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青蒿素诱导胆囊癌细胞的细胞周期阻滞和抑制细胞凋亡的体外实验研究

Artemisinin inhibits proliferation of gallbladder cancer cell lines through triggering cell cycle arrest and apoptosis

摘要:

目的 探讨青蒿素对胆囊癌细胞增殖、细胞周期和细胞凋亡的影响.方法 体外培养胆囊癌细胞株(GBC-SD和NOZ).采用MTT法检测不同浓度青蒿素对两株细胞增殖的影响;通过流式细胞仪检测20 μmol/L青蒿素处理24h对细胞周期的影响;通过FITC-Annexin V双染法检测20 μmol/L青蒿素处理24 h对细胞凋亡的影响;通过免疫印迹法检测20 μmol/L青蒿素处理24 h后p-ERK1/2、CDK4、cyclin D1、p16、cytochrome C、caspase-3在细胞内的表达水平变化.两组间比较采用t检验,两组以上比较采用单因素方差分析.结果 WST-1实验结果显示,青蒿素可以抑制GBC-SD和NOZ细胞增殖,且该抑制作用具有浓度依赖性,青蒿素对GBC-SD和NOZ细胞的半数抑制浓度分别为14.05 μmol/L和12.42 μmol/L.细胞周期分析结果显示,GBC-SD细胞[(74.60±2.24)%]和NOZ细胞[(78.86±2.02)%]G1期比例高于对照组[(54.26±1.61)%、(58.44±1.83)%](F=0.658,P=0.000;F =0.055,P=0.000).免疫印迹法检测结果显示,青蒿素作用24 h后,细胞周期调控相关蛋白CDK4和cyclin D1蛋白表达水平显著降低,而p16蛋白表达水平显著增高(P值均<0.05).Annexin V/PI双染凋亡检测结果显示,GBC-SD细胞[(15.67±2.40)%]和NOZ细胞[(16.51±2.20)%]的凋亡比例高于对照组[(6.28±1.18)%、(6.94±1.23)%](F=1.110,P=0.004;F=1.273,P=0.003).免疫印迹法检测结果显示,20 μmol/L青蒿素处理24h后,细胞色素C在线粒体中的含量显著降低,而在胞质中的含量显著增高(P值均<0.05).结论 青蒿素可能通过抑制ERK1/2信号转导通路干扰细胞增殖,通过抑制CDK4和cyclin D1,诱导p16表达造成G1期阻滞,通过激活线粒体途径介导细胞凋亡.

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abstracts:

Objective To evaluate the effects of artemisinin on proliferation,cell cycle and apoptosis of gallbladder cancer cells.Methods Gallbladder carcinoma cell lines (GBC-SD and NOZ) were cultured in vitro.The effects of artemisinin in different concentration on proliferation of the two cell lines in vitro were examined using MTT assay.The cell cycle distribution of GBC-SD and NOZ cells 24 h after treatments with artemisinin(20 μ mol/L) were examined using flow cytometry.The apoptosis of GBC-SD and NOZ cells 24 h after treatments with artemisinin (20 μ mol/L) were examined using Annexin V/PI staining.The expressions of p-ERK1/2,CDK4,cyclin D1,p16,cytochrome C and caspase-3 were examined by Western blot assay.t-test and one way ANOVA were used to evaluate the differences between two groups and more than two groups,respectively.Results The cell proliferation was significantly inhibited by artemisinin,the ICS0 of artemisinin against GBC-SD and NOZ cells were 14.05 μmol/L and 12.42 μmol/L,respectively.Artemisinin induced cycle arrest,and G1 population of GBC-SD and NOZ cells increased to 74.60% and 78.86%.Cell apoptosis and apoptotic population of GBC-SD and NOZ cells were increased to 15.67% and 16.51% after dealt with artemisinin,respectively.In addition,expression of pl6 was increased,and expressions of p-ERK1/2,CDK4 and cyclin D1 were down-regulated by artemisinin(all P <0.05).Cytochrome C was released from mitochondria to cytoplasm leading to the activation of caspase-3 and PARP after dealt with artemisinin (P < 0.05).Conclusion The inhibition effect of artemisinin on the proliferation gallbladder cancer cells is accompanied by down-regulation of ERK1/2 signaling pathway,G1 phase arrest and triggering caspase-3-mediate apoptosis.

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作者: 贾建光 [1] 张立功 [1] 郭晨旭 [1] 王亚国 [1] 陈邦领 [1] 王益民 [1] 钱军 [1]
第一作者: 贾建光
期刊: 《中华外科杂志》2016年54卷3期 222-227页 MEDLINEISTICPKUCSCD
栏目名称: 论著
DOI: 10.3760/cma.j.issn.0529-5815.2016.03.014
发布时间: 2016-03-31
基金项目:
安徽省高校省级青年人才基金重点项目(2013SQRL052ZD) Key Program of Foundation for Youth Talents in Colleges and Universities of Anhui Province
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