Shen Zhong-Ying, Xu Li-Yan, Chen Min-Hua, Shen Jian, Cai Wei-Jia, Zeng Yi
Department of Tumor Pathology, Medical College of Shantou University, Guandong Province, China.
World J Gastroenterol. 2002 Dec;8(6):976-81. doi: 10.3748/wjg.v8.i6.976.
To investigate the progressive transformation of immortal cells of human fetal esophageal epithelium induced by human papillomavirus, and to examine biological criteria of sequential passage of cells, including cellular phenotype, proliferative rate, telomerase, chromosome and tumorigenicity.
The SHEE cell series consisted of immortalized embryonic esophageal epithelium which was in malignant transformation when cultivated over sixty passages without co-carcinogens. Cells of the 10th, 31st, 60th and 85th passages were present in progressive development after being transfected with HPV. Cells were cultivated in a culture flask and 24-hole cultural plates. Progressive changes of morphology, cell growth, contact-inhibition, and anchorage-dependent growth characteristics were examined by phase contrast microscopy. The cell proliferation rate was assayed by flow cytometry. The modal number of chromosomes was analyzed. HPV18E(6)E(7) was detected by Western blot methods and activities of telomerase were analyzed by TRAP. Tumorigenicity of cells was detected with soft agar plates cultivated and with tumor formation in SCID mice.
In morphological examination the 10th passage cells were in good differentiation, the 60th and 85th passages cells were in relatively poor differentiation, and the 31st passage cells had two distinct differentiations. The characteristics of the 85th and 60th passage cells were weakened at contact-inhibition and anchorage-dependent growth. Karyotypes of four stages of cells belonged to hyperdiploid or hypotriploid, and bimodal distribution of chromosomes appeared in the 31st and 60th passage cells. All of these characteristics combined with a increasing trend. The activities of telomerase were expressed in the latter three passages. Four fourths of SCID mice in the 85th passage cells and one fourth of SCID mice in the 60th passage cells developed tumors, but the cells in the 10th and 31st passage displayed no tumor formation.
In continual cultivation of fetal esophageal epithelial cells with transduction of HPV18E(6)E(7), cells from the 10th to the 85th passage were changed gradually from preimmortal, immortal, precancerous to malignantly transformed stages. All of these changes were in a dynamic progressive process. The establishment of a continuous line of esophageal epithelium may provide a in vitro model of carcinogenesis induced by HPV.
研究人乳头瘤病毒诱导的人胎儿食管上皮永生细胞的渐进性转化，并检测细胞连续传代的生物学指标，包括细胞表型、增殖率、端粒酶、染色体和致瘤性。
SHEE细胞系由永生化的胚胎食管上皮组成，在无共致癌物的情况下培养60代以上时发生恶性转化。第10、31、60和85代细胞在转染HPV后呈渐进性发展。细胞在培养瓶和24孔培养板中培养。通过相差显微镜观察形态、细胞生长、接触抑制和贴壁依赖性生长特性的渐进性变化。通过流式细胞术检测细胞增殖率。分析染色体众数。采用蛋白质免疫印迹法检测HPV18E(6)E(7)，采用端粒重复序列扩增法分析端粒酶活性。用软琼脂平板培养和SCID小鼠成瘤检测细胞的致瘤性。
形态学检查显示，第10代细胞分化良好，第60和85代细胞分化相对较差，第31代细胞有两种不同的分化。第85和60代细胞的接触抑制和贴壁依赖性生长特性减弱。四个阶段细胞的核型属于超二倍体或亚三倍体，第31和60代细胞出现染色体双峰分布。所有这些特征呈上升趋势。端粒酶活性在后三代中表达。第85代细胞中有四分之三的SCID小鼠和第60代细胞中有四分之一的SCID小鼠发生肿瘤，但第10和31代细胞未形成肿瘤。
在转导HPV18E(6)E(7)的情况下持续培养胎儿食管上皮细胞，第10至85代细胞逐渐从前永生、永生前期、癌前期转变为恶性转化阶段。所有这些变化都处于动态渐进过程中。食管上皮连续细胞系的建立可为HPV诱导的致癌作用提供体外模型。
