【摘要】 人乳头瘤病毒(human papillomavirus,HPV)是一种无包膜的环状闭合双链DNA病毒,具有严格的嗜人组织的特性.为探讨不同启动子驱动HPVE6/E7癌蛋白在裸鼠体内不同组织的表达效率,构建了带有角蛋白(K14)启动子和带有巨细胞病毒(CMV)启动子的E6/E7腺病毒载体(pAd-K14-E6/E7和pAd-CMV-E6/E7),pAd-K14-E6/E7和pAd-CMV-E6/E7、以及作为对照的重组腺病毒空载体pAdtrack-K14和pAd-CMV同源重组后,分别在293细胞中包装,收集重组病毒Ad-K14-E6/E7、Ad-CMV-E6/E7、Adtrack-K14和Ad-CMV,通过尾缘静脉注射到随机分组的裸鼠体内,并每d向裸鼠腹腔注射0.05 mg雌激素.采用RT-PCR和Western免疫印迹检测不同实验组E6/E7 mRNA和蛋白表达水平,免疫组化法检测P53和Bcl-2蛋白表达.结果显示,注射病毒Ad-K14-E6/E7(实验组1)裸鼠子宫体中E6/E7 mRNA、E6蛋白质、P53和Bcl-2蛋白高表达,而其它组织中低表达;注射病毒Ad-CMV-E6/E7(实验组2)裸鼠各组织E6/E7 mRNA、E6蛋白质、P53和Bcl-2蛋白均低表达.研究表明,在裸鼠体内角蛋白K14启动子可以调控E6/E7在子宫体中表达,CMV启动子未能诱导E6/E7在子宫体中表达.更多还原

【Abstract】 Human papillomaviruses(HPVs),one kind of double-stranded covalently closed circular DNA(cccDNA) viruses,have a specific infection property in human tissues.To explore the expression ability of different promoters on HPV E6/E7 oncoproteins in different tissues of nude mice,the vectors of pAd-K14-E6/E7 containing K14 promoter and pAd-CMV-E6/E7 containing CMV promoter,were constructed,respectively.The replication-deficient adenovirus Ad-K14-E6/E7,Ad-CMV-E6/E7,Adtrack-K14 and Ad-CMV were generated and amplified after the vectors were transfected into HEK293 cells.The virus supernatant was collected and injected into nude mice grouped randomly by trail vein.The Ad-K14-E6/E7 and Ad-CMV-E6/E7 were as experimental groups,while the Ad-CMV and Adtrack-K14 were as control groups.These nude mice were treated with 0.05 mg estrogen by intraperitoneal injection every day.E6 and E7 mRNA and protein expression of different groups were detected through RT-PCR and Western blot,and P53 and Bcl-2 protein expression were detected by immunohistochemistry.The results showed that E6/E7 mRNA,E6 protein,P53 and Bcl-2 proteins were all highly expressed in uterine body of experimental group 1(injected Ad-K14-E6/E7+estrogen),while they showed low expression in other tissues.In experimental group 2(injected Ad-CMV-E6/E7+estrogen),those proteins and mRNA were expressed at quite low levels in all tissues.The conclusion was that the K14 cytokeratin promoter could drive E6/E7 expression.in uterine body of nude mice,while the CMV promoter could not.更多还原