外源性人乳头状瘤病毒16型E7基因对子宫颈癌细胞周期的影响  

Construction and package of the expression plasmid pAdEasy-1 system encoding the human papillomavirns 16 E7 gene and the gene's influence on HeLa cells

作　　者：戴淑真[1] 孔守芳[1] 黄丽丽[1] 罗兵[2] 

机构地区：[1]青岛大学医学院附属医院妇产科,266003 [2]青岛大学医学院附属医院微生物学教研室,266003

出　　处：《中华妇产科杂志》2006年第9期612-617,共6页Chinese Journal of Obstetrics and Gynecology

摘　　要：目的了解外源性人乳头状瘤病毒16型(HPV16)E7基因对子宫颈癌细胞周期的影响。方法从宫颈癌标本中经 PCR 扩增回收 HPV16 E7基因片段,将正确的 E7基因片段插入到穿梭质粒 pAdrTrack-CMV 中,与骨架质粒 pAdEasy-1在大肠埃希菌 Bj5183中同源重组,重组腺病毒质粒转染人胚肾细胞株 HEK-293细胞,包装表达 E7基因的腺病毒,然后转染 HPV18阳性的宫颈癌细胞株HeLa 细胞,四甲基偶氮唑蓝(MTT)比色法检测转染后 HeLa 细胞的增殖情况,以吸光度(A)值表示;流式细胞仪检测转染前后 HeLa 细胞的细胞周期和细胞周期蛋白 D1表达的变化。结果在 HEK-293细胞中,可以收获高转染效率的腺病毒。在荧光显微镜下,可以观察到 HEK-293细胞和 HeLa 细胞中均有绿色荧光蛋白的表达。提取 HEK-293细胞 RNA,RT-PCR 技术可以检测到 E7基因的表达。MTT 比色法检测结果显示,转染后 HeLa 细胞的 A 值最低为0.38±0.02,最高为0.96±0.01,分别与转染前的0.27±0.03比较,差异均有统计学意义(P<0.01)。流式细胞仪检测结果显示,转染12h后,S 期细胞占(36.0±2.0)%,转染24h 后为(49.9±4.2)%,分别与转染前的(26.0±0.4)%比较,差异均有统计学意义(P<0.05);转染前、后细胞周期蛋白 D1阳性表达率分别为22.4%、55.2%,两者比较,差异有统计学意义(P<0.01)。结论携带 HPV16 E7基因的腺病毒可转染子宫颈癌 HeLa细胞,并在 HeLa 细胞内表达,HPV16 E7基因的表达进一步影响 HeLa 细胞的细胞周期蛋白的表达,促进 HeLa 细胞增殖,可望用于治疗宫颈癌。Objective To investigate the effect of human papillomavirus 16 E7 gene on cell cycle of cervical cancer HeLa cell, through construction and expression of human papillomavirus 16 E7 gene with adenovirus vector. Methods Recombinant adenovirus which expressed E7 gene was constructed and packed. Flow cytometry（FCM） was used to detect the changes of cell cycle phase and cyclin D1 between cells infected and uninfected by recombinant adenovirus. The 3-（ 4, 5-dimethylthiazol -2-yl ）-2, 5- diphennyhetrazolium bromide （MTT） colorimetric assay was used in the detection of the alteration of cell growth. Results The recombinant adenovirus had stable efficiency of infection and E7 genes could be expressed stably. The result of MTT showed the multiplication of HeLa cells was accelerated from 0. 27 ± 0. 03 before infection to 0. 38 ± 0.02 after infection （ P 〈 0. 01 ）. FCM showed the number of cells in S phase increased from （ 26.0 ± 0. 4 ） % to （ 36. 0 ± 2.0 ） % at 12 hours and （49. 9 ± 4. 2 ） % at 24 hours after infection （ P 〈 0. 05 ）. Cyclin D1 expression was 22.4% before infection, and decreased to 55. 2% after infection （P 〈 0. 01 ）. Conclusions The recombinant adenovirus expressing E7 gene could infect target cells. E7 gene can influence cell-cycle of HeLa cells, which can be used to restrain cervical cancer.

关 键 词：宫颈肿瘤 癌基因蛋白质类 病毒性 转染 HELA细胞 

分 类 号：R737.33[医药卫生—肿瘤]