1332 HEREDITAS (Beijing) 2007 29 [2~4] EC9706 [5],,, 1 1.1 EC9706, (RPMI 1640, 15% ) ml 14, 96, 50 µl( 0.7 / ), 4 h, 50 µl, 5% CO 2 37,, 24 1.2, 0.01

HEREDITAS (Beijing) 2007 11, 29(11): 1331 1335 ISSN 0253-9772 www.chinagene.cn 研 究 报 告 DOI: 10.1360/yc-007-1331 EC9706 韩 亚 玲, 冯 彦 斌, 罗 曼 莉, 徐 昕, 蔡 岩, 王 明 荣 100021 : 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 100021, 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 : 2007 05 29; 修 回 日 期 : 2007 09 18 基 金 项 目 : ( 2004CB518705) ( 30470969) [Supported by the Grant from National Basic Research Program of China (No.2004CB518705), and the National Natural Science Foundation of China (No.30470969)] 作 者 简 介 : (1968 ),,,, Tel: 010-87788425; E-mail: hyl1228@yahoo.com.cn 通 讯 作 者 : (1956 ),,,, Tel: 010-87788425; E-mail: wangmr04@126.com

1332 HEREDITAS (Beijing) 2007 29 [2~4] EC9706 [5],,, 1 1.1 EC9706, (RPMI 1640, 15% ) ml 14, 96, 50 µl( 0.7 / ), 4 h, 50 µl, 5% CO 2 37,, 24 1.2, 0.01 µg/ml, 37 1 h,,, (0.075 mol/l KCl) 10 ml,, 37 30 min ( : =3 1, ),, 1 000 r/min, 10 min, 10 ml,, 4, 30 min 3,, 20 1.3 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, 1.4, 2 000 96, 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, 100 60 mm, 3, 14 d 20 min,, Giemsa,, 1.6, 10% FBS RPMI-1640 1 000 r/min 10 min, 70% 20 30 min, 1 000 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 / ), 37 16 h, PBS,, / (1 1) 20 min 0.5% 5 min, 3 5, 5, 1.8 20 20 mm 35 mm, 10 5, 1.5 ml, 5% CO 2 37, PBS 2,, PV-9000, Ki-67 1 500, 30 min, PBS PV9000 1( )37, 20 min; 2( / IgG )37, 30 min; DAB,, ; 5~10 s, 70% 85% 100% 3 min,,

11 : EC9706 1333 2 EC2 EC3 25 37 39 50 52 Ki-67 EC2 EC3 2.1 EC2 EC3 3, EC1 EC2 EC3 10, EC1, EC2 102 122, 117; EC3 59 62, 62 EC2 EC3 3 8 10 17, 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 EC2 3 100 39 45 41, 41 ; EC3 3 57 65 62, 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 37 2.3 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

1334 HEREDITAS (Beijing) 2007 29,, 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 )

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