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为了证实HPV18E6E7基因诱导的人胚食管上皮永生化细胞 (SHEE) 6 1代 (SHEE6 1)部份细胞 (SHEE6 1A)已经恶性转化 ,以寻找监控细胞早期恶性转化的方法。对培养的SHEE第 10代 (SHEE10 )和 6 1代 (SHEE6 1)细胞 ,用光学显微镜和电子显微镜观察细胞形态及生长形式 ;用流式细胞仪分析细胞周期 ;用染色体G带核型分析和间期核染色体 1、7、8号着丝粒探针荧光原位杂交 (FISH)检测细胞染色体改变 ;用增殖优势克隆优选法选出生长快的细胞群体 ,接种裸小鼠和SCID小鼠。结果 :光学显微镜下见SHEE6 1细胞大小不等 ,重叠生长 ;电子显微镜下见细胞核多型性和核仁增大等增殖表型 ;流式细胞仪检测 ,SHEE6 1增殖指数和DNA >4n细胞高于SHEE10 ;SHEE6 1细胞染色体众数出现 5 7～ 6 0、6 3～ 6 5两亚群 ,1、7、9、13、17等染色体出现较多 3体、4体或 5体 ;FISH间期核 1、7号着丝粒数增多 ;SHEE6 1克隆优选的细胞SHEE6 1A接种SCID小鼠成瘤。这表明 :SHEE6 1细胞形态及生长特性有了改变 ,接触抑制减弱 ,高倍体和不整倍体细胞增多 ,染色体数目明显改变 ,SHEE6 1A接种SCID小鼠产生浸润性肿瘤 ,可以判定SHEE6 1部份细胞已恶性转化。用克隆优选法筛选和染色体检测可以监控永生化细胞早期恶性转化。
In order to confirm that the HPV18E6E7 gene-induced human embryonic esophageal epithelial immortalized cell (SHEE) 61 (SHEE61) cells (SHEE6 1A) cells have been malignantly transformed to find ways to monitor early malignant transformation of the cells. The cultured SHEE generation 10 (SHEE10) and 61st generation (SHEE61) cells were observed by light microscopy and electron microscopy for cell morphology and growth patterns; the cell cycle was analyzed by flow cytometry; and chromosome G banding analysis and Interphase chromosome 1,7,8 centromeric probe fluorescence in situ hybridization (FISH) was used to detect cell chromosome changes; cell populations with fast growth were selected by the method of proliferative advantage cloning, nude mice and SCID mice were inoculated. RESULTS: Under the light microscope, the size of SHEE61 cell was unequal and overlapped; the proliferative phenotype such as nuclear polymorphism and enlarged nucleolus was seen under electron microscope; the proliferation index of SHEE61 and the high DNA >4n were detected by flow cytometry. In the SHEE10; SHEE1 cells, the chromosomes appeared in the subpopulations of 57-600, 63-65, and chromosomes 1, 7, 9, 13, and 17 appeared more three, four, or five. Number of centromeric No. 1 and No. 7 centromeres increased; SHEE6 1 clone-preferred cells, SHEE6 1A, were inoculated into SCID mice for tumorigenesis. This indicated that the morphology and growth characteristics of SHEE61 cells were changed, contact inhibition was weakened, hyperploidy and aneuploid cells were increased, and the number of chromosomes was significantly changed. SCEE mice were inoculated with SHEE6 1A and infiltrating tumors were identified. Has been malignant transformation. Screening by colony optimization and chromosome detection can monitor the early malignant transformation of immortalized cells.