载体介导的RNAi技术对HL-60细胞hTERT基因和细胞增殖的影响.docVIP

精品论文 参考文献 载体介导的RNAi技术对HL-60细胞hTERT基因和细胞增殖的影响 高吉照 许 伟 郭 雷 李 艳 卢立慧 薛天阳 　　徐州医学院附属医院儿科医院 江苏徐州 221002 　　摘要：目的 探讨由载体介导的靶向hTERT基因的RNAi技术对白血病HL-6细胞hTERT基因和细胞增殖的影响。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；以空质粒、转染试剂及空白组作对照，将转染后24h、72h、120h的细胞作为三个实验组，用RT-PCR检测细胞hTERT基因mRNA的表达，MTT检测细胞增殖活性。结果 三个实验组HL-60细胞hTERTmRNA表达水平低于各对照组，细胞增殖活性抑制率高于各对照组（P＜0.05），实验组组间比较差异无统计学意义（P＞0.05）；三个对照组组间比较差异无统计学意义（P＞0.05）。结论 载体介导的靶向hTERT基因的RNAi技术能在体外抑制HL-60细胞hTERT基因的表达，抑制细胞增殖。 　　关键词：端粒酶逆转录酶；RNA干扰；HL-60细胞；hTERT基因表达；细胞增殖抑制率 　　Abstract Objective To explore the effect of RNAi technology targeting hTERT by vector on hTERT gene expression and cell proliferative of leukemia HL-60 cell. Methods hTERT-siRNA plasmid that has been structured was transfered into HL-60 cells by RNAi-Mate，the empty plasmid，transfering reagent and blank cells were used as control.Cells were collected at 24h，72h，120h after transfered，and RT-PCR，Western-blot were used to detect the expression of hTERT mRNA and hTERT protein，flow cytometry was used to observe the apoptosis，MTT assay was used to observe the proliferative effects of cells. Results hTERT mRNA expression of test groups at 24h，72h，120h after transfered were 0.31plusmn;0.08，0.28plusmn;0.05，0.32plusmn;0.06 respectively，expression level of empty plasmid，transfering reagent and blank groups were 0.55plusmn;0.13，0.49plusmn;0.08，0.58plusmn;0.19 respectively.Compared with control groups，hTERT mRNA in test groups was significantl low（P＜0.05）；compared with blank group，hTERT mRNA in the empty plasmid and transfering reagent groups had no significant change（P＞0.05）.The antiproliferative effects of RNAi in HL-60 cells in 24h，72h，120h test groups were（23.7plusmn;4.0）%、（35.1plusmn;5.9）%、（29.6plusmn;3.8）%，they were（3.7plusmn;0.8）%、（2.1plusmn;0.5）%、（2.7plusmn;0.9）% in the empty plasmid group，（4.0plusmn;1.8）%、（2.6plusmn;1.3）%、（2.2plusmn;1.1）% in transfering reagent group and in blank group，all 0.The antiproliferative effects of cells in test groups were higher