人乳头状瘤病毒18型E6E7基因诱导人胚食管上皮永生化

目的为研究病毒和肿瘤的关系，用人乳头状瘤病毒（ＨＰＶ）１８型Ｅ６Ｅ７基因感染胎儿食管上皮，建立一株新的人食管上皮永生化细胞株（ＳＨＥＥ）。方法ＨＰＶ１８Ｅ６Ｅ７腺病毒伴随病毒（ＨＰＶ１８Ｅ６Ｅ７ＡＡＶ））载体的构建；胚胎食管组织培养，ＨＰＶ１８Ｅ６Ｅ７ＡＡＶ感染，继续培养传代。用光镜、电镜检查其形态；聚合酶链反应（ＰＣＲ）、荧光原位杂交（ＦＩＳＨ）检查该病毒片段；用软琼脂培养和裸鼠接种检查致瘤性。结果经过长时间的传代培养，ＳＨＥＥ的表型仍保留原代上皮细胞培养的特征，表现为单层生长和锚锭依赖性生长，在软琼脂培养不形成克隆，接种裸鼠未成瘤。ＳＨＥＥ细胞系电镜检查可见张力原纤维，免疫组织化学检查细胞角质蛋白阳性，证实为鳞状上皮来源。ＦＩＳＨ和ＰＣＲ检测显示有ＨＰＶ１８Ｅ６Ｅ７基因。结论用ＨＰＶ１８Ｅ６Ｅ７基因建立食管上皮永生化细胞株ＳＨＥＥ，支持ＨＰＶ１８可能和食管癌病因有关的观点，可进一步用以研究食管癌的病因和发病机制。

Immortalization of human fetal esophageal epithelial cells induced by E 6 and E 7 genes of human papilloma virus 18

Objective For studying the relationship between HPV and esophageal carcinoma, an immortalized human fetal esophageal epithelial cell line (SHEE) was established. Methods The human fetal esophageal tissues were cultured and infected with HPV 18 E6E7 AAV. It was examined by lightand electronmicroscope for morphological changes, by PCR and FISH for detection of HPV E6E7 and by soft agar culture and nude mice inoculatior for dectecting tumor transformation. Results The cell line has become immortal and has propagated cntinuously for more than 50 passages. After a longterm cultrue, the phenotype keeps the characteristics of primary epithelial cells. They showed as monolayer growth and anchorage dependent growth without forming colonies in softagar. They were nontumorigenic in nude mice. SHEE cells contained tonofilaments in its cytoplasm by electron microscopic examination and showed cytokeratin positive in immunohistochemical procedure. So it shows that the cells are squamous epithelium in origin. The cell line contained the HPV 18 E6and E7 genes by FISH and PCR assay. Conclusion Establishment of the esophageal epithelial cell line SHEE successfully immortalized with HPV 18,E6E7, supports that the HPV18 may be related to the etiology of esophageal carcinoma. It will facilitate further research on etiology and pathogenesis of esophageal carcinoma.