食管鳞癌紫杉醇耐药细胞株的建立及耐药机制探讨  

Establishment of esophageal squamous cell carcinoma paclitaxe-resistant cell line and investigation of its drug resistance mechanism

作　　者：惠怡然 杨珍珍 李岳衡 高盼 许培荣[3] 范天黎 HUI Yiran;YANG Zhenzhen;LI Yueheng;GAO Pan;XU Peirong;FAN Tianli(Department of Pharmacology,School of Basic Medical Sciences,Zhengzhou University,Zhengzhou 450001;Department of Pharmacy,People~ Hospital of Zhengzhou,Zhengzhou 450003;School of Pharmaceutical Sciences,Zhengzhou University,Zhengzhou 450001)

机构地区：[1]郑州大学基础医学院药理学教研,室郑州450001 [2]郑州人民医院药剂科,郑州450003 [3]郑州大学药学院,郑州450001

出　　处：《郑州大学学报（医学版）》2018年第5期552-557,共6页Journal of Zhengzhou University(Medical Sciences)

基　　金：河南省高等学校科技创新人才项目(15HASTIT037); 河南省基础与前沿技术研究计划项目(152300410052)

摘　　要：目的:建立食管鳞癌紫杉醇(PTX)耐药细胞株EC1/PTX,并对其耐药机制进行初步探讨。方法:采用大剂量间歇冲击结合时间递增的方法构建食管鳞癌耐药细胞株EC1/PTX。倒置显微镜下观察EC1/PTX形态变化;细胞计数法绘制EC1和EC1/PTX的生长曲线并计算倍增时间;CCK-8法检测PTX、多柔比星、五氟尿嘧啶、顺铂对细胞的IC50及其耐药指数;Transwell实验检测细胞迁移、侵袭能力;流式细胞术检测细胞周期及凋亡率;平板克隆形成实验检测细胞克隆形成能力;Western blot法检测细胞中P-糖蛋白(P-gp),凋亡相关蛋白Bcl-2、Bax的表达。结果:历时8个月成功构建了EC1/PTX。EC1/PTX细胞多呈聚团生长状态;对PTX的耐药指数为12. 82,并表现出多药耐药。与EC1相比,EC1/PTX生长倍增时间延长;迁移和侵袭能力增强;细胞周期分布改变,G0/G1、S期细胞增多,G2/M期细胞减少;细胞凋亡率下降;平板克隆形成能力降低;P-gp、Bcl-2蛋白表达升高,Bax表达降低(P <0. 05)。结论:成功建立了食管鳞癌耐药细胞株EC1/PTX,其耐药机制可能与P-gp及Bcl-2家族蛋白的表达变化有关。Aim：To establish paclitaxel-resistant cell line of esophageal squamous cell carcinoma（ EC1/PTX） and to investigate the drug resistance mechanism. Methods：High-dose intermittent shock combined with time-increasing methods was used to construct esophageal squamous cell carcinoma paclitaxel（ PTX）-resistant cell line named EC1/PTX. The morphology of EC1/PTX was observed under an inverted microscope. The growth curves of EC1 and EC1/PTX were plotted by cell counting method and the doubling time was calculated. The IC50 of PTX,ADM,5-FU,and CDDP was detected by CCK-8 method and resistance index was calculated. The migration and invasion of EC1 and EC1/PTX were detected by Transwell assay,cell cycle and apoptosis were detected by flow cytometry,and the colony forming ability was detected by plate colony formation assay. The expressions of P-glycoprotein（ P-gp）,and apoptosis-related proteins Bcl-2 and Bax were detected by Western blot. Results：EC1/PTX was successfully constructed after 8 months. EC1/PTX was mostly agglomerated.The resistance index to PTX was 12. 82,and multidrug resistance was observed. Compared with EC1 cells,the doubling time of EC1/PTX was prolonged; migration and invasion capability were enhanced; the distribution of cell cycle was changed,presenting as the cell number of G0/G1 and S phases increased,and that of G2/M phase decreased; the apoptosis rate and plate colony formation ability were lower; P-gp and Bcl-2 protein expressions increased,while Bax expression was reduced（ P〈0. 05）. Conclusion：EC1/PTX has been successfully established. The mechanism of drug resistance may be related to the expressions of P-gp and Bcl-2 family proteins.

关 键 词：食管癌 多药耐药 紫杉醇 

分 类 号：R735.1[医药卫生—肿瘤]