人乳头状瘤病毒16型E6、E7基因诱导的人子宫颈永生化上皮细胞系的建立及其生物学特性的鉴定

Immortalization of human embryonic cervical epithelial cells induced by E6,E7 genes of human papillomavirus 16

目的 建立人宫颈上皮的永生化细胞系 ,并进行生物学特性进行鉴定。方法 用含人乳头状瘤病毒 (HPV) 16型E6、E7基因的腺病毒伴随病毒感染人胎儿宫颈上皮细胞 ,培养、传代。采用激光共聚焦免疫荧光和PCR技术检测HPV16型E6、E7基因的表达情况 ;取 2 0代细胞 ,采用软琼脂培养、染色体核型分析和Scid小鼠皮下接种等方法检测细胞系的生物学特性 ;采用电子显微镜观察细胞的形态。结果 2 0代细胞的表型仍保留原代上皮细胞的特征 ,表现为单层生长和锚锭依赖性生长。激光共聚焦免疫荧光和PCR技术检测显示 ,细胞内有HPV16型E6、E7基因的表达 ,其基因片断长度为 82 9bp。 2 0代细胞进行软琼脂培养不形成克隆 ;scid小鼠皮下接种未成瘤 ;染色体核型分析为二倍体和多倍体 ,11号染色体可能为HPV16型E6、E7基因整合的部位。电子显微镜观察可见张力原纤维 ,证实细胞为鳞状上皮来源。结论 成功建立了HPV16型E6、E7基因诱导的宫颈上皮永生化细胞系 ,且其生物学特性稳定 ,可进一步用以宫颈癌病因和发病相关机制的研究。

Objective To establish an immortalized cell line derived from the embryonic cervical epithelium by infection with the recombinant adeno-associated virus(rAAV) containing human papillomavirus(HPV)16 E6, E7, and to study the biological features of cervical cancer cell line. Methods Human embryonic cervical tissues were cultured in keratinocyte free serum (K-FS) medium and infected with rAAV containing HPV16 E6, E7. Morphological features and growth rate were examined by light, electronic and fluorescence microscopies. The fragments of E6, E7 were detected by polymerase chain reaction(PCR) and laser confocal microscopy. The biological characteristics of human cervical epithelium were observed by soft agar culture, scid mice inoculation and chromosome analysis. Cell proliferative dynamics was plotted by flow cytometry. Results After a long-term culture, the phenotype kept the characteristics of primary epithelial cells. They showed monolayer, anchorage-dependent and attachment-inhibited growth without forming colonies in soft agar culture. They were non-oncogenic when inoculated into scid mice. The tonofilament expression in the cervical cancer cells was inspected by electronic microscopy, demonstrating that the cells were squamous epithelium in origin. The cell line contained HPV16 E6, E7 genes by PCR and laser confocal detection. Chromosome analysis disclosed that the karyotype was diploid or polyploid. The 11th chromosome was assumed to be the integration site by rAAV containing HPV16 E6, E7. Conclusions Establishment of the immortalized cervical epithelial cell line by infection with rAAV containing HPV16 E6, E7, supports that HPV16 E6, E7 may be the primary etiology of cervical cancer. It will facilitate further research on the etiology and pathogenesis of cervical cancer.