【作者】 郭爱林； 杨素清； 董嵩； 朱建权； 黄玉娟； 黄逸生； 吴红穗； 吴一龙；

【Author】 Guo Ai-Lin Yang Su-Qing Dong Song Zhu Jian-Quan Huang Yv-Juan Huang Yi-Sbeng Wu Hong-Sui Wu Yi-Long Cancer Center, Medical Research Center, Guangdong Provincial People’ s Hospital, Guangzhou 510080, P.R.China.

【机构】 广东省人民医院肿瘤中心医学研究中心广东省肺癌研究所；

【摘要】 目的:体外培养和建立人肺癌细胞系对于研究肺癌癌变,侵袭转移,多药耐药的分子机理,生物学特征,以及开发抗肺癌新药等均有十分重要的理论和临床意义。本研究目的在于建立人肺腺癌细胞系,为肺腺癌体外实验提供研究材料。方法:将患者胸水离心,取沉淀用PBS悬浮,淋巴细胞分离液分离肿瘤细胞, 悬浮于含10％胎牛血清的 RPMI1640培养基中,待细胞长满瓶后,胰酶消化传代。将患者肿瘤组织,裸鼠移植瘤组织,培养细胞常规制备样本, 进行光镜观察。培养细胞进行电镜观察。对培养的肿瘤进行细胞增殖周期测定及染色体分析。应用甲基纤维素测算克隆形成率。RT—PER检测EGFR基因mRNA表达,免疫组化检测肿瘤标志物的表达。采用透射电镜,地依红染色及肉汤培养法检测培养液及细胞内支原体。裸鼠接种观察成瘤情况。结果:成功建立肺腺癌细胞系,取名LYD,成功传代至70代。该细胞系生长快,稳定传代,形态学特征符合腺上皮恶性肿瘤细胞特征。染色体均数70条,分布范围在56— 78之间。EGFR基因mRNA阳性,免疫组化检测CK7(+), TTF1(+),CD44S(+),ECD(+),EGFR(+),CEA(-),CK20(-), 细胞倍增时间为25小时,克隆形成率为20％,致瘤率100 ％,移植瘤病理学特征与原细胞系相似。结论:成功建立人肺腺癌细胞系,为开展人类肺腺癌基础和临床研究提供了理想实用的模型。更多还原

【Abstract】 Objective: Lung cancer is currently the most frequently diagnosed cancer and the most common cause of cancer mortality in males worldwide. The purpose of this study was to characterize cell cultures and xenografts derived from patients with Lung adenocarcinoma. Methods: Cell lines were established from pathologically proven Lung adenocarcinoma. 1000 ml of ascites or pleu-ral effusion was then processed in the laboratory in the following manner. Tumor cells were isolated from neo-plastic effusions by centrifugation. Dissociated tumor cells were then resuspended in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FCS and glutamine (2 mM). The cells remained in culture until sufficiently confluent for a first tissue culture passage. A cell culture was considered established if it could be carried through at least 5 in vitro passages. However, for establishment of a cell line, at least 15 passages were required. Immunoperoxidase staining was performed both on formalin- fixed tissue sections and also on cells prepared from the growing cultured cell lines.When enough material was available, specimens were inoculated into Swiss BALB/nude mice s.c. to obtain xenografts. Cell cultures were suspended in RPMI 1640 at a concentration of 5 - 105 cells/ml and cytospinned. After a 1-min cytospin at 750 rpm, the slides were fixed in cold acetone for 5 min at 20 ℃ and stored at - 20 ℃ until processing. Normal LUNG epithelial cells were cultured in vitro and used as control for the immu-nochemistry experiments. The DNA index and the distribution of cells in specific phases of the cell cycle were determined by standard flow cytometry methods, using an Epics Elite Analyzer. Cell Slides were dried at 57 ℃ before staining with Giemsa for examination Chromosome analysis. Results: Several features of the cell line were investigated, including growth characteristics, electron microscopic features, cloning efficiency in soft agar, expression of various antigenic markers, chromosomal and DNA analysis. On the basis of morphological and immu-nohistochemical analysis of Mehr-80, it is possible to conclude that this cell line is characterized by features similar to those reported for Lung adenocarcinoma Conclusion: This cell line will be a valuable in vitro tool for further studieson lung cancers.更多还原