国际肿瘤学杂志

国际肿瘤学杂志 ›› 2016, Vol. 43 ›› Issue (8): 646-650.doi: 10.3760/cma.j.issn.1673422X.2016.09.002

• 论著 • 上一篇    下一篇

结肠癌COLO耐药株的构建及其与肿瘤干细胞的关系

 甘亚平, 郭晓花, 张君彧, 许晴晴, 吴疆, 王任勇, 邱敏, 蒋汝刚, 刘复兴, 宁志丰   

  1. 437100 咸宁，湖北科技学院基础医学院病理学教研室（甘亚平、邱敏、刘复兴），基础医学院（郭晓花、张君彧、许晴晴、吴疆、王任勇），预防医学教研室（蒋汝刚），解剖学教研室（宁志丰）
  • 出版日期:2016-09-08 发布日期:2016-08-04
  • 通讯作者: 宁志丰，Email: ningzhifeng1976@163.com E-mail:ningzhifeng1976@163.com
  • 基金资助:

    国家级大学生创新创业项目（201310927001）；湖北省自然科学基金（2015CFB470）

Construction of colonic cancer drugresistant cell line COLO and its relationship with tumor stem cells

Gan Yaping, Guo Xiaohua, Zhang Junyu, Xu Qingqing, Wu Jiang, Wang Renyong, Qiu Min, Jiang Rugang, Liu Fuxing, Ning Zhifeng   

  1. Department of Pathology, Basic Medical College of Hubei University of Science and Technology, Xianning 437100, China
  • Online:2016-09-08 Published:2016-08-04
  • Contact: Ning Zhifeng E-mail:ningzhifeng1976@163.com
  • Supported by:

    National Undergraduate Innovative and Business Program (201310927001); Natural Science Foundation of Hubei Province of China (2015CFB470)

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摘要： 【摘要】目的构建结肠癌COLO耐药株，探索其功能特性及其与肿瘤干细胞的关系。方法通过逐渐递增化疗药5氟尿嘧啶（5FU）浓度的方法，构建了耐0.10 μmol/ml和0.20 μmol/ml 5FU的结肠癌COLO耐药株各1株，分别命名为COLO/5FU1、COLO/5FU2。从增殖能力、克隆形成能力、迁移和侵袭能力、微球体形成能力、干性基因的表达、交叉耐药等方面与亲本结肠癌细胞株COLO进行对比。结果在存活检测中，常规培养后第4天，结肠癌耐药株COLO/5FU2、COLO/5FU1、亲本COLO细胞的A值分别为0.61±0.13、0.54±0.07、0.41±0.09，差异有统计学意义（F=63.43，P=0.033）。随着耐5FU药物浓度的增加，COLO耐药株的克隆形成率逐渐增强，COLO/5FU2、COLO/5FU1、亲本COLO的克隆形成率依次为(87.6±12.7)%、(65.3±9.7)%、(38.5±7.6)%，差异有统计学意义（F=33.64，P=0.017）。COLO/5FU2、COLO/5FU1、亲本COLO每高倍视野中迁移穿过基底膜的细胞数分别为（482±39）个、（434±45）个、（373±38）个，侵袭穿过基底膜的细胞数分别为（174±42）个、（112±31）个、（87±29）个，差异均有统计学意义（F=109.61，P=0.009；F=67.31，P=0.032）。与亲本结肠癌细胞株COLO比较，耐药株表达更高的干性基因，差异有统计学意义（F=47.31，P=0.042）；且耐药株对其他化疗药如米托蒽醌存在交叉耐药［如给药96 h，米拖蒽醌对亲本COLO的抑制率高于COLO/5FU1、COLO/5FU2 （0.749±0.042、0.423±0.024、0.342±0.018），差异有统计学意义 （F=12.61, P=0.028）］。COLO/5FU2、COLO/5FU1、亲本COLO细胞的微球体形成率依次为（8.90±0.97）%、（6.20±0.75）%、（3.90±0.32）%，差异有统计学意义（F=164.32，P=0.006）。结论结肠癌COLO耐药株有肿瘤干细胞样细胞的特点，是富集了的肿瘤干细胞。

关键词: 结肠肿瘤, 抗药性, 肿瘤, 肿瘤干细胞, 耐药株

Abstract: 【Abstract】ObjectiveTo construct a colon cancer chemotherapyresistant cell line COLO, and study its characteristics and its relationship with tumor stem cells. MethodsWe constructed two 5fluorouraci (5FU)resistant colon cancer cell line COLO/5FU1 and COLO/5FU2, which were resistant to 0.10 μmol/ml and 0.20 μmol/ml 5FU respectively through gradiently increased drug concentration. The characteristics of 5FUresistant cell lines were compared with parental colon cancer cell line COLO related to proliferation, colony forming ability, migration and invasion, sphere forming ability, expression of stemness genes and cross drugresistance. ResultsIn the cell viability assay, 4 days after regular training, the absorbancy of colon cancer 5FUresistant cell lines COLO/5FU2, COLO/5FU1 and parental colon cancer cell line COLO were 0.61±0.13, 0.54±0.07 and 0.41±0.09 respectively, with significant difference (F=63.43, P=0.033). With the increased concentration of 5FU, 5FUresistant cell lines presented increasing clonality. The cloning efficiency of COLO/5FU2, COLO/5FU1 and parental colon cancer cell line COLO were (87.6±12.7)%, (65.3±9.7)% and (38.5±7.6)% respectively, with significant difference (F=33.64, P=0.017). In each high power field of vision, the cell numbers of migration through the basement membrane of COLO/5FU2, COLO/5FU1 and parental colon cancer cell line COLO were 482±39, 434±45 and 373±38 respectively; and the cell numbers of invasion through the basement membrane were 174±42, 112±31 and 87±29 respectively, with significant differences (F=109.61, P=0.009; F=67.31, P=0.032). Compared with parental colon cancer cell line COLO, 5FUresistant cell lines had higher expression of stemness genes (F=47.31, P=0.042). 5FUresistant cell lines were crossresistant to other chemotherapeutic drugs such as mitoxantrone. For example, after incubation for 96 hours, inhibition rate of mitoxantrone to parent colon cancer cell line COLO was higher significantly than COLO/5FU1 and COLO/5FU2 (0.749±0.042, 0.423±0.024, 0.342±0.018), with significant difference (F=12.61, P=0.028). The microsphere forming rates of COLO/5FU2, COLO/5FU1 and parental colon cancer cell line COLO were (8.90±0.97)%, (6.20±0.75)% and (3.90±0.32)% respectively, with significant difference (F=164.32, P=0.006). ConclusionColon cancer drugresistant cell line COLO possess tumor stem celllike characteristics, which are enriched in cancer stem cells.

Key words: Colonic neoplasms, Drug resistance, neoplasms, Neoplastic stem cells, Drugresistant cell line