CAJ | 학술논문

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-HPV 16 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-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 HPV16 E6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,bDaxx高表达可下调HPV16 E6蛋白这种作用.
목적 탐토외원인유두류병독16형E6단백(HPV16 E6)여인Daxx단백(hDaxx)재HeLa세포내적아세포정위급유도HeLa세포조망적영향.방법 Western인적법검측융합단백DsRed-HPV 16 E6화EGFP-hDaxx적표체.격광공취초현미경관찰HPV16 E6화hDaxx적아세포정위.장HeLa세포분위대조조、TNF-α처리조、전염공재체조、전염HPV16 E6조、공전염HPV16 E6화hDaxx조.후4조균경TNF-α유도.용류식세포의측정세포조망솔,분광광도법검측Caspase-8여Caspase-3적상대활성.결과 융합단백DsRed-HPV16E6화EGFP-hDaxx재세포내표체병분포우포질여포핵,출현공정위현상,차부분hDaxx종포핵전이지포질.전염E6조조망솔(21.4％±1.1％)저우전염공재체조(27.0％±0.9％,P＜ 0.01)；여전염E6조상비교,공전염조조망솔(32.5％±2.1％)현저승고(P＜0.01).전염E6조Caspase-8화Caspase-3상대활성분별위0.057±0.003、0.054±0.006,균저우공재체조(0.092±0.012、0.093±0.005,균P＜0.01)；여전염E6조상비교,공전염조Caspase-8화Caspase-3상대활성(0.109±0.013、0.110±0.004)균현저승고(P치균＜ 0.01).결론 HPV16 E6사부분hDaxx종포핵전위지포질,이자발생공정위.HPV16E6단백가억제TNF-α유도적HeLa세포조망,bDaxx고표체가하조HPV16 E6단백저충작용.
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-CI/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-ot 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 pDsRedmonomer-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/HPVl6 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.