摘要
背景与目的:人乳头状瘤病毒(HPV)感染及其引发的抑癌基因功能丧失和细胞周期调控失调是宫颈癌发病的重要因素。HPV16诱发宫颈癌的机制主要与其两个早期开放读码框架E6、E7转化基因密切相关。HPVE6、E7引起细胞生长和增殖异常,同时伴有Cyclin A、Cyclin D1、CDK4等细胞周期蛋白的表达异常。本研究采用体外基因转染技术将HPV16 E7基因转入靶细胞,通过对目的基因瞬时表达的检测,进而观察在E7病毒癌蛋白的影响下调控G1/S检验点的几种细胞周期调节蛋白的表达,以探讨HPV16型E7基因外源性表达对子宫颈癌HeLa细胞细胞周期调节因子cdc25A及细胞周期蛋白E(Cyclin E)的影响。方法:从宫颈癌标本中经PCR扩增回收HPV16 E7基因片段,将其插入腺病毒穿梭质粒pAdTrack-CMV,与腺病毒骨架质粒pAdEasy-1构建重组腺病毒基因组Ad-E7。用脂质体介导Ad-E7导入包装细胞人胚肾细胞系HEK-293细胞,包装出完整腺病毒颗粒。测定病毒上清液滴度,感染体外培养的HeLa细胞。采用RT-PCR方法检测转染前后HeLa细胞cdc25A的mRNA表达的差异;激光扫描共聚焦显微镜(LSCM)检测转染前后Cyclin E表达的差异。结果:荧光显微镜下,转染后HEK-293细胞和HeLa细胞表达绿色荧光蛋白。RT-PCR结果显示转染后,HeLa细胞cdc25A的mRNA含量较转染前显著增加[感染前灰度值0.23±0.10,感染后0.87±0.22(P<0.01)],LSCM检测结果显示转染后Cyclin E表达较转染前明显增加。结论:重组腺病毒可以成功介导外源性HPV16 E7基因在HeLa细胞内表达,HPV16 E7基因促进HeLa细胞周期调节因子cdc25A和Cyclin E的表达。
Background and purpose： Carcinoma of the uterine cervix is one of the most common malignancies among women. Human papillomaviruses （HPV） have been identified as the major etiological factor in cervical carcinogenesis and HPV16 is one of the most prevalent subtypes. The high-risk-HPV oncoproteins, E6 and ET, induce the growth and proliferation of cervical keratinocytes, usually accompanied by abnormal expression of Cyclin A ,Cyclin D1 and CDK4. The aim of this study was to investigate the effect of the human papillomavirus 16 E7 gene on cdc25A and Cyclin E by transfecting recombinant adenovirus HPV16 E7 into Hela cell line. Methods： Polymerase chain reaction （PCR） was performed to obtain Human HPV 16 E7 gene fragment from human cervical carcinoma tissues, then the human papillomavlrus （HPV） 16 E7 oncogene was cloned into the shuttle vector pAdTrack-CMV, the resultant plasmid was linearized with restriction endonuclease Pme I, and subsequently cotransformed into E. coli. BJ5183 cells with adenovlral backbone plasmid pAdEasy-1. Finally, the recombinant plasmid was transfected into adenovlrus packaging cell lines HEK-293 cells. The viral titer was detected and calculated with the aid of GFP expression. The recombinant adenovlrus was transfected in HeLa cells, reverse transcription polymerase chain reaction （RT-PCR） techniques were used to evaluate the expression of cdc25A in HeLa cell and cyclln E expression was estimated by laser scanning confocal microscope （LSCM） before and after transfection. Resuits： HPV16 E7 was expressed efficiently in HEK-293 and HeLa cells. The expression of cdc25A mRNA was increased significantly after the Hela cells were transfected with the gray numerical values of 0.23 ±0.10 and 0.87 ±0.22, respectively （P 〈 0.01）. LSCM analysis showed that Cyclin E protein was significantly increased after the Hela cells were transfected by the recombinant adenovirus Ad-ET. Conclusions： In the present study, the recombinant adenoviruses carrying the HPV 16 E7 was successfully construeled.HPV 16 E7 oncogene could enhance the transcription of cde25A and the expression of Cyclin E.
出处
《中国癌症杂志》
CAS
CSCD
2007年第2期130-134,共5页
China Oncology
