二甲双胍对人食管癌Eca109细胞的增殖与凋亡的影响及其机制  

Effects of metformin on cell proliferation and apoptosis in human esophageal squamous cancer cell line Eca109

作　　者：梁凤[1,2] 王昌成[2] 费素娟[3] 

机构地区：[1]徐州医学院,江苏省徐州市221000 [2]徐州医学院附属淮安医院消化科,江苏省淮安市223002 [3]徐州医学院附属医院消化科,江苏省徐州市221000

出　　处：《世界华人消化杂志》2013年第36期4075-4083,共9页World Chinese Journal of Digestology

摘　　要：目的:研究二甲双胍对人食管鳞状细胞癌Eca109细胞的增殖、凋亡的影响及其机制.方法:倒置相差显微镜观察二甲双胍作用于Eca109细胞后细胞形态学的改变;二甲双胍或二甲双胍联合5-氟尿嘧啶(5-fluorouracil,5-FU)干预细胞24、48、72 h后,MTT检测细胞抑制率;Hoechst33258荧光染色法观察凋亡细胞核的形态学变化;药物干预后,流式细胞仪检测细胞周期的改变,抽提mRNA并以RTPCR(reverse transcription PCR)检测相关基因cyclinD1、p27的转录情况.结果:二甲双胍处理后倒置相差显微镜观察其大体形态和荧光显微镜观察其细胞核均呈现凋亡的形态学变化;MTT结果显示,不同浓度二甲双胍作用于食管癌Eca109细胞后,相关分析结果显示,食管癌Eca109细胞生长抑制率与二甲双胍浓度呈正相关(r=0.968,P<0.05),与作用时间呈正相关(r=0.914,P<0.05);与5-FU联合使用时(联合用药组24 h:t=6.943,P<0.05,48 h:t=7.764,P<0.05,72 h:t=14.554,P<0.05 vs单用二甲双胍组),但根据金正均法判断,三组的q值0.85-1.15,表示两药有单纯相加作用,并无协同作用;流式测细胞周期结果显示,二甲双胍阻滞细胞于G0/G1期细胞;RT-PCR结果显示周期相关基因cyclinD1mRNA显著下调(P<0.05),p27 mRNA显著上调(P<0.05).结论:二甲双胍能促进人食管癌细胞系Eca109的凋亡,与5-FU联合用药时无明显协同作用,阻滞细胞周期于G0/G1期,其机制可能与上调或下调某些细胞周期相关基因的表达有关.AIM： To investigate the effects of metformin on cell proliferation, apoptosis and cell cycle pro- gression in human esophageal cancer cell line Ecal09 in vitro, to explore the possible mecha- nisms, and to observe whether there is a syner- gistic effect between metformin and 5-fluoroura- cil （5-FU） METHODS： MTT assay was used to detect cell inhibition rate after treatment with metfor- min alone or in combination with 5-FU. Mor- phological changes of cells were observed by Hoechest33258 staining. The changes in cell cy- cle progression were examined by flow cytom- etry （FCM）. The expression of p27 and cyclin D1 mRNAs in E^al09 cells was detected by reverse transcription-PCR. RESULTS： Apoptotic features including nuclear pyknosis, chromatin margination and apop- totic bodies were observed in Ecal09 ceils after treatment with metformin by inverted phase contrast microscopy and Hoechest33258 stain- ing. Metformin significantly inhibited the pro- liferation of Ecal09 cells in a dose- （r = 0.968, P 〈 0.05 ） and time-dependent （r = 0.914, P 〈 0.05） manner. Metformin treatment enhanced 5-Fu-mediated cell growth inhibition （24 h： t -- 6.943, P 〈 0.05; 48 h： t = 7.764, P 〈 0.05; 72 h： t = 14.554, P 〈 0.05 vs metformin alone）. However, metformin and 5-FU had no synergistic anti- proliferative effect in esophageal ceils. Flow cytometry analysis showed that metformin in- creased the percentage of cells in G0/G1 phase in a dose-dependent manner. The expression of cyclin D1 mRNA was down-regulated, while the expression of p27 mRNA was up-regulated after metformin treatment. CONCLUSION： Metformin inhibits cell prolif- eration, promotes apoptosis and blocks the cell cycle at G0/G1 phase, which may be attribut- able to down-regulation of cyclin D1 and up- regulation of p27. Metformin and 5-FU have no synergistic anti-proliferative effect in Ecal09 cells.

关 键 词：二甲双胍 5-氟尿嘧啶 ECA109细胞 协同 作用 细胞周期 抑制率 

分 类 号：R735.1[医药卫生—肿瘤]