安徽医药
安徽醫藥
안휘의약
ANHUI MEDICAL AND PHARMACEUTICAL JOURNAL
2014年
12期
2267-2270
,共4页
葛畅%许春伟%王鲁平%方园%张玉萍
葛暢%許春偉%王魯平%方園%張玉萍
갈창%허춘위%왕로평%방완%장옥평
DNA甲基化状态%5′-氮杂-2′-脱氧胞苷%HT-29%LoVo%p16基因
DNA甲基化狀態%5′-氮雜-2′-脫氧胞苷%HT-29%LoVo%p16基因
DNA갑기화상태%5′-담잡-2′-탈양포감%HT-29%LoVo%p16기인
DNA methylation%5′-Aza-CdR%HT-29%LoVo%p16 gene
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。
目的：探討甲基化酶抑製劑5′-氮雜-2′-脫氧胞苷（5′-Aza-2′-deoxycytidine，5′-Aza-CdR）對結直腸癌細胞株HT-29和Lo-Vo中p16基因甲基化狀態、mRNA及蛋白錶達的影響。方法應用TaqMan探針為基礎的實時定量PCR法、SYBR Green PCR法及蛋白印跡實驗（ Western blot ）檢測不同濃度5′-Aza-CdR處理前後HT-29和LoVo細胞中p16基因的甲基化狀態、mRNA和蛋白錶達。結果 TaqMan 探針為基礎的實時定量PCR法檢測HT-29和LoVo細胞中p16蛋白在藥物作用後異常甲基化得到逆轉；實時熒光定量PCR和Western Blot檢測到0．5、1．0、1．5μM 5′-Aza-CdR處理後p16基因mRNA和蛋白均重新錶達，具有統計學意義（P均＜0．05）。結論結直腸癌細胞株HT-29和LoVo中p16啟動子甲基化可能是導緻該基因錶達下調甚至失活的主要原因。5′-Aza-CdR能夠較成功的逆轉結直腸癌細胞株HT-29和LoVo中p16基因的甲基化狀態，併能恢複mRNA及蛋白重新錶達。
목적：탐토갑기화매억제제5′-담잡-2′-탈양포감（5′-Aza-2′-deoxycytidine，5′-Aza-CdR）대결직장암세포주HT-29화Lo-Vo중p16기인갑기화상태、mRNA급단백표체적영향。방법응용TaqMan탐침위기출적실시정량PCR법、SYBR Green PCR법급단백인적실험（ Western blot ）검측불동농도5′-Aza-CdR처리전후HT-29화LoVo세포중p16기인적갑기화상태、mRNA화단백표체。결과 TaqMan 탐침위기출적실시정량PCR법검측HT-29화LoVo세포중p16단백재약물작용후이상갑기화득도역전；실시형광정량PCR화Western Blot검측도0．5、1．0、1．5μM 5′-Aza-CdR처리후p16기인mRNA화단백균중신표체，구유통계학의의（P균＜0．05）。결론결직장암세포주HT-29화LoVo중p16계동자갑기화가능시도치해기인표체하조심지실활적주요원인。5′-Aza-CdR능구교성공적역전결직장암세포주HT-29화LoVo중p16기인적갑기화상태，병능회복mRNA급단백중신표체。
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 .
