Apollon反义核酸促进肝癌HepG2细胞凋亡机制的探讨

doi:10.3969/j.issn.1673-8640.2013.09.027

检验医学 ›› 2013, Vol. 28 ›› Issue (9): 845-850.DOI: 10.3969/j.issn.1673-8640.2013.09.027

Apollon反义核酸促进肝癌HepG₂细胞凋亡机制的探讨

黎毓光,何金花,王莉,谢杏仪,陈顺仪,陈武嘉,黄国贤

广州市番禺区中心医院检验科,广东广州511400

收稿日期:2013-09-25 修回日期:2013-09-25 出版日期:2013-09-15 发布日期:2013-09-25
通讯作者: 何金花,联系电话:020-34858850。
作者简介:黎毓光,男,1970年生,学士,主任技师,主要从事医学分子生物学研究。
基金资助:
广州市番禺区科技计划局科技计划项目(2011-Z-03-24)

Investigation on the apoptosis mechanisms of liver cancer HepG₂ cells induced by Apollon antisense oligodeoxynucleotide

LI Yuguang,HE Jinhua, WANG Li, XIE Xingyi,CHEN Shunyi, CHEN Wujia,HUANG Guoxian.

Department of Clinical Laboratory,Panyu Central Hospital,Guangdong Guangzhou 511400,China

Received:2013-09-25 Revised:2013-09-25 Online:2013-09-15 Published:2013-09-25

摘要/Abstract

摘要：

目的研究凋亡抑制蛋白(IAP)家族成员Apollon 反义核酸(ASO)对人肝癌HepG₂细胞增殖、凋亡的影响,并探讨其机制。方法将化学合成的Apollon ASO经脂质体包裹后作用于肝癌HepG₂细胞48 h,采用WST-8法检测不同浓度的Apollon ASO对肝癌细胞增殖抑制作用,实时荧光定量逆转录聚合酶链反应(PCR)检测细胞Apollon mRNA 的表达水平; 流式细胞术检测细胞早期凋亡率; Hochest33258染色观察HepG₂细胞凋亡形态;线粒体膜电位检测细胞凋亡;比色法测定半胱氨酸蛋白酶(Caspase-3、Caspase-9)活性的改变;蛋白质免疫印记技术检测细胞色素C蛋白的表达水平。结果 Apollon ASO转染HepG₂细胞48 h能明显抑制细胞增殖,并呈浓度依赖关系。Apollon ASO 能促进HepG₂细胞凋亡。不同浓度的Apollon ASO能显著下调Apollon mRNA的表达水平,同时Caspase-3、Caspase-9的活性明显增加,细胞色素C蛋白表达水平也增加。经荧光显微镜观察,ASO 组可见核高强度荧光的细胞,并见凋亡小体。结论 Apollon ASO可下调 Apollon mRNA表达水平,抑制HepG₂细胞增殖,其诱导细胞早期凋亡可能通过线粒体介导的途径。

关键词: 反义核酸, Apollon, 增殖, 凋亡, HepG₂细胞, 肝癌

Abstract:

Objective To study the influence of inhibitor apoptosis protein (IAP) Apollon antisense oligodeoxynucleotide (ASO) on the proliferation and apoptosis on human liver cancer HepG₂ cells, and investigate the mechanisms. Methods Apollon ASO was incubated by liposome with human liver cancer HepG₂ cell for 48 h, and the proliferation inhibition was detected by WST-8. The expression of Apollon mRNA was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (PCR). The early apoptosis rates were determined by flow cytometry. The morphology of HepG₂ cell apoptosis was determined by Hochest 33258 staining. The cell apoptosis was determined by mitochondrial membrane potential. The activities of cysteine proteinases (Caspase-3 and Caspase-9) were determined by colorimetric method. The expression of cytochrome C was analyzed by Western blot. Results After being treated by Apollon ASO on HepG₂ cells for 48 h, it was found that Apollon ASO could all suppress the proliferation of HepG₂ cells, which was depended on the concentration. Apollon ASO could induce HepG₂ cell apoptosis. The different concentrations of Apollon ASO reduced significantly the expression level of Apollon mRNA. The activities of Caspase-3 and Caspase-9 and the protein expression levels of cytochrome C increased. Through observing by fluorescence microscopy assay, nucelus high fluorescence cells were found, and the apoptosis cytoryctes were also observed. Conclusions Apollon ASO can reduce the expression level of Apollon mRNA and inhibit the proliferation of liver cancer HepG₂ cells, and it can induce the apoptosis by the way of mitochondria mediation.

Key words: Antisense oligodeoxynucleotide, Apollon, Proliferation, Apoptosis, HepG₂ cell, Liver cancer

中图分类号:

R446.62

黎毓光,何金花,王莉,谢杏仪,陈顺仪,陈武嘉,黄国贤. Apollon反义核酸促进肝癌HepG₂细胞凋亡机制的探讨[J]. 检验医学, 2013, 28(9): 845-850.

LI Yuguang,HE Jinhua, WANG Li, XIE Xingyi,CHEN Shunyi, CHEN Wujia,HUANG Guoxian.. Investigation on the apoptosis mechanisms of liver cancer HepG₂ cells induced by Apollon antisense oligodeoxynucleotide[J]. , 2013, 28(9): 845-850.

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Apollon反义核酸促进肝癌HepG₂细胞凋亡机制的探讨

Investigation on the apoptosis mechanisms of liver cancer HepG₂ cells induced by Apollon antisense oligodeoxynucleotide

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