CAJ | 학술논문

目的探讨5-氮杂-2’-脱氧胞苷(5-Aza-dC)联合曲古抑菌素A(TSA)对分化程度不同的人胃腺癌细胞株MKN-45、SGC-7901与MGC-803的细胞存活率和抑癌基因E-cadherin启动子区甲基化以及mRNA表达的影响. 方法培养3个细胞株,实验分为空白对照组、5-Aza-dC组(10μmol/L 5-Aza-dC处理72 h)、TSA组(1μmol/L TSA处理48 h)和联合组(10μmol/L5-Aza-dC处理24 h后,再加入1μmol/L TSA处理48 h).观察三株细胞形态的改变,用台盼蓝染色法检测活细胞数.采用甲基化特异性聚合酶链反应(MSP)和反转录聚合酶链反应(RT-PCR)分别检测3株细胞的E-cadherin基因启动子区甲基化水平与mRNA表达. 结果 MKN-45细胞中,5-Aza-dC组、TSA组与联合组的细胞存活率分别为(73.03±1.92)％、(64.48±1.28)％和(52.44±1.45)％;SGC-7901细胞分别是(63.33±1.92)％、(52.15±1.44)％和(44.44±1.45)％;MGC-803细胞分别是(57.33±1.61)％、(45.48±1.29)％和(33.11±1.12)％.3株细胞的单药组的细胞存活率均减少,联合组分别比5-Aza-dC和TSA处理组明显下降(均P＜0.05).E-cadherin基因启动子区甲基化水平,5-Aza-dC组、TSA组及联合组的甲基化产物与空白对照组相比较均降低,其中联合组比单药组降低明显.空白对照、5-Aza-dC、TSA及二者联合处理组的E-cadherin的mRNA相对表达量在MKN-45细胞株中分别为0.17±0.01、0.32±0.01、0.29±0.02和0.57±0.02;在SGC-7901细胞株中分别为0.22±0.01、0.46±0.01、0.43±0.01和0.61±0.01;在MGC-803细胞株中分别为0.24±0.02、0.49±0.01、0.44±0.01和0.73±0.01.在单药处理组中比空白对照组升高(P＜0.05),并且二药联合组比单药组升高(P＜0.01). 结论 5-Aza-dC和TSA单独处理均降低分化程度不同的胃癌细胞存活率,使E-cadherin基因的甲基化水平下降,并增加其mRNA表达.3株细胞的联合组的影响效果更明显,两药之间具有协同作用.其中MKN-45的存活率,甲基化水平和mRNA表达的改变均较SGC-7901与MGC-803明显. 目的探討5-氮雜-2’-脫氧胞苷(5-Aza-dC)聯閤麯古抑菌素A(TSA)對分化程度不同的人胃腺癌細胞株MKN-45、SGC-7901與MGC-803的細胞存活率和抑癌基因E-cadherin啟動子區甲基化以及mRNA錶達的影響. 方法培養3箇細胞株,實驗分為空白對照組、5-Aza-dC組(10μmol/L 5-Aza-dC處理72 h)、TSA組(1μmol/L TSA處理48 h)和聯閤組(10μmol/L5-Aza-dC處理24 h後,再加入1μmol/L TSA處理48 h).觀察三株細胞形態的改變,用檯盼藍染色法檢測活細胞數.採用甲基化特異性聚閤酶鏈反應(MSP)和反轉錄聚閤酶鏈反應(RT-PCR)分彆檢測3株細胞的E-cadherin基因啟動子區甲基化水平與mRNA錶達. 結果 MKN-45細胞中,5-Aza-dC組、TSA組與聯閤組的細胞存活率分彆為(73.03±1.92)％、(64.48±1.28)％和(52.44±1.45)％;SGC-7901細胞分彆是(63.33±1.92)％、(52.15±1.44)％和(44.44±1.45)％;MGC-803細胞分彆是(57.33±1.61)％、(45.48±1.29)％和(33.11±1.12)％.3株細胞的單藥組的細胞存活率均減少,聯閤組分彆比5-Aza-dC和TSA處理組明顯下降(均P＜0.05).E-cadherin基因啟動子區甲基化水平,5-Aza-dC組、TSA組及聯閤組的甲基化產物與空白對照組相比較均降低,其中聯閤組比單藥組降低明顯.空白對照、5-Aza-dC、TSA及二者聯閤處理組的E-cadherin的mRNA相對錶達量在MKN-45細胞株中分彆為0.17±0.01、0.32±0.01、0.29±0.02和0.57±0.02;在SGC-7901細胞株中分彆為0.22±0.01、0.46±0.01、0.43±0.01和0.61±0.01;在MGC-803細胞株中分彆為0.24±0.02、0.49±0.01、0.44±0.01和0.73±0.01.在單藥處理組中比空白對照組升高(P＜0.05),併且二藥聯閤組比單藥組升高(P＜0.01). 結論 5-Aza-dC和TSA單獨處理均降低分化程度不同的胃癌細胞存活率,使E-cadherin基因的甲基化水平下降,併增加其mRNA錶達.3株細胞的聯閤組的影響效果更明顯,兩藥之間具有協同作用.其中MKN-45的存活率,甲基化水平和mRNA錶達的改變均較SGC-7901與MGC-803明顯.
목적 탐토5-담잡-2’-탈양포감(5-Aza-dC)연합곡고억균소A(TSA)대분화정도불동적인위선암세포주MKN-45、SGC-7901여MGC-803적세포존활솔화억암기인E-cadherin계동자구갑기화이급mRNA표체적영향. 방법 배양3개세포주,실험분위공백대조조、5-Aza-dC조(10μmol/L 5-Aza-dC처리72 h)、TSA조(1μmol/L TSA처리48 h)화연합조(10μmol/L5-Aza-dC처리24 h후,재가입1μmol/L TSA처리48 h).관찰삼주세포형태적개변,용태반람염색법검측활세포수.채용갑기화특이성취합매련반응(MSP)화반전록취합매련반응(RT-PCR)분별검측3주세포적E-cadherin기인계동자구갑기화수평여mRNA표체. 결과 MKN-45세포중,5-Aza-dC조、TSA조여연합조적세포존활솔분별위(73.03±1.92)％、(64.48±1.28)％화(52.44±1.45)％;SGC-7901세포분별시(63.33±1.92)％、(52.15±1.44)％화(44.44±1.45)％;MGC-803세포분별시(57.33±1.61)％、(45.48±1.29)％화(33.11±1.12)％.3주세포적단약조적세포존활솔균감소,연합조분별비5-Aza-dC화TSA처리조명현하강(균P＜0.05).E-cadherin기인계동자구갑기화수평,5-Aza-dC조、TSA조급연합조적갑기화산물여공백대조조상비교균강저,기중연합조비단약조강저명현.공백대조、5-Aza-dC、TSA급이자연합처리조적E-cadherin적mRNA상대표체량재MKN-45세포주중분별위0.17±0.01、0.32±0.01、0.29±0.02화0.57±0.02;재SGC-7901세포주중분별위0.22±0.01、0.46±0.01、0.43±0.01화0.61±0.01;재MGC-803세포주중분별위0.24±0.02、0.49±0.01、0.44±0.01화0.73±0.01.재단약처리조중비공백대조조승고(P＜0.05),병차이약연합조비단약조승고(P＜0.01). 결론 5-Aza-dC화TSA단독처리균강저분화정도불동적위암세포존활솔,사E-cadherin기인적갑기화수평하강,병증가기mRNA표체.3주세포적연합조적영향효과경명현,량약지간구유협동작용.기중MKN-45적존활솔,갑기화수평화mRNA표체적개변균교SGC-7901여MGC-803명현.
Objective To observe the effects of 5-aza-2 '-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) alone and combination on cell survival rate and promoter methylation and mRNA expression level of tumor suppressor gene E-cadherin in three human gastric cancer cell lines MKN-45,SGC-7901 and MGC-803 with different differentiation degree.Methods MKN-45,SGC 7901 and MGC-803 cells were routinely cultured,and were divided into blank control,5-Aza-dC (10μmol/L for 72 h),TSA (1 μmol/L for 48h) and the combination (10μmol/L 5-Aza-dC for 72 h plus 1 μmol/L TSA for last 48 hours) groups.Morphological changes of the gastric cells were observed.The number of live cells was detected by trypan blue staining,and the cell survival rate was calculated.The promoter methylation and mRNA expression of E-cadherin gene were detected by methylation-specific PCR (MSP) and RT-PCR methods,respectively.Results The survival rates in groups of 5-Aza-dC,TSA and combination were (73.03±1.92)％,(64.48±1.28)％ and (52.44±1.45)％ for MKN-45 cells,and (63.33±1.92)％,(52.15±1.44)％ and (44.44±1.45)％ for SGC-7901 cells,and (57.33±1.61)％,(45.48±1.29)％ and (33.11±1.12)％ for MGC-803 cells.It was more significant that the cell survival rates in the three cell lines were significant decreased in the combination group than that in single drug group (all P＜0.05).The promoter methylation level of E-cadherin gene were lower in each drug group than that in blank control group,and the combination group showed more remarkable decrease than single drug groups.The relative mRNA expression of E-cadherin in control,5-Aza-dC,TSA and combination groups was (0.17 ± 0.01),(0.32 ± 0.01),(0.29±0.02) and (0.57±-0.02) in MKN-45 cells,(0.22±0.01),(0.46±0.01),(0.43±0.01)and (0.61±0.01) in SGC-7901 cells,(0.24±0.02),(0.49±0.01),(0.44±0.01) and (0.73±0.01) in MGC-803 cells,respectively.There was significant decrease of E-cadherin mRNA level in single drug groups,as compared with blank control group,and E-cadherin mRNA level was more decreased in combination group than that in single-drug groups.Conclusions The 5-Aza-dC and TSA single-drugs treatment can alone reduce the cell survival rate in the three gastric cancer cell lines and E-cadherin gene methylation,and increase the expression of E-cadherin mRNA,and the 5-Aza-dC and TSA combination treatment has a more potent efficacy and synergistic effect.The changes in survival rates and promoter methylation and mRNA levels of E-cadherin gene are more obvious in cell line MKN-45 than in cell lines SGC-7901 and MGC-803.Our results suggest that 5-Aza-dC and TSA have potentials used in the clinical treatment of gastric cancer.