人乳头瘤病毒关键基因在裸鼠体内表达的初步研究  

Preliminary study of HPV critical gene expression in nude mice in vivo

作　　者：潘巍巍[1,2] 徐营[2] 易发平[1,3] 曹利仙[2] 卜友泉[1,3] 赖国旗[4] 宋方洲[1,3] 

机构地区：[1]重庆医科大学生物化学与分子生物学教研室,重庆400016 [2]嘉兴学院医学院,浙江嘉兴314001 [3]教育部临床检验诊断学重点实验室,重庆400016 [4]重庆医科大学动物实验中心,重庆400016

出　　处：《中国病理生理杂志》2009年第2期209-214,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30371479);教育部"春晖计划"资助项目(No.Z2004-1-55103);嘉兴市科技局科研重点资助项目(No.2007AZ2013);嘉兴市科技局科研项目(No.2008AYZ039-3);浙江省实验动物科研计划项目(2008F80025)

摘　　要：目的:初步研究HPV16E6/E7基因在裸鼠体内不同组织引起的病变。方法:将重组腺病毒Ad-CMV-E6/E7、Ad-K14-E6/E7病毒上清以及作为对照的重组腺病毒空载体pAd-CMV和pAdtrack的上清液,经过纯化后,通过裸鼠的尾缘静脉注射到裸鼠体内,并每天注射0.05mg雌激素到注射病毒的裸鼠,同时做阴性对照。12周后给裸鼠注射2.5%阿佛丁进行麻醉,并将其耳朵、头颈部皮肤、口腔黏膜、结肠、直肠、子宫颈-阴道移行区等组织取出,浸泡在3.75%多聚甲醛中4℃固定过夜,然后做石蜡包埋切片、HE染色、免疫组化检测P53蛋白和Bcl-2的表达,取裸鼠的各个组织提取DNA、RNA和蛋白质,然后分别检测有无重组腺病毒的感染、病毒表达的情况及E6蛋白的表达。结果:实验组1注射腺病毒Ad-K14-E6/E7的裸鼠组织HE染色结果显示裸鼠子宫颈-阴道移行处的增生较明显。SP法免疫组化发现突变型P53和Bcl-2在该组裸鼠的子宫颈-阴道移行处表达都增加(P<0.05),并且RT-PCR检测子宫颈-阴道移行处组织也有E6/E7的表达,Western blotting检测该组织也有E6蛋白的表达。结论:K14启动子增加了E6/E7在裸鼠子宫颈-阴道移行处的表达,并且使子宫颈-阴道移行处的上皮组织发生病变。AIM： To study HPV16 E6/E7 gene expression in different tissue of nude mice. METHODS： After purified, recombined adenovirus Ad- CMV- E6/E7, Ad- K14 -E6/E7 were used as experimental group, while pAd - CMV and pAdtrack were used as control group. Four of them were injected through one main vein of nude mice tail. Then, these mice were treated with 0. 05 mg 17β - estradiol over 12 weeks. At the same time, the negative comparison was done. Mice were anesthetized with 2. 5% avertin. The ear, skin of head -neck, mucous membrane of oral cavity, colon, rectum ,cervicovaginal transformation zone were dissected and postfixed in 3.75% paraformaldehyde overnight at 4℃, paraffinembedded, sectioned and HE stained. The P53 and Bcl -2 protein were identified via immunohistochemistry. Infection of recombined adenovirus was verified by DNA extraction. The expression of E6/E7 gene and E6 protein were verified via RT - PCR method and Western blotting method in different tissues of nude mice. RESULTS ： HE staining showed evident hyperplasy in cervico - vaginal transformation zone in experimental group 1 （ treated with recombined adenovirus Ad - K14 - E6/E7 ）. Mutant 1＇53 and Bcl - 2 expressed obviously via immunohistochemical S - P method in cervico - vaginal transformation zone （P 〈0. 05）. RT - PCR method was used to detect the expression of E6/E7 in cervico - vaginal transformation zone of experimental group 1. Western blotting showed that E6 protein was expressed obviously. CONCLU- SION： Human cytokeratin promotersl4 promotes the expression of E6/E7 gene and pathological changes are detected in epithelial tissue of cervico - vaginal transformation zone.

关 键 词：乳头状瘤病毒 人 腺病毒 基因 E6/E7 小鼠 裸 

分 类 号：R373.39[医药卫生—病原生物学]