中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (6): 400-403.

• 论著 • 上一篇    下一篇

HPV16 E6蛋白与hDaxx在HeLa细胞的定位及对凋亡的影响

陈苏芳1,朱翠明1,唐双阳1,余敏君2,粟盛敏1,阳帆1,万艳平1   

  1. 1. 南华大学病原生物学研究所
    2. 衡阳南华大学医学院微生物学教研室
  • 收稿日期:2011-07-11 修回日期:2011-08-09 出版日期:2012-06-15 发布日期:2012-05-31
  • 通讯作者: 万艳平 E-mail:wanyy1991@yahoo.com.cn
  • 基金资助:

    国家自然科学基金资助项目

Localization of human papillomavirus type 16 E6 protein and hDaxx in a human cervical carcinoma cell line HeLa and their effects on cell apoptosis

  • Received:2011-07-11 Revised:2011-08-09 Online:2012-06-15 Published:2012-05-31

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摘要：

【摘要】 目的 探讨外源人乳头瘤病毒16型E6蛋白（HPV16 E6）与人Daxx蛋白（hDaxx）在HeLa细胞内的亚细胞定位及诱导HeLa细胞凋亡的影响。方法 Western印迹法检测融合蛋白DsRed-HPV16 E6和EGFP-hDaxx的表达。激光共聚焦显微镜观察HPV16 E6和hDaxx的亚细胞定位。将HeLa细胞分为对照组、TNF-α处理组、转染空载体组、转染HPV16 E6组、共转染HPV16 E6和hDaxx组。后4组均经TNF-α诱导。用流式细胞仪测定细胞凋亡率，分光光度法检测Caspase-8与Caspase-3的相对活性。结果 融合蛋白DsRed-HPV16 E6 和EGFP-hDaxx在细胞内表达并分布于胞质与胞核，出现共定位现象，且部分hDaxx从胞核转移至胞质。转染E6组凋亡率（21.4% ± 1.1%）低于转染空载体组（27.0% ± 0.9%，P < 0.01）；与转染E6组相比较，共转染组凋亡率（32.5% ± 2.1%）显著升高（P < 0.01）。转染E6组Caspase-8和Caspase-3相对活性分别为0.057 ± 0.003、0.054 ± 0.006，均低于空载体组（0.092 ± 0.012、0.093 ± 0.005，均P < 0.01）；与转染E6组相比较，共转染组Caspase-8和Caspase-3相对活性（0.109 ± 0.013、0.110 ± 0.004）均显著升高（P值均 < 0.01）。结论 HPV16 E6使部分hDaxx从胞核转位至胞质，二者发生共定位。HPV16 E6蛋白可抑制TNF-α诱导的HeLa细胞凋亡，hDaxx高表达可下调HPV16 E6蛋白这种作用。

关键词: HeLa细胞

Abstract:

【Abstract】 Objective To determine the subcellular localization of exogenous human papillomavirus type 16 E6 protein （HPV16 E6） and hDaxx in HeLa cells and their effects on tumor necrosis factor （TNF）-α- induced apoptosis. Methods HeLa cells were transfected with plasmids pDsRed-monomer-C1/HPV16 E6, pEGFP-C1/hDaxx, pEGFP-C1 and pDsRed-monomer-C1 respectively. Subsequently, Western blot was carried out to quantify the expression of fusion proteins DsRed-HPV16E6 and EGFP-hDaxx in transfected cells, and laser scanning confocal microscopy to observe the subcellular distribution of HPV16 E6 protein and hDaxx. Some HeLa cells were divided into 5 groups: untransfected （control group）, untransfected and treated with TNF-α （TNF-α group）, transfected with pcDNA3.1（-） and treated with TNF-α （empty vector group）, transfected with pcDNA3.1（-）/HPV16 E6 and treated with TNF-α （HPV16 E6 group）, cotransfected with pcDNA3.1（-）/HPV16 E6 and pcDNA3.1（-）/hDaxx and treated with TNF-α （cotransfected group）. After additional culture, the cells were collected and subjected to flow cytometry （FCM） to evaluate the apoptosis of cells as well as spectrophotometry to determine the relative activity of Caspase-8 and Caspase-3. Results Western blot showed that both DsRed-HPV16 E6 and EGFP-hDaxx were expressed in HeLa cells. In Hela cells transfected with pDsRed-monomer-C1/HPV16 E6 or pEGFP-C1/hDaxx alone, the red fluorescence of HPV16 E6 was observed in the nucleus and cytoplasm, while the green fluorescence of hDaxx only in the nucleus; in those cotransfected with pDsRed-monomer-C1/HPV16 E6, HPV16 E6 and hDaxx proteins were regionally aggregated near the nuclear membrane in nuclei, and hDaxx was partly translocated from the nucleus to the cytoplasm. The apoptosis rate and relative activity of Caspase-8 and Caspase-3 were statistically lower in HPV16 E6 group than in the empty vector group and cotransfected group （21.4% ± 1.1% vs. 27.0% ± 0.9% and 32.5% ± 2.1%, 0.057 ± 0.003 vs. 0.092 ± 0.012 and 0.109 ± 0.013, 0.054 ± 0.006 vs. 0.093 ± 0.005 and 0.110 ± 0.004, all P < 0.01）. Conclusions HPV16 E6 protein induces the partial translocation of hDaxx from the nucleus into the cytoplasm and colocalizes with hDaxx in the cells. The apoptosis of HeLa cells induced by TNF-α can be suppressed by HPV16 E6 protein, while the overexpression of hDaxx can attenuate the suppressing effect of HPV16 E6 protein on apoptosis in Hela cells.

Key words: HeLa cells

中图分类号: 

  • R737.33