1332 HEREDITAS (Beijing) [2~4] EC9706 [5],,, EC9706, (RPMI 1640, 15% ) ml 14, 96, 50 µl( 0.7 / ), 4 h, 50 µl, 5% CO 2 37,, , 0.01

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1 HEREDITAS (Beijing) , 29(11): ISSN 研究报告 DOI: /yc EC9706 韩亚玲, 冯彦斌, 罗曼莉, 徐昕, 蔡岩, 王明荣 : 用限制性稀释法对食管癌 EC9706 细胞系进行单细胞克隆分离, 建立了 3 个亚系, 分别命名为 EC1 EC2 和 EC3 细胞遗传学分析显示,EC1 仍为混合克隆,EC2 和 EC3 的染色体众数分别为 117 条和 62 条对 EC2 和 EC3 的生物学特性进行比较, 生长曲线平皿集落形成能力实验流式细胞分析和细胞运动能力实验均表明, 两者的恶性程度有显著差别,EC2 较低,EC3 较高成功分离的 2 个食管癌单克隆细胞亚系, 可为食管癌发生发展的分子机制研究基因治疗研究耐药机制研究以及抗肿瘤药物筛选提供实验材料 : 食管癌 ; 细胞系 ; 荧光原位杂交 ; 免疫细胞化学 Isolation and comparative study of clones from human esophageal carcinoma cell line EC9706 HAN Ya-Ling, FENG Yan-Bin, LUO Man-Li, XU Xin, CAI Yan, WANG Ming-Rong State Key Laboratory of Molecular Oncology, Cancer Institute, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing , China Abstract: Cell line is one of the important materials for cancer research. In the present study, single-cell clones were isolated from esophageal carcinoma cell line EC9706 by limited dilution method. Three sublines were obtained, namely EC1, EC2 and EC3. Cytogenetic analysis showed that EC1 was a mixed clone, and the chromosome number of sublines EC2 and EC3 were 117 and 62, respectively. There were significant differences in the growth rate, cell cycle distribution, cell mobility, and colony formation ability between EC2 and EC3. The degree of malignancy was significantly higher for EC3 than for EC2. The monoclonal sublines EC2 and EC3 with different malignant potential provide useful materials for exploring the pathogenesis and gene therapy of human esophageal carcinoma, investigating multidrug resistance, and screening anti-tumor medications. Keywords: esophageal squamous cell carcinoma; cell line; fluorescence in situ hybridization; immunocytochemistry,, 4 [1], 收稿日期 : ; 修回日期 : 基金项目 : ( 2004CB518705) ( ) [Supported by the Grant from National Basic Research Program of China (No.2004CB518705), and the National Natural Science Foundation of China (No )] 作者简介 : (1968 ),,,, Tel: ; [email protected] 通讯作者 : (1956 ),,,, Tel: ; [email protected]

隆,EC2 和 EC3 的染色体众数分别为 117 条和 62 条对 EC2 和 EC3 的生物学特性进行比较, 生长曲线平皿集落形成能力实验流式细胞分析和细胞运动能力实验均表明, 两者的恶性程度有显著差别,EC2 较低,EC3 较高成功分离的 2 个食管

2 1332 HEREDITAS (Beijing) [2~4] EC9706 [5],,, EC9706, (RPMI 1640, 15% ) ml 14, 96, 50 µl( 0.7 / ), 4 h, 50 µl, 5% CO 2 37,, , 0.01 µg/ml, 37 1 h,,, (0.075 mol/l KCl) 10 ml,, min ( : =3 1, ),, r/min, 10 min, 10 ml,, 4, 30 min 3,, Bio-Primer DNA labeling system, h, stop buffer,,, 4, 75 8 min,, h 43 50% /2 SSC 15 min,, DAPI, 1.4, , 6, 5 96, 5% CO 2 37, 24 h, 10 µl Cell Counting kit-8, 1 h 450 nm (OD 450 ), OD 450, 1.5, mm, 3, 14 d 20 min,, Giemsa,, 1.6, 10% FBS RPMI r/min 10 min, 70% min, r/min 10 min, PI (PBS, 0.1% Triton X-100, 50 μg/ml PI, 100 μg/ml RNase A), 30 min, 1.7 Transwell (8-µm ), : 3, 600 µl, 100 µl ( 10 / ), h, PBS,, / (1 1) 20 min 0.5% 5 min, 3 5, 5, mm 35 mm, 10 5, 1.5 ml, 5% CO 2 37, PBS 2,, PV-9000, Ki , 30 min, PBS PV9000 1( )37, 20 min; 2( / IgG )37, 30 min; DAB,, ; 5~10 s, 70% 85% 100% 3 min,,

3 Bio-Primer DNA labeling system, 3 8 10 17 37 16 h, stop buffer,,, 4, 75 8 min,, 37 16 h 43 50% /2 SSC 15 min,, DAPI, 1.

3 11 : EC EC2 EC Ki-67 EC2 EC3 2.1 EC2 EC3 3, EC1 EC2 EC3 10, EC1, EC , 117; EC , 62 EC2 EC , EC2 EC3 ( 1), EC2 EC3 图 2 EC2 与 EC3 第 37 代细胞的生长曲线 Fig. 2 Growth curve of EC2 and EC3 at passage 37 图 1 第 25 代细胞的荧光原位杂交 3, 8, 10, 17 Fig. 1 Chromosome aberrations of clones EC2 and EC3 at passage 25 as detected by FISH Red, blue, yellow and green fluorescence signals indicate chromosomes 3, 8, 10 and 17, respectively. 2.2 EC2 EC3 6 OD, 5 d, OD,, EC2, EC3 ( 2), EC2 EC3(P=0.02, 3), 37 EC , 41 ; EC , 61 EC3 EC2 图 3 EC2 与 EC3 第 37 代细胞的平皿集落形成能力分析 Fig. 3 Colony formation assay of EC2 and EC3 at passage EC2 EC3, EC2 G1 65.5%, EC3 G1 47.8%, EC3, EC2 G1 EC3 S 26%, EC2 19.3%, EC3 EC2, EC3 EC2 ( 4) 2.4 EC3 EC2 Transwell 3, 5 (40 ), 15

2 Growth curve of EC2 and EC3 at passage 37 图 1 第 25 代细胞的荧光原位杂交 3, 8, 10, 17 Fig.

4 1334 HEREDITAS (Beijing) ,, EC2 101, EC3 170 ( 5), EC3 EC2 2.5 Ki-67 EC3 EC2 Ki-67, 300, Ki-67, EC2 Ki-67 75%, EC3 Ki-67 95%( 6), EC3 EC2 图 4 Fig. 4 EC2 与 EC3 第 37 代细胞的流式细胞检测 Cell cycle profile of EC2 and EC3 at passage 37 by flow cytometry 图 5 EC2 与 EC3 第 39 代细胞运动能力检测 (40 ) Fig. 5 Transwell assay of EC2 and EC3 at passage 39 (40 ) 图 6 EC2 与 EC3 第 39 代 Ki 67 免疫细胞化学检测 (10 ) Fig. 6 Immunostaining of Ki-67 in EC2 and EC3 at passage 39 (10 )

5 11 : EC , EC9706 EC3 EC2, EC2 EC3 G1, EC3 S EC2 Ki-67, C1 S M C2, Ki67, EC2 EC3,,,, EC2 EC3, [6], [7] EC9706,,, (References): [1] LI Lian-Di, LU Feng-Zhu, ZHANG Si-Wei, MU Ren, SUN Xiu-Di, XIA Yi. The death epidemiology analysis of malignancies in China in Chin J Oncol, 1996, 18(6): ,,,,, , 1996, 18(6): [2] WANG Xiu-Qin, XIAO Feng, WANG Ming-Rong, ZHOU Chuan-Nong, WU Min. Establishment of two human esophageal carcinomas cell lines and their cytogenetic analysis. Chin J Oncol, 1998, 20(1): 5 8.,,,, 旻.., 1998, 20(1): 5 8. [3] Tang JC, Wan TS, Wong N, Pang E, Lam KY. Establishment and characterization of a new xenograft-derived human esophageal squamous cell carcinoma cell line SLMT-1 of Chinese origin. Cancer Genet Cytogenet, 2001, 124(1): [DOI] [4] Hu Y, Lam KY, Wan TS, Fang WG. Establishment and characterization of HKESC-1, a new cancer cell line from human esophageal squamous cell carcinoma. Cancer Genet Cytogenet, 2000, 118(1): [DOI] [5] HAN Ya-Ling, WEI Fang, XU Xin, CAI Yan, CHEN Bao-Sheng, WANG Jie, XIA Shu-Hua, HU Hai, HUANG Xiao-Ping, HAN Yu-Sheng, WU Min, WANG Ming-Rong. Establishment and comparative genomic hybridization analysis of human esophageal carcinomas cell line EC9706. Chin J Med Genet, 2002, 19(6): ,,,,,,,,,, 旻,. EC9706., 2002, 19(6): [6] Yen CC, Chen YJ, Chen JT, Yen CC, Chen YJ, Chen JT, Hsia JY, Chen PM, Liu JH, Fan FS, Chiou TJ, Wang WS, Lin CH. Comparative genomic hybridization of esophageal squamous cell carcinoma: correlations between chromosomal aberrations and disease progression/prognosis. Cancer, 2001, 92(11): [DOI] [7] SU Yuan, WANG Xiu-Qin, WANG Zhi-Hua, ZHOU Chuan-Nong, WANG Jun, WU Min. Evolution of human esophageal cancer cell in the progress of cell line establishment. Chin J Oncol, 1988, 10(6): ,,,,, 旻.., 1988, 10(6):

Establishment of two human esophageal carcinomas cell lines and their cytogenetic analysis. Chin J Oncol, 1998, 20(1): 5 8.,,,, 旻.., 1998, 20(1): 5 8. [3] Tang JC, Wan TS, Wong N, Pang E, Lam KY.

王声文等 :GNC62 数控系统组态和设备调试设计 2 GNC62 系统组态设计原理 2 2 [1] 2 A I/O 1 293

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