人乳头状瘤病毒16型E6E7肿瘤性转化人永生化支气管上皮细胞

HPV16 E6E7 fragments transform immortalized human bronchial epithelial cells into neoplastic cells

目的 探讨人乳头状瘤病毒 (HPV) 16型E6E7片段对人永生化支气管上皮细胞系TR细胞的作用。方法 将E6E7片段构建入逆转录病毒载体 ,导入TR细胞 ,观察生长特性和致瘤性的改变 ;并用免疫沉淀 (IP) Westernblot检测p2 7蛋白功能及FAK、桩蛋白数量及磷酸化状况。结果 嘌呤霉素抗药性克隆TR/E6E7有E6E7的存在和稳定表达 ;TR/E6E7细胞系细胞生长加快 ,软琼脂集落形成能力增强 ,并具有裸鼠致瘤性 ;TR/E6E7细胞中与cyclinE/CDK结合的p2 7数量减少 ,即cyclinE/CDK活性增强。结论 转染E6E7的TR细胞可在裸鼠体内产生肿瘤 ,说明HPV感染在肺癌的发生中起一定作用。E6E7加快细胞增殖可能与它减少p2

Objective To study the effects of HPV16 E6E7 fragments on the biological behavior of immortalized human bronchial epithelial cells TR. Methods E6E7 fragments were constructed into retrovirus expression vector and then transfected into TR cells to observe changes in growth character and tumorigenesis in nude mice. IP Western blots were done to analyze function of p27 protein as well as expression and phosphorylation of FAK and paxillin. Results Southern, Northern, Western blot confirmed the stable expression of E6E7 fragments in puromycin resistant cell strain TR/E6E7. TR/E6E7 proliferated faster, showed stronger growth ability in soft agar and eventually formed tumor after being inoculated into nude mice. IP Western blot also showed sequestering of p27 protein from cyclin E/CDK2 complex, which implied increased activity of cyclin E/CDK2 and promoting G1 to S cell cycle. Conclusion TR/E6E7 cells formed tumor in nude mice indicating that HPV infection played a role in pulmonary carcinogenesis. Sequestering of p27 protein from cyclin E/CDK2 complex may be responsible for increased proliferation in cells transfected by HPV16 E6E7.