人乳头状瘤病毒协同^(60)钴照射促进食管上皮细胞恶性转化

Human Papillomavirus in Synergy with^(60)Cobalt Radiation Promotes Malignant Transformation of Esophageal Epithelial Cells

为探明人乳头状瘤病毒(HPV)和促癌剂对食管上皮致癌作用,人胚食管上皮细胞转染HPV协同60钴(60Co)放射观察其恶性转化。用HPV18E6E7AAV转染的人胚食管上皮(SHEE),培养至13代,分为4组,实验组分别用60Co2、4、8Gy照射,每周1次共4周;SHEE未经照射为对照组。细胞形态用相差显微镜观察;细胞DNA合成和定量用3H TdR掺入和用流式细胞仪分析;染色体众数用常规方法分析;致瘤性用软琼脂培养和裸小鼠接种;HPVDNA用PCR检测。经60Co照射后细胞呈凋亡和坏死(危象期)。8周后SHEE4Gy组细胞增殖,增殖指数(34%)和3H TdR摄入增高,软琼脂培养和裸鼠接种出现致瘤性。对照组SHEE组细胞增殖指数24%,伴有少数3H TdR掺入,裸鼠未成瘤。染色体众数:对照组,58～62;4Gy组,63～65;两组HPV18E6E7PCR呈阳性条带。此结果表明,用HPV18E6E7协同60Coγ射线可以使人胚食管上皮恶性转化,60Coγ射线有加速食管上皮细胞恶性转化作用。

To explore the effect of human papillomavirus and tumor promoter on the tumorigenicity of esophageal epithelium,the human embryonic esophageal epithelial cells were infected with human papillomavirus(HPV)in synergy with^(60)cobalt radiation to observe their malignant transformation.The cultured esophageal epithelial cells(SHEE)was tansfected with HPV18E_6E_7 AAV and propagated for 13 passages.The transfected cells were then divided into four groups,three experimental groups were exposed to^(60)Co radiation(2,4,8Gy respectively)once a week for 4 weeks at the 13th passage of the SHEE cells and one control group was cultured in the same medium without radiation.The morphology of the cells was observed under phase contrast microscope,the DNA content during the cell cycle synthesis was analyzed by flow cytometry and the modal number of chromosomes were analyzed by routine method.The tumorigenicity was assessed by colony formation after cultivating in soft agar and transplanting the cells into nude mice.HPV18E_6E_7 DNA was assayed by PCR.The results revealed that cell necrosis and apoptosis(crisis)stages appeared after cobalt radiation.At 8 weeks after radiation,cells of 4Gy group proliferated with higher proliferative index(34%).Through soft-agar cultivation and injection into nude mice,SHEE cells became tumorigenic.In control group,proliferative index was 24% and no tumor formation appeared in nude mice.The modal number of chromosome was 58-62 in control group and 63-65 in 4Gy group.In these two cell groups,HPV18E_6E_7 DNA was positively detected by PCR.The data above suggest that the malignant transformation of human embryonic esophageal epithelial cells was induced in vitro by HPV18E_6E_7 in synergy with cobalt radiation and cobalt radiation is the promotive factor to accelerate malignant transformation of esophageal epithelial cells.