目的：探讨甲基化酶抑制剂5′-氮杂-2′-脱氧胞苷（5′-Aza-2′-deoxycytidine，5′-Aza-CdR）对结直肠癌细胞株HT-29和Lo-Vo中p16基因甲基化状态、mRNA及蛋白表达的影响。方法应用TaqMan探针为基础的实时定量PCR法、SYBR Green PCR法及蛋白印迹实验（ Western blot ）检测不同浓度5′-Aza-CdR处理前后HT-29和LoVo细胞中p16基因的甲基化状态、mRNA和蛋白表达。结果 TaqMan 探针为基础的实时定量PCR法检测HT-29和LoVo细胞中p16蛋白在药物作用后异常甲基化得到逆转；实时荧光定量PCR和Western Blot检测到0．5、1．0、1．5μM 5′-Aza-CdR处理后p16基因mRNA和蛋白均重新表达，具有统计学意义（P均＜0．05）。结论结直肠癌细胞株HT-29和LoVo中p16启动子甲基化可能是导致该基因表达下调甚至失活的主要原因。5′-Aza-CdR能够较成功的逆转结直肠癌细胞株HT-29和LoVo中p16基因的甲基化状态，并能恢复mRNA及蛋白重新表达。%Objective To explore the impact of methylation enzyme inhibitor 5′-Aza′-deoxycytidine on colorectal cancer cell line HT-29 and LoVo levels of methylation of p16 gene,mRNA and protein expression.Methods Treatment was conducted with different concen-trations of 5′-Aza-CdR in colorectal cancer cell line HT-29 and LoVo.Real-time quantitative PCR using TaqMan probe based method , SYBR Green PCR and Western blot test ( Western blot ) before and after drug treatment HT-29 and LoVo cells were used to detect the methylation of p16 gene,mRNA and protein expression .Results Methylight HT-29 and LoVo cells and abnormal methylation of p 16 protein in drug action were reversed;real-time fluorescent quantitative PCR and Western Blot test showed that p 16 gene mRNA and pro-tein expressed again with 0.5,1.0,1.5 μm 5′-Aza-CdR,(P<0.05),which was statistically significant.Conclusions Colorectal car-cinoma cell line HT-29 and LoVo methylation of p16 promoter may be the main reasons leading to inactivation of the gene expression . 5′-Aza-CdR can successfully reverse the methylation of p 16 gene in colorectal cancer cell line HT-29 and LoVo and restore the expres-sion of mRNA and protein .
展开▼
