目的: 建立食管癌耐长春新碱（vincristine, VCR）细胞株 Eca-109/VCR, 并对其耐药机制进行初步探讨。方法: 采用大剂量间歇冲击给药结合时间递增的方法构建食管癌耐药株Eca-109/VCR。倒置显微镜下观察 Eca-109/VCR 形态变化;细胞计数法绘制Eca-109 和 Eca-109/VCR 的生长曲线并计算倍增时间;CCK-8 法检测VCR、fluorouracil（5-FU）、paclitaxel（PTX）对细胞的inhibitory concentration 50（IC50）及其耐药指数;流式细胞术检测细胞周期及凋亡率;免疫细胞化学、Western blot 检测Eca-109/VCR 细胞P-glycoprotein（P-gp）、multi-drug resistance related protein（MRP）的表达。结果: 历时6 个月成功构建了Eca-109/VCR。Eca-109/VCR 细胞多呈聚团生长状态, 对VCR 的耐药指数为17. 60, 并表现出多药耐药, 对5-FU、PTX 的耐药指数分别为6. 59、5. 60（P<0. 01）。与Eca-109 相比, Eca-109/VCR 生长倍增时间延长, 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（P<0. 05）;P-gp、MRP 蛋白表达升高（P<0. 05）。结论: 建立了食管癌耐长春新碱细胞株Eca-109/VCR, 其多药耐药机制可能与 P-gp及MRP 表达上调有关。
Objective: To establish a vincristine-resistant cell line of esophageal cancer（Eca-109/VCR）and to investigate the drug-resistance mechanism. Methods: The vincristine（VCR）-resistant esophageal cancer cell line（Eca-109/VCR）was constructed by high-dose intermittent shock combined with time-increasing methods. The morphology of Eca-109/VCR cells were observed under the inverted microscope. The growth curves of Eca-109 and Eca-109/VCR were plotted by cell counting method, and the doubling time was calculated. The IC50 and resistance index of VCR/5-FU/PTX were detected by CCK-8 method. The cell cycle and apoptosis were detected using flow cytometry. The expressions of P-glycoprotein（P-gp）, and multidrug resistance related protein（MRP） were detected by immunocytochemical staining and Western blot. Results: Eca-109/VCR cells were successfully constructed after 6 months. Eca-109/VCR cells mostly grew in clusters. The resistance index to VCR was 17. 60, and presenting multidrug resistance, the resistance index to 5-Fu and PTX were 6. 59 and 5. 60 respectively （P<0. 01）. Compared with Eca-109 cells, the growth doubling time of Eca-109/VCR cells were prolonged;migration and invasion capability were enhanced, and the distribution of cell cycle was changed, as the cell number of G0/G1 and S were observed increased, and that of G2/M phase decreased;the apoptosis rate was lower;Immunocytochemistry and Western blot showed that the relative expression of P-gp and MRP in Eca-109/VCR was higher than that in Eca109 cells（P<0. 05）. Conclusion: Eca-109/VCR cell line has been successfully established. The mechanism of multiple drug-resistance may be related to the increased expression of P-gp and MRP proteins.
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