【摘要】 研究中期永生化食管上皮细胞的表型、细胞遗传学和部份基因的改变 ,以阐明癌前病变的特征。SHEE是该院用人乳头状瘤病毒HPV18E6E7诱导的永生化上皮 ,传至 6 1代已开始有少量细胞恶性转化。对永生化中期 31代培养细胞用相差显微镜检查细胞形态改变和细胞生长状态 (细胞接触抑制和锚定生长 ) ;流式细胞仪检测细胞周期 ;做染色体众数分析 ;多重PCR检查c-myc、p5 3、bcl- 2和ras等基因。免疫组化检查细胞角蛋白和鬼臼毒素荧光标记检查肌动蛋白F(F -actin)。软琼脂培养的集落细胞接种SCID小鼠检查成瘤性。Westernblot方法检测细胞内HPVE6表达蛋白。结果 :培养细胞有两种不同分化形态 ,分化差的基底细胞和分化好的鳞状上皮 ;前者角蛋白和F -actin极少 ,后者含量丰富 ;细胞接触抑制和锚锭生长特性减弱。分析 10 0个细胞染色体众数分二干系 ,5 6条 (占 30 % )和 6 1条 (占 2 4% )染色体 ,核型分析属于超二倍体 ,亚三倍体。多重PCR检查 :c -myc、p5 3基因上调 ,bcl- 2和ras基因阳性。用优选生长在软琼脂上的克隆细胞接种SCID小鼠 ,未成瘤 ;HPV18E6表达蛋白阳性。以HPV18E6E7诱导的食管永生化上皮 31代细胞形态出现了双向分化 ,根据其形态表型 ,双染色体众数的细胞遗传学改变和某些癌基因上调等特性 ,可以认为SHEE更多还原

【Abstract】 In order to explore the characters of precancerous phase,the changes of phenotype,cytogenetics and some oncogenes of the immortalized esophageal epithelium in the middle phase were investigated The immortalized human esophageal epithelium SHEE cell line induced by HPV18E 6E 7 genes was malignantly transformed in partial cells at its 61th passage in our laboratory The cultured SHEE cells at its 31th passage,in the middle phase of immortalization,were observed under phase contrast microscope for morphology and cell growth pattern(contact-inhibition and anchorage-dependent growth),analyzed by flow cytometry for cell cycle,calculated the modal number of chromosome,and assayed c-myc,p53,bcl-2 and ras genes by multi-PCR method The cytokeratin was examined immunohistochemically and the F-actin was assayed by fluorescence labeled phalloidin Tumorigenicity of the SHEE cells,which were the colony cells cultured on the soft agar,were assayed by using SCID mice subcutaneously injected with the cells The expressed protein of HPV18E 6 was detected by Western blotting method The results showed that the cultured SHEE31 cells were grouped into both the undifferentiated basal epithelium and the differentiated squamous epithelium,the cytokeratin and F-actin were empty in the former and rich in the latter The contact-inhibition and anchorge-dependent growth of these cells weakened The modal number of chromosome in 100 mitotic cells revealed two stemlines,56 chromosomes(30%)and 61 chromosomes(24%) By karyotype analysis,SHEE31 were heteroploidy belonging to hyperdiploidy and hypotriploidy c-myc and p53 genes were upregulated and ras gene were positive The colony cells (the optimum cells) sought from the soft-agar culture,could not grow in SCID mice The expression of HPV18E 6 was positive The immortalized esophageal epithelium,induced by HPV18E 6E 7 genes,at its 31th passage displayed biphasically differentiated phenotype Based on these findings of morphological phenotype,the double chromosomal modal number of cytogenetics and the upregulation of some oncogenes,it is suggested that SHEE31 cell line is in the precancerous phase更多还原