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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。
Supernatants were obtained after packaging the recombinant adenovirus Ad-CMV-E6 / E7 and Ad-K14-E6 / E7 in 293 cells. Ad-CMV-E6 / E7 and Ad-K14-E6 / E7 virus supernatant And the control recombinant adenovirus vector pAd-CMV and pAdtrack-K14 empty supernatant, after purification, through the tail vein of nude mice injected into the nude mice in vivo injection of 0.05 mg estrogen daily injection of the virus to the naked Rats, at the same time as a control. Twelve weeks later, nude mice were injected with 2.5% Averdin for anesthesia. The tissues of uterus, vagina, ovary and mammary gland were immersed in 3.75% paraformaldehyde overnight at 4 ° C. Paraffin-embedded sections were made and HE staining Immunohistochemistry was used to detect the expression of P53 protein and Bcl-2 protein. DNA, RNA and protein were extracted from various tissues of nude mice, and then the presence or absence of recombinant adenovirus infection, virus expression and E6 protein expression were detected. The results of HE staining showed that the nude mice injected with adenovirus Ad-K14-E6 / E7 (experimental group 2) had obvious hyperplasia of cervical-uterine body. SP immunohistochemical detection of mutant P53 and Bcl-2 in this group of nude mice increased uterine stromal cells, and RT-PCR detection of uterine tissue in this group also have E6 / E7 expression Western Blot detection of the uterine tissue There are also E6 protein expression. Animal experiments showed that the K14 promoter increased the expression of E6 / E7 in the uterus of nude mice, and also found that the uterine cervix - uterine body in this group had hyperplastic changes.