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

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