.05). Conclusion Vector-mediated RNAi technology targeting hTERT gene can inhibit the expression of hTERT gene and protein in HL-60 cells in vitro and increase apoptosis.