人乳头状瘤病毒16型DNA诱导的永生化人喉上皮细胞系的建立

摘要

[Objective]To establish immortalized human laryngeal epithelial cell fines induced by HPV16 DNA and explore the relationship between HPV infection and laryngeal carcinoma.[Methods]Plasmid with HPV16 DNA carded by lipofectin was transfected into primary cultured human laryngeal epithelial cells and the cells were sub-cultured. At the 20th passage, the specific fragments of HPV16 DNA were detected by polymerase chain reaction(PCR), expression of HPV16 E6 and E7 proteins was tested by immunohistochemistry.The cellular biological char-acterizations were examined by the reverse microscope, growth curve, flow cytometry, immunohistochemistry for cy-tokeratin, electron microscope and soft agar culture.[Results]3 cell lines keeping proliferative potency were cul-tured for over 20 passages and contained the specific fragments of HPV16 DNA and expressed E6 and E7 proteins.They showed monolayer, anchorage-dependent and attachment-inhibited growth, the growth curve showed a typical"S" shape and the proliferative index was 48％, the cells expressed cytokemtin and contained tonofilaments, there were no colonies formed in soft agar culture.[Conclusions]Immortalized human laryngeal epithelial cell lines in-duced by HPV16 DNA have been successfully established,which provides ideal models for further research on the etiology and pathogenesis of laryngeal carcinoma.And this proves that detected HPV 16 DNA in specimens of laryn-geal carcinoma must play an important role in the multistage process of carcinogenesis.%目的 建立人乳头状瘤病毒(HPV)16型DNA诱导的永生化人喉上皮细胞系.确证HPV与喉癌的发生有无关系.方法 采用脂质体介导法,将pSV HPV16 DNA导入原代培养的人喉上皮细胞,继续培养、传代,取20代细胞.用PCR检测细胞是否含有病毒的特异片段,用免疫组化染色检测E6、E7蛋白的表迭,用倒置显微镜、生长曲线、流式细胞仪、细胞角蛋白免疫组化染色、透射电镜及软琼脂克隆形成试验检测细胞的生物学特性.结果 有3株细胞已连续培养传代超过20代,细胞含有HPV16 DNA的特异片段并有E6、E7蛋白的表达,细胞呈锚着依赖性、接触抑制性单层平铺生长.生长曲线呈典型的"S"型,细胞增殖指数为48％,所有细胞均表达角蛋白,胞浆含张力原纤维,软琼脂培养克隆形成试验阴性.结论 成功建立了HPV16 DNA诱导的永生化人喉上皮细胞系,为喉癌研究提供了新的理想模型,HPV16对人喉上皮有致癌作用,在喉癌组织标本中检测到的HPV16 DNA必定在其多步癌变过程中发挥了重要作用.