HPV16 E6蛋白与hDaxx在HeLa细胞的定位及对凋亡的影响  

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

作　　者：陈苏芳[1] 朱翠明[1] 唐双阳[1] 余敏君[1] 粟盛梅[1] 阳帆[1] 万艳平[1] 

机构地区：[1]南华大学病原生物学研究所,湖南衡阳421001

出　　处：《中华皮肤科杂志》2012年第6期400-403,共4页Chinese Journal of Dermatology

基　　金：基金项目：国家自然科学基金（30973402）;湖南省高校创新团队经费资助（湘教通2010-212）

摘　　要：目的探讨外源人乳头瘤病毒16型E6蛋白（HPV16E6）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—HPV16E6和EGFP—hDaxx的表达。激光共聚焦显微镜观察HPV16E6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HPV16E6组、共转染HPV16E6和hDaxx组。后4组均经TNFTNF-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—HPV16E6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HPV16E6使部分hDaxx从胞核转位至胞质，二者发生共定位。HPV16E6蛋白可抑制TNF-α诱导的HeLa细胞凋亡，hDaxx高表达可下调HPV16E6蛋白这种作用。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 transfeeted 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 transfeeted ceils, and laser scanning eonfoeal microscopy to observe the subcellular distribution of HPV16 E6 protein and hDaxx. Some HeLa cells were divided into 5 groups： untransfeeted （control group）, untransfeeted and treated with TNF-α （TNF-α group）, transfeeted with peDNA3.1 （-） and treated with TNF-α （empty vector group）, transfeeted with peDNA3.1 （-）/HPV16 E6 and treated with TNF-α （HPV16 E6 group）, cotransfeeted with peDNA3.1 （-）/ HPV16 E6 and peDNA3.1 （-）/hDaxx and treated with TNF-α （eotransfected group）. After additional culture, the cells were collected and subjected to flow eytometry （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 transfeeted with pDsRedmonomer-C1/HPVl6 E6 or pEGFP-C1/hDaxx alone, the red fluorescence of HPVI6 E6 was observed in the nucleus and cytoplasm, while the green fluorescence of hDaxx only in the nucleus; in those eotransfeeted 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% TNF-α 1.1% vs. 27.0% TNF-α 0.9% and 32.5% ±2.1%, 0.057 TNF-α 0.003 vs. 0.

关 键 词：人乳头瘤病毒16 乳状瘤病毒E6蛋白质类 细胞凋亡 HELA细胞 

分 类 号：R737.33[医药卫生—肿瘤]