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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 蛋白的表达,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。
Objective To establish an immortalized human laryngeal epithelial cell line induced by human papillomavirus (HPV) 16 DNA and confirm whether HPV is associated with the occurrence of laryngeal cancer. Methods The pSV HPV16 DNA was introduced into primary cultured human laryngeal epithelial cells by liposome-mediated method. The cells were passaged and passaged for 20 passages. The cells were examined by PCR to detect whether the cells contained specific fragments of virus. The expression of HPV16 DNA was detected by immunohistochemistry The expression of E6 and E7 proteins were detected by inverted microscope, growth curve, flow cytometry, cytokeratin immunohistochemistry, transmission electron microscopy and soft agar colony formation assay. Results Three of the cells had been subcultured for more than 20 passages. The cells contained specific fragments of HPV16 DNA and expressed E6 and E7 proteins. The cells were anchored and anchored to contact inhibition monolayers. The growth curve was typical “S ” type, cell proliferation index was 48%, all cells expressed keratin, cytoplasmic containing fibril, soft agar culture cloning test was negative. Conclusion The HPV16 DNA-induced immortalized human laryngeal epithelial cell line was successfully constructed and provided a new ideal model for the study of laryngeal cancer. HPV16 has an oncogenic effect on human laryngeal epithelium. The HPV16 DNA detected in laryngeal cancer tissue must be in its Multi-step carcinogenesis plays an important role.