人乳头状瘤病毒18E6E7和TPA协同诱发人胚食管上皮细胞恶性转化的研究

HUMAN PAPILLOMA VIRUS 18E6E7 IN SYNERGY WITH TPA INDUCED MALIGNANT TRANSFORMATION OF HUMAN EMBRYONIC ESOPHAGEAL EPITHELIAL CELLS

为了研究病毒和促癌物在食管癌形成中的作用，用带有人乳头状瘤病毒１８型Ｅ６Ｅ７片段的载体腺病毒（简称ＨＰＶ１８Ｅ６Ｅ７ＡＡＶ）感染人胚食管上皮细胞，然后加ＴＰＡ协同作用，观察细胞转化。将人胚食管切碎，与ＨＰＶ１８Ｅ６Ｅ７ＡＡＶ同孵育２小时，在加有１０％小牛血清的１９９培养液培养和传代，形成永生化细胞株，即人胚食管上皮细胞汕头株（ＳＨＥＥ）。实验分两组：一组ＳＨＥＥ细胞在传代至第５和１３代时，两次在培养基中加入ＴＰＡ（１２Ｏｔｅｔｒａｄｅｃａｎｏｙｌｐｈｏｒｂｏｌ１３ａｃｅｔａｔｅ）５ｎｇ／ｍｌ，每次诱导２周，所获得的细胞株称为人胚食管上皮癌细胞汕头株１号（ＳＨＥＥＣ１）；另一组ＳＨＥＥ细胞培养条件相同，未加ＴＰＡ，为对照组。细胞转化的形态表型由光学显微镜、电子显微镜和荧光显微镜检查；ＤＮＡ含量和细胞周期用流式细胞仪检测；用３５ｍｍ软琼脂培养皿接种１０３细胞（第２０代），每组５碟，计算集落形成率；裸鼠皮下接种１０６细胞检测致瘤性；用荧光原位杂交（ＦＩＳＨ）和ＰＣＲ检测ＨＰＶ１８Ｅ６Ｅ７。结果表明：细胞ＤＮＡ合成和增殖指数（ＰＩｘ），ＳＨＥＥＣ１组（４５％）高于ＳＨＥＥ组（３４％）；高倍体细胞数，ＳＨＥ?

In order to investigate the role of HPV and promoters in the formation of esophageal carcinoma, HPV18E6E7 AAV infected human embryonic esophageal epithelial cell (SHEE) in synergy with TPA and the malignant transformation of the cells (SHEEC1) was observed. Human embryonic esophageal tissue, which were cut into small pieces, were incubated with HPV18 E6 E7 AAV for two hours, cultured and passaged in normal 199 medium. Cultured cells (SHEE) were exposed to media with TPA (5ng/ml) for 2 weeks at 5th and 13th passages. Control group (SHEE) was cultured in the same media without TPA. The morphological phenotype of transformation was assessed by microscopy (including light , electron and fluorescent microscope). DNA content and cell cycle were detected by flow cytometry. Morphologically transformed foci were assayed by plating 10 3 cells at passage 20 on 35mm soft agar dishes (5 dishes for each group). The tumorigenicity was assayed in nude mice injected with 10 6cells/mouse subcutaneously in 6 animals of each group. HPV 18 E6 E7 gene was detected by PCR and FISH (at 20th passage of SHEEC1 and SHEE). SHEEC1 showed that DNA synthesis and PIx increased more than that of SHEE group. The poly ploid of DNA in SHEEC1 (5.70%) was more than that in SHEE (1.53%). Scoring foci in soft agar dishes, large colony (as positive transformed foci) with dense, multilayered cells were found more in TPA group (4.0%) and few in control group (0.1%). Tumorigenesis was observed in all six nude mice of TPA group (SHEEC1) and non tumorigenic in control group (SHEE). E6E7 gene was found in nucleus of two cell groups by FISH. Also, the HPV18 DNA was positive in two groups of cell line and in transplanted tumor of nude mice by PCR procedure. The malignant transformation of human embryonic esophageal epithelial cells were induced in vitro by HPV18 E6 E7 in synergy with TPA. It is a good proof for the close relationship between the HPV and the etiology and pathogenesis of esophageal carcinoma. Also, it is a reliable model for investigating cellular and molecular mechanisms of carcinogenesis of esophageal carcinoma.