人乳头瘤病毒16型E7过表达细胞的鉴定及其迁移效应的影响

Identification of Transfected Cells with Early Protein 7(E7)of Human Papilloma Virus Type 16 and Impact on Cell Migration

鉴定人乳头瘤病毒16型早期蛋白7(HPV16E7)过表达细胞及其迁移效应的影响,为后续基于HPV16E7靶向分子作用机制的研究奠定工作基础。脂质体转染法将本室保存的pcDNA3.1⁃HPV16E7重组真核表达质粒分别转染人胚肾293T细胞(HPV16型DNA阴性)、宫颈癌SiHa细胞株(HPV16 DNA阳性),转染48 h后收集细胞,提取RNA,RT⁃PCR扩增相应的目的基因,Western Blotting和间接免疫荧光实验检测HPV16E7目的蛋白在细胞中的表达。转染24 h的细胞进行细胞划痕和Transwell实验,检测过表达细胞迁移行为的变化。RT⁃PCR结果显示:分别从E7质粒转染的293T、SiHa细胞的cDNA中,均可扩增到250 bp的目的条带;Western Blotting分析结果显示:以HPV16E7单克隆抗体为检测抗体,转染细胞的裂解液中均能在相对分子质量(Mr)约为15000处出现特异性目的条带;间接免疫荧光结果显示:转染细胞中均能检测到目的绿色荧光,且分布于胞浆及细胞核周围;细胞划痕和Transwell实验结果显示:转染E7细胞的迁移效应显著提高。本研究证实了HPV16E7转染细胞后可成功表达,且过表达细胞明显促进了细胞迁移行为,为后续基于HPV16E7迁移相关分子机制及靶向干预等研究奠定了前期工作基础。

We wished to identify transfected cells with early protein 7(E7)of human papilloma virus type 16(HPV16 E7)and the impact on cell migration to lay a foundation for subsequent mechanistic research of cervical cancer targeting HPV16 E7. Recombinant plasmid pcDNA3.1-HPV16 E7 was transfected into 293 T cells(HPV16 DNA-negative)or SiHa cells(HPV16 DNA-positive),respectively. After 48 hours of culture,cells were collected to extract RNA. Reverse transcription-polymerase chain reaction(RT-PCR)was undertaken to amplify target genes. Western Blotting and indirect immunofluorescence analyses were carried out to measure expression of target protein HPV16 E7 in cells. After 24 hours of serum-free culture,wound healing test and Transwell^(TM) assays were carried out to detect cell migration. RT-PCR using the cDNA of pcDNA3.1-HPV16 E7 plasmid-transfected 293 T,or-transfected SiHa cells as the template,respectively,could produce a specific band of 250 bp. Western blotting revealed a specific band of relative Mr 15,000 in both types of transfected cells using HPV16 E7 monoclonal antibody as the primary antibody,and E7-specific green fluorescence was also observed in the cytoplasm and around the nucleus in transfected cells according to the indirect-immunofluorescence test. Tests for wound healing of cells and Transwell assays showed that migration of transfected cells was improved significantly. Our study showed that HPV16 E7 could be transfected into cell lines. Further studies on targeted therapy and related molecular mechanism based on HPV16 E7 are underway.