藻红蛋白对HeLa细胞生长和凋亡的影响

Effect of phycoerythrin on growth and apoptosis of HeLa cells

目的探讨藻红蛋白(PE)对HeLa细胞生长和凋亡的影响及其机制。方法从龙须菜中获得高纯度PE,用PE 0、5、10、20、40 mg/L处理宫颈癌HeLa细胞,采用MTT法检测细胞生长,Hoechst 33342染色检测细胞核凋亡,流式细胞术测定细胞凋亡,细胞膜电位探针JC-1染色检测线粒体膜电位,实时荧光定量PCR和Western blot法检测细胞凋亡相关基因和蛋白表达情况。结果与PE 0 mg/L比较,PE 10、20、40 mg/L呈浓度依赖性地抑制HeLa细胞生长,增加晚期凋亡率和总凋亡率,上调Caspase-3、Caspase-9和Bax mRNA表达和剪切型Caspase-3蛋白表达(P<0.05)。与PE 0 mg/L比较,PE 20、40 mg/L处理24 h后细胞核凋亡率升高,PE 10、20和40 mg/L处理48 h后细胞核凋亡率升高,且48 h比24 h更明显(P<0.05)。与PE 0、10 mg/L比较,PE 20、40 mg/L处理后HeLa细胞线粒体膜电位降低,Bcl-2 mRNA和蛋白表达下调,CytC蛋白表达上调,且PE 40 mg/L较PE 20 mg/L更明显(P<0.05)。结论PE能呈浓度依赖性地抑制HeLa细胞生长,诱导细胞凋亡,其机制可能与降低细胞线粒体膜电位和调控线粒体凋亡通路相关蛋白的表达有关。

Objective To investigate the effect of phycoerythrin(PE)on the growth and apoptosis of HeLa cells and underlying mechanism.Methods High purity PE was obtained from asparagus.Cervical cancer HeLa cells were treated with PE in different concentrations of 0,5,10,20 and 40 mg/L.The cell growth was detected by MTT assay,cell nucleus apoptosis was detected by Hoechst 33342 staining,and cell apoptosis was detected by flow cytometry.The mitochondrial membrane potential was detected by cell membrane potential probe JC-1 staining.The apoptosis-related genes and proteins were detected by real-time fluorescence quantitative PCR and Western blot.Results Compared with PE 0 mg/L,PE 10,20 and 40 mg/L inhibited HeLa cell growth,increased late apoptosis rate and total apoptosis rate,increased mRNA expressions of Caspase-3,Caspase-9 and Bax,and cleaved Caspase-3 protein expression in a dose-dependent manner(P<0.05).Compared with PE 0 mg/L,PE 20 and 40 mg/L increased cell nuclear apoptosis rate after treatment for 24 hours,and PE 10,20 and 40 mg/L increased cell nuclear apoptosis rate after treatment for 48 hours,which was more obvious at 48 hours than 24 hours(P<0.05).Compared with PE 0 and 10 mg/L,PE 20 and 40 mg/L decreased mitochondrial membrane potential of HeLa cells,downregulated Bcl-2 mRNA and protein expressions,and upregulated CytC protein expression,which were more obvious after treated with PE 40 mg/L than those treated with PE 20 mg/L(P<0.05).Conclusion Different concentrations of PE can inhibit the growth of HeLa cells and induce cell apoptosis,which may be related to the reduction of mitochondrial membrane potential and the regulation of protein expressions associated with mitochondrial apoptosis pathway.