陈 凌, 沈 柱①, 伍津津. HPV 16型E6 蛋白诱导PHK 多倍体的形成与消除细胞纺锤体检查点无关*[J]. 中国肿瘤临床, 2009, 36(1): 38-41. DOI: 10.3969/j.issn.1000-8179.2009.01.011
引用本文: 陈 凌, 沈 柱①, 伍津津. HPV 16型E6 蛋白诱导PHK 多倍体的形成与消除细胞纺锤体检查点无关*[J]. 中国肿瘤临床, 2009, 36(1): 38-41. DOI: 10.3969/j.issn.1000-8179.2009.01.011
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CHEN Ling1, SHEN Zhu2, WU Jinjin1. HPV16E6 Protein Induces Polyploidy Through a Mechanism that Does Not Involve Abrogating Mitotic Spindle Checkpoints in Primary Human Keratinocytes[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2009, 36(1): 38-41. DOI: 10.3969/j.issn.1000-8179.2009.01.011
Citation: CHEN Ling1, SHEN Zhu2, WU Jinjin1. HPV16E6 Protein Induces Polyploidy Through a Mechanism that Does Not Involve Abrogating Mitotic Spindle Checkpoints in Primary Human Keratinocytes[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2009, 36(1): 38-41. DOI: 10.3969/j.issn.1000-8179.2009.01.011
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HPV 16型E6 蛋白诱导PHK 多倍体的形成与消除细胞纺锤体检查点无关*

HPV16E6 Protein Induces Polyploidy Through a Mechanism that Does Not Involve Abrogating Mitotic Spindle Checkpoints in Primary Human Keratinocytes

    摘要
  • 摘要: 目的：人乳头瘤病毒16型（humanpapillo mavirus，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HPV 16致瘤的重要机制之一。本研究对HPV 16型E6 蛋白诱导人原代角质形成细胞（primaryhumankeratinocytes，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HPV 16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HPV 16E6 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：HPV 16型E6 成功感染PHK ，并呈功能性表达。HPV 16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：HPV 16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HPV 16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。

     

    Abstract: Objective:Human Papillomavirus type16(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 HPV16E6 on polyploidy and mitot-ic spindle checkpoints in primary human keratinocytes (PHK). Methods:PHK were isolated from human pre-puce and cultured by routine methods. The pBabe- 16E6 vector was first transfected into the retrovirus pack -aging cell line PA 317 using lipofectamine transduction. G 418 -resistant colonies were picked and assayed for virus titration. PHK were then infected with this retrovirus expressing HPV16E6. Polyploidy was detected by flow cytometry and the mitotic index was analyzed with phospho-histone H3 staining and flow cytometry at dif -ferent time points. Results: PHK were successfully infected by HPV 16E6. HPV 16E6 can induce polyploidy in PHK. PHK infected by pBabe- 16E6 or empty vector entered into mitosis with similar kinetics. Conclusion: HPV16E6 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 in -duce polyploidy in HPV-related neoplasms is needed.

     

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