中国药理学通报
中國藥理學通報
중국약이학통보
CHINESE PHARMACOLOGICAL BULLETIN
2014年
7期
973-978,979
,共7页
项梦琦%刘丽璇%邓巍%周小涛%陈沛锐%郭益添%叶艳清%蒲泽锦%吴灵飞
項夢琦%劉麗璇%鄧巍%週小濤%陳沛銳%郭益添%葉豔清%蒲澤錦%吳靈飛
항몽기%류려선%산외%주소도%진패예%곽익첨%협염청%포택금%오령비
腺苷%同型半胱氨酸%5-氮杂-2′-脱氧胞苷%甲基化%细胞凋亡%DNA甲基化酶%线粒体膜电位
腺苷%同型半胱氨痠%5-氮雜-2′-脫氧胞苷%甲基化%細胞凋亡%DNA甲基化酶%線粒體膜電位
선감%동형반광안산%5-담잡-2′-탈양포감%갑기화%세포조망%DNA갑기화매%선립체막전위
adenosine%homocysteine%5-Aza-CdR%methylation%apoptosis%DNA methyltransferase%mito-chondrial membrane potentials
目的：探索去甲基化机制在腺苷（ADO）及同型半胱氨酸（HCY）诱导肝癌 HepG2细胞凋亡中的作用。方法CCK8法检测不同浓度腺苷（1.0、2.0、4.0 mol·L－1）单独或者联合同型半胱氨酸作用肝癌HepG2细胞不同时间（6、12、24 h）后细胞存活率；亦使用DNA甲基化酶抑制剂5-Aza-CdR观察其对细胞生长的影响。AnnexinV-FITC／PI 双染法检测细胞凋亡率，流式细胞术检测细胞内线粒体膜电位：实时荧光定量PCR及Western免疫印迹法分别检测caspase-3、caspase-8、caspase-9、MDM-2、p53、Cytochrome C、DNMT1、DN-MT3a、DNMT3b等因子的表达量。结果 ADO联合HCY明显抑制 HepG2细胞增殖，呈剂量和时间依赖性；不同浓度ADO联合HCY（1.0、2.0、4.0 mol·L－1）作用HepG2细胞24 h，细胞凋亡率分别为（18.63±1.25）％，（29.42±2.37）％，（42.47±3.09）％，均明显高于阴性对照组（1.30±0.82）％， P＜0.01；联合用药组线粒体膜电位明显下降，分别从空白对照组（674.15±82.8）％下降为（428.38±54.5）％、（297.78±30.5）％、（74.45±5.73）％，P＜0.01；ADO联合HCY导致caspase-3、caspase-8、caspase-9、p53、Cytochrome C 表达上调，MDM-2表达下调。ADO 联合 HCY 或5-Aza-CdR 处理HepG2细胞均可抑制DNA（甲基化酶DNMT1、DNMT3a、DN-MT3b）mRNA表达量。结论 ADO与HCY联合作用引起了细胞内甲基化改变，低甲基化作用激活了p53及线粒体等凋亡通路，最终导致肝癌HepG2细胞凋亡。
目的：探索去甲基化機製在腺苷（ADO）及同型半胱氨痠（HCY）誘導肝癌 HepG2細胞凋亡中的作用。方法CCK8法檢測不同濃度腺苷（1.0、2.0、4.0 mol·L－1）單獨或者聯閤同型半胱氨痠作用肝癌HepG2細胞不同時間（6、12、24 h）後細胞存活率；亦使用DNA甲基化酶抑製劑5-Aza-CdR觀察其對細胞生長的影響。AnnexinV-FITC／PI 雙染法檢測細胞凋亡率，流式細胞術檢測細胞內線粒體膜電位：實時熒光定量PCR及Western免疫印跡法分彆檢測caspase-3、caspase-8、caspase-9、MDM-2、p53、Cytochrome C、DNMT1、DN-MT3a、DNMT3b等因子的錶達量。結果 ADO聯閤HCY明顯抑製 HepG2細胞增殖，呈劑量和時間依賴性；不同濃度ADO聯閤HCY（1.0、2.0、4.0 mol·L－1）作用HepG2細胞24 h，細胞凋亡率分彆為（18.63±1.25）％，（29.42±2.37）％，（42.47±3.09）％，均明顯高于陰性對照組（1.30±0.82）％， P＜0.01；聯閤用藥組線粒體膜電位明顯下降，分彆從空白對照組（674.15±82.8）％下降為（428.38±54.5）％、（297.78±30.5）％、（74.45±5.73）％，P＜0.01；ADO聯閤HCY導緻caspase-3、caspase-8、caspase-9、p53、Cytochrome C 錶達上調，MDM-2錶達下調。ADO 聯閤 HCY 或5-Aza-CdR 處理HepG2細胞均可抑製DNA（甲基化酶DNMT1、DNMT3a、DN-MT3b）mRNA錶達量。結論 ADO與HCY聯閤作用引起瞭細胞內甲基化改變，低甲基化作用激活瞭p53及線粒體等凋亡通路，最終導緻肝癌HepG2細胞凋亡。
목적：탐색거갑기화궤제재선감（ADO）급동형반광안산（HCY）유도간암 HepG2세포조망중적작용。방법CCK8법검측불동농도선감（1.0、2.0、4.0 mol·L－1）단독혹자연합동형반광안산작용간암HepG2세포불동시간（6、12、24 h）후세포존활솔；역사용DNA갑기화매억제제5-Aza-CdR관찰기대세포생장적영향。AnnexinV-FITC／PI 쌍염법검측세포조망솔，류식세포술검측세포내선립체막전위：실시형광정량PCR급Western면역인적법분별검측caspase-3、caspase-8、caspase-9、MDM-2、p53、Cytochrome C、DNMT1、DN-MT3a、DNMT3b등인자적표체량。결과 ADO연합HCY명현억제 HepG2세포증식，정제량화시간의뢰성；불동농도ADO연합HCY（1.0、2.0、4.0 mol·L－1）작용HepG2세포24 h，세포조망솔분별위（18.63±1.25）％，（29.42±2.37）％，（42.47±3.09）％，균명현고우음성대조조（1.30±0.82）％， P＜0.01；연합용약조선립체막전위명현하강，분별종공백대조조（674.15±82.8）％하강위（428.38±54.5）％、（297.78±30.5）％、（74.45±5.73）％，P＜0.01；ADO연합HCY도치caspase-3、caspase-8、caspase-9、p53、Cytochrome C 표체상조，MDM-2표체하조。ADO 연합 HCY 혹5-Aza-CdR 처리HepG2세포균가억제DNA（갑기화매DNMT1、DNMT3a、DN-MT3b）mRNA표체량。결론 ADO여HCY연합작용인기료세포내갑기화개변，저갑기화작용격활료p53급선립체등조망통로，최종도치간암HepG2세포조망。
Aim To investigate the mechanism of demethylation on adenosine (ADO )and homocysteine (HCY)-induced apoptosis in human hepatoma HepG2 cells .Methods HepG 2 cells were treated with differ-ent concentrations of ADO (1.0、2.0、4.0 mol · L-1 ) alone or in combination with HCY for 6h,12h and 24h,5-aza-2-deoxycytidine (5-Aza-CdR)as a positive control.Cell viabilities were assessed by CCK8 assay. Cell apoptosis was observed by AnnexinV-FITC/PI staining.The mitochondrial membrane potentials(ΔΨ) were measured by flow cytometry.The mRNA and pro-tein expressions of caspase-3,caspase-8,caspase-9, MDM-2,p53,Cytochrome C,DNMT1,DNMT3a,DN-MT3 b were detected by RT-qPCR and Western blot re-spectively.Results ADO alone or in combination with HCY significantly reduced the viability of HepG2 cells in a dose and time-dependent manner.The apoptotic rates of HepG2 cells after combination treatment with ADO and HCY at 1 .0,2.0,4.0 mol · L-1 for 24 h were (1 8.63 ± 1.25 )%,(29.42 ±2.37 )% and (42.47 ±3.09 )%,compared with the control group (1.30 ±0.82 )%,P <0.01;and the mitochondrial membrane potentials were decreased from 674.15 ± 82.8%(black control group)to (428.38 ±54.5)%, (297.78 ±30.5)%,(74.45 ±5.73)%,P<0.01, respectively.The expressions of caspase-3,caspase-8, caspase-9,MDM-2,p53,Cytochrome C were up-regula-ted and MDM-2 were down-regulated after combination treatment of ADO and HCY.The mRNA expressions of DNMT1 ,DNMT3 a and DNMT3 b were down-regulated after combination treatment with ADO and HCY or 5-Aza-CdR alone.Conclusion Combination treatment of ADO and HCY can cause cellular methylation chan-ges.The effects of demethylation of ADO and HCY may activate p53 gene and mitochondrial pathway, which at last leads to HepG2 cell apoptosis.
