CAJ | 학술논문

目的通过真核表达载体介导人乳头瘤病毒(HPV)16型E2基因在HpG2细胞中的表达,探讨E2蛋白的促凋亡作用是否为非HPV病毒依赖性.方法利用pcDNA3.1V5 His构建HPV16型E2基因的真核表达载体并转染HpG2细胞,逆转录-聚合酶链反应(RT-PCR)和Western b1ot检测转染后E2基因mRNA和蛋白的变化,AnnexinV-FTTC/PI双染后利用流式细胞仪和激光共聚焦显微镜检测转染后细胞的凋亡.结果转染携带HPV16型E2质粒后,HpG2细胞可表达E2蛋白.流式细胞仪检测结果显示,转染后细胞凋亡率(21.25±5.05)%与转染空载体细胞(10.91±2.70)%和未转染细胞(7.35±0.79)%比较差异有统计学意义(P＜0.05).激光共聚焦显微镜结果显示,转染后细胞凋亡明显增多.结论 E2蛋白可以明显诱导HPV感染无关的HepG2细胞凋亡,其凋亡作用不依赖HPV病毒.
목적 통과진핵표체재체개도인유두류병독(HPV)16형E2기인재HpG2세포중적표체,탐토E2단백적촉조망작용시부위비HPV병독의뢰성.방법 이용pcDNA3.1V5 His구건HPV16형E2기인적진핵표체재체병전염HpG2세포,역전록-취합매련반응(RT-PCR)화Western b1ot검측전염후E2기인mRNA화단백적변화,AnnexinV-FTTC/PI쌍염후이용류식세포의화격광공취초현미경검측전염후세포적조망.결과 전염휴대HPV16형E2질립후,HpG2세포가표체E2단백.류식세포의검측결과현시,전염후세포조망솔(21.25±5.05)%여전염공재체세포(10.91±2.70)%화미전염세포(7.35±0.79)%비교차이유통계학의의(P＜0.05).격광공취초현미경결과현시,전염후세포조망명현증다.결론 E2단백가이명현유도HPV감염무관적HepG2세포조망,기조망작용불의뢰HPV병독.
Objective The human papillomavirus (HPV) type 16 early gene 2 which regulates both viral DNA replication and transcription. This study was to investigate whether the HPV16 E2 protein could induce apoptosis in HepG2 cells without other HPV 16 genome products. Methods The sequence of HPV16 E2 was cloned into the eukaryotic expression vector pcDNAV5His. HepG2 cells were transfected with the recombinated plasmid containing E2. Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) were performed to document the changes of mRNA and protein of E2 gene. Flow cytometry and confocal laser scanning microscopy (CLSM) were performed to study the apoptosis of transfected HepG2 cells. Results Flow cytometric analysis of presidium iodide stained showed that the apoptosis rate of HepG2/pcDNA3. 1V5HisE2 was significantly higher than that of HepG2/pcDNA3. 1 V5His and HepG2 cell [(21.25 ±5.05)% vs (10.91 ±2.70)%, (7.35 ±0.79)% ,P ＜0.05]. The result of CLSM displayed the apoptosis cells were increased noticeably after transfection. Conclusion The F2 protein could induce apoptosis of HepG2 cells significantly.