CAJ | 학술논문

目的探讨DNA甲基转移酶抑制剂--5-氮杂脱氧胞苷(5-Aza-CdR)对子宫内膜癌细胞的生长及RASSF1A基因表达的影响.方法 5-Aza-CdR作用后,采用锥虫蓝染色法、流式细胞仪分别检测子宫内膜癌细胞株HEC-1B细胞的生长及凋亡情况,采用RT-PCR技术、甲基化特异性PCR(MSP)技术分别检测HEC-1B细胞中RASSF1A mRNA的表达和RASSF1A基因启动子的甲基化状态.结果 (1)HEC-1B细胞的生长及凋亡情况:5-Aza-CdR对HEC-1B细胞生长的抑制作用呈明显的剂量和时间依赖性(P＜0.01);且HEC-1B细胞的凋亡率也呈明显的剂量依赖性(P＜0.01).(2)HEC-1B细胞中BASSF1A mRNA的表达:未经5-Aza-CdR作用的HEC-1B细胞中BASSF1A mRNA表达缺失;不同浓度5-Aza-CdR(分别为0.05、0.1、1、5、10 nmol/ml)作用后,HEC-1B细胞中RASSF1AmRNA表达不同程度地上升,其相对表达水平分别为0.074±0.004、0.105±0.004、0.167±0.006、0.334±0.005、0.484±0.007,分别与未经5-Aza-CdR作用的细胞(为0)比较,差异均有统计学意义(P＜0.01).(3)HEC-1B细胞中RASSF1A基因启动子的甲基化状态:未经5-Aza-CdR作用的HEC-1B细胞中RASSF1A基因启动子呈高甲基化状态;加入低浓度(即0.05、0.1、1、5 nmol/ml)的5-Aza-CdR后,HEC-1B细胞中RASSF1A基因启动子的甲基化水平下降,呈半甲基化状态(即同时出现甲基化和非甲基化条带);当5-Aza-CdR浓度为10 nmol/ml时,HEC-1B细胞中RASSF1A基因启动子呈非甲基化状态.结论 (1)5-Aza-CdR可抑制HEC-1B细胞增殖、促进HEC-1B细胞凋亡.(2)5-Aza-CdR能使HEC-1B细胞重新表达RASSF1A mRNA,能逆转RASSF1A基因启动子的高甲基化状态.
목적 탐토DNA갑기전이매억제제--5-담잡탈양포감(5-Aza-CdR)대자궁내막암세포적생장급RASSF1A기인표체적영향.방법 5-Aza-CdR작용후,채용추충람염색법、류식세포의분별검측자궁내막암세포주HEC-1B세포적생장급조망정황,채용RT-PCR기술、갑기화특이성PCR(MSP)기술분별검측HEC-1B세포중RASSF1A mRNA적표체화RASSF1A기인계동자적갑기화상태.결과 (1)HEC-1B세포적생장급조망정황:5-Aza-CdR대HEC-1B세포생장적억제작용정명현적제량화시간의뢰성(P＜0.01);차HEC-1B세포적조망솔야정명현적제량의뢰성(P＜0.01).(2)HEC-1B세포중BASSF1A mRNA적표체:미경5-Aza-CdR작용적HEC-1B세포중BASSF1A mRNA표체결실;불동농도5-Aza-CdR(분별위0.05、0.1、1、5、10 nmol/ml)작용후,HEC-1B세포중RASSF1AmRNA표체불동정도지상승,기상대표체수평분별위0.074±0.004、0.105±0.004、0.167±0.006、0.334±0.005、0.484±0.007,분별여미경5-Aza-CdR작용적세포(위0)비교,차이균유통계학의의(P＜0.01).(3)HEC-1B세포중RASSF1A기인계동자적갑기화상태:미경5-Aza-CdR작용적HEC-1B세포중RASSF1A기인계동자정고갑기화상태;가입저농도(즉0.05、0.1、1、5 nmol/ml)적5-Aza-CdR후,HEC-1B세포중RASSF1A기인계동자적갑기화수평하강,정반갑기화상태(즉동시출현갑기화화비갑기화조대);당5-Aza-CdR농도위10 nmol/ml시,HEC-1B세포중RASSF1A기인계동자정비갑기화상태.결론 (1)5-Aza-CdR가억제HEC-1B세포증식、촉진HEC-1B세포조망.(2)5-Aza-CdR능사HEC-1B세포중신표체RASSF1A mRNA,능역전RASSF1A기인계동자적고갑기화상태.
Objective To investigate the effects and mechanisms of 5-aza-2'-deoxycytidine (5-Aza-CdR) on endometrial cancer cell.Methods In vitro experiments of 5-Aza-CdR were done using human endometrial cancer cell line HEC-1B.Evaluation of cellular proliferation and apoptosis was ascertained respectively using trypan blue exclusion and flow cytometry.RT-PCR and methylation specific PCR(MSP) was done to detect the expression of RASSF1 A mRNA and methylation status of RASSF1 A promoter of HEC-1B cell line.Results (1) The status of cellular growth and apeptosis of HEC-1 B cell line:the growth inhibition effects of 5-Aza-CdR on HEC-1B cell line were both concentration-dependent (P ＜ 0.01) and time-dependent(P ＜0.01),as well as the apoptosis rate of HBC-1-B cell line depended on the dose of 5-Aza-CdR obviously(P ＜0.01).(2)The expression of RASSF1A mRNA of HEC-1B cell line:RASSF1A mRNA was expressed in HEC-1B cell after 5-Aza-CdR treatment,but it was undetectable before the treatment.In the groups with different concentration of 5-Aza-CdR (0.05,0.1,1,5,10 nmol/ml),the expression of RASSF1A mRNA was respectively 0.074±0.004,0.105±0.004,0.167±0.006,0.334±0.005,0.484±0.007,which were remarkably different from the group without 5-Aza-CdR(the expression of RASSF1A mRNA was 0;P ＜ 0.01).(3) The hypermethylation of RASSF1A promoter of HEC-1B cell line:the hypermethylation of RASSF1A promoter was detected in HEC-1B cell line.The status of hypermethylation was decreased after treatment with 5-Aza-CdR of 0.05,0.1,1,5 nmol/ml,meanwhile,both methylation bands and demethylation bands were observed by methylation specific PCR.After the treatment with 5-Aza-CdR of 10 nmol/ml the hypermethylation was absent absolutely.Conclusions (1) In HEC-1B cell line,5-Aza-CdR can inhibit cell proliferation and induce cell apopotosis.(2) 5-Aza-CdR can renew the expression of RASSF1A mRNA of HEC-1B cell line and reverse the hypermethylation of RASSF1A promoter.