【作者】 严春光； 张冬梅； 李俊； 陈钧辉；

【Author】 YAN Chun-guang ZHANG Dong-mei LI Jun CHEN Jun-hui ( State Key Laboratory of Pharmaceutical Biotechnology,Department of Biochemistry,Nanfing University,Nanjing Jiangsu 210093,China)

【机构】 南京大学生物化学系医药生物技术国家重点实验室；

【摘要】 目的研究醋酸铜对人宫颈癌细胞(Hela 细胞)存活,生长,周期和凋亡的影响及 Bcl-2,Fas 基因表达的变化,探讨铜离子引起细胞凋亡的分子机制。方法体外培养 Hela 细胞,不同剂量醋酸铜(0～40μg/ml)作用72 h,苔盼蓝拒染法测细胞存活率,噻唑蓝法(MTT)测醋酸铜对细胞生长的抑制作用。不同剂量的醋酸铜(0～60μg/ml)作用36 h,碘丙啶(PI)单染测铜离子对细胞周期的影响；作用24、36和48 h,Annexin V/PI 双染法测细胞凋亡；作用24 h,RT-PCR 法检测与凋亡有关基因 Bcl-2,Fas 表达的变化。结果苔盼蓝拒染法显示,随着浓度醋酸铜的增加,细胞存活率降低；MTT 法显示,醋酸铜对细胞生长有强烈的抑制作用。PI 单染证明铜离子对细胞周期无影响。Annexin V/PI 双染法的结果显示,随着醋酸铜浓度及作用时间的增加,细胞凋亡率显著升高。基因表达分析显示 Bcl-2表达量随铜离子增加而降低,而 Fas 与 Bcl-2相反。结论醋酸铜降低 Hela 细胞的存活率,抑制 Hela 细胞的生长,这主要是由细胞凋亡引起的。凋亡的发生可能与 Bcl-2 mRNA 水平的下降及 Fas mRNA 水平的升高有关。更多还原

【Abstract】 Objective To investigate the effect of copper acetate[Cu（AC）₂ ] on cell viability,growth,cell cycle and apoptosis in Hela cells,and to evaluate the pathway associated with the regulation of Bcl-2 and Fas.Methods After Hela cells were treated with Cu（AC）₂ at different concentrations（0～40μg/ml）for 72h,cell viability and cytotoxic potential of the copper（Ⅱ）was determined respectively by the trypan blue dye exclusion test and MTT assay.PI single staining method was performed to examine the influence of the substance on cell cycle.In addition,the Annexin V-FITC Apoptosis Detection Kit I was applied to detect apoptosis by flow cytometry after Hela ceils were exposed to various concentrations of Cu（AC）₂（0～60μg/ml）for 24,36 and 48h.Moreover,the levels of Bcl-2 and Fas mRNA expression were determined by RT-PCR method,when Hela cells were treated with a variety of concentrations of Cu （AC）₂（0～60μg/ml）（24h exposure）.Results Both dye exclusion test and MTT assay separately demonstrated that cell viability was decreased and cell growth was intensely inhibited by Cu（AC）₂ in a dose-dependent manner.PI single staining showed that the substance had no pronounced influence on cell cycle.However,the results of Annexin V/PI assay indicated that the apoptosis rates of Hela cells increased in a time- and dose-dependent manner.Additionally,the determination of the levels of Bcl-2 mRNA expression by RT-PCR specified that treatment with Cu（Ⅱ）down regulated Bcl-2 mRNA expression in a concentration-dependent fashion,which was in contrast with Fas mRNA expression.Conclusion Cell viability and growth was influenced by Cu（AC）₂,which was triggered by apoptosis. Moreover,the apoptosis was pertinent to decreased expression of Bcl-2 gene and increased expression of Fas genes.更多还原