人乳头瘤病毒引起裸鼠生殖系统病变的研究  

HPV Causex Pathological Changes in Genital System of Mice

作　　者：潘巍巍[1,2] 曹利仙[2] 易发平[1,3] 徐营[2] 卜友泉[1,3] 赖国旗[4] 马永平[1,3] 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]重庆医科大学分子医学与肿瘤研究中心,400016 [4]重庆医科大学动物实验中心,重庆400016

出　　处：《病毒学报》2009年第1期35-40,共6页Chinese Journal of Virology

基　　金：国家自然科学基金资助项目(30371479);教育部"春晖计划"资助项目(NOZ2004-1-55103);浙江省实验动物科研计划项目(2008F80025)[Y3];嘉兴市科技局科研重点项目(2007AZ2013);嘉兴市科技局科研计划项目(2008AY2039-3)

摘　　要：重组的腺病毒Ad-CMV-E6/E7和Ad-K14-E6/E7在293细胞中包装后得到上清液,将Ad-CMV-E6/E7和Ad-K14-E6/E7病毒上清液以及做为对照的重组腺病毒空载体pAd-CMV和pAdtrack-K14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,每天注射0.05 mg雌激素给注射病毒的裸鼠,同时做对照。12周后给裸鼠注射2.5%的阿佛丁进行麻醉,取其子宫、阴道、卵巢、乳腺等组织,浸泡在3.75%的多聚甲醛中4℃固定过夜,做石蜡包埋切片、HE染色、免疫组化检测P53蛋白和Bcl-2蛋白的表达,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。HE染色结果显示注射腺病毒Ad-K14-E6/E7的裸鼠(实验组2)子宫颈-子宫体移行组织增生明显。SP法免疫组化检测突变型P53和Bcl-2在该组裸鼠的子宫基质细胞表达增加,并且RT-PCR检测该组裸鼠子宫组织也有E6/E7的表达,Western Blot检测该组子宫组织也有E6蛋白的表达。动物实验结果表明K14启动子增加了E6/E7在裸鼠子宫组织的表达,同时也发现该组裸鼠的子宫颈-子宫体移行组织有增生改变。The recombined adenovirus DNA was transfected into 293 cells for packing and amplification of replication-deficient Ad-CMV-E6/ET, Ad-K14 -E6/E7 virus was purified by CsC1 density gradient centrifugation , recombined adenovirus Ad-CMV-E6/ET, Ad-K14 -E6/E7 were used as experimental group, while pAd-CMV and pAdtrack-K14 were used as control group. Four of them were injected through one main vein of nude mice tail respectively. These mice were then treated with 0.05rag 1713-estradiol over 12 weeks. Mice were anaesthesiaed with 2.5 % Avertint and the vagina,mammary gland,ovaries and uterus were dissected and fixed in 3.75 % paraformaldehyde overnight at 4℃. Paraffin-embedded sections, HE staining and identification of P53 and Bcl-2 protein via immunohistochemistry were performed. The expression of E6/E7 was verified by RT-PCR in different tissue of nude mice. HE staining showed evident hyperplasy in cervixuterus transformation zone of experimental group 2. The expression of mutant P53 and Bcl-2 were higher than control group via immunohistochemical S-P method in uterus stroma-cell. Western blotting also showed that E6 protein was expressed. The expression of E6/E7 was higher than control group by human cytokeratin promoter 14 and hyperlasy changes were detected in epithelial tissue of cervix-uterus transformation zone.

关 键 词：人乳头瘤病毒 腺病毒 E6/E7基因 裸鼠 

分 类 号：R373.9[医药卫生—病原生物学]