摘要
目的:人乳头瘤病毒16型(human papillomavirus,HPV 16)与包括宫颈癌在内的多种肿瘤的发生明确相关。诱导宿主基因组不稳定性可能是HPV16致瘤的重要机制之一。本研究对HPV16型E6蛋白诱导人原代角质形成细胞(primary human keratinocytes,PHK)形成多倍体以及对PHK有丝分裂纺锤体检查点的影响进行初步的探索。方法:取正常人包皮组织,分离表皮,常规方法培养PHK。脂质体介导法将pBabe-16E6质粒转染逆转录病毒包装细胞PA317。挑选G418抗性克隆,检测病毒滴度,将表达HPV16型E6蛋白的高滴度逆转录病毒感染PHK。通过逆转录PCR法和免疫印迹法证实HPV16E6成功感染PHK。PHK经Nocodazole处理后,利用流式细胞仪分析多倍体形成,于不同时间点收集细胞、固定,进行抗磷酸化组蛋白染色,利用流式细胞仪分析细胞有丝分裂指数差异。结果:HPV16型E6成功感染PHK,并呈功能性表达。HPV16型E6可诱导PHK形成多倍体,表达E6蛋白的PHK和对照细胞以相似的动力学进入和退出有丝分裂。结论:HPV16型E6对PHK有丝分裂过程中的纺锤体检查点无直接影响,鉴于有丝分裂后期检查点在细胞有丝分裂过程中较持久和严格的作用,本研究结果提示,对有丝分裂后期检查点的作用可能是HPV16型E6蛋白诱导宿主细胞多倍体形成的重要机制。为进一步探讨HPV相关肿瘤的分子发生机制奠定了一定基础。
Objective: Human Papillomavirus type 16 (HPV 16) is involved in the pathogenesis of several neoplasms, including cervical cancer. Induction of genomic instability may be one of the main mechanisms in- volved in this process. The aim of this study is to investigate the impact of HPV 16 E6 on polyploidy and mitotic spindle checkpoints in primary human keratinocytes (PHK). Methods: PHK were isolated from human prepuce and cultured by routine methods. The pBabe-16 E6 vector was first transfected into the retrovirus packaging cell line PA317 using lipofectamine transduction. G418-resistant colonies were picked and assayed for virus titration. PHK were then infected with this retrovirus expressing HPV16 E6. Polyploidy was detected by flow cytometry and the mitotic index was analyzed with phospho-histone H3 staining and flow cytometry at different time points. Results: PHK were successfully infected by HPV16 E6. HPV16 E6 can induce polyploidy in PHK. PHK infected by pBabe-16 E6 or empty vector entered into mitosis with similar kinetics. Conclusion: HPV 16 E6 protein induces polyploidy through a mechanism that is not related to abrogating mitotic spindle checkpoints in PHK. These results suggest further exploration of the mechanism by which HPV oncogenes induce polyploidy in HPV-related neoplasms is needed.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2009年第1期38-41,共4页
Chinese Journal of Clinical Oncology
基金
国家自然科学基金资助(编号:30572112,30600558)~~