摘要
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 Eca-109 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.
作者
孙晓冉
辛运超
孙剑经
李多
张林西
SUN Xiao-ran;XIN Yun-chao;SUN Jian-jing;LI Duo;ZHANG Lin-xi(The First Affiliated Hospital of Hebei North University,Zhangjiakou 075000,China;Hebei North University,Zhangjiakou 075000,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2021年第3期446-451,共6页
Chinese Journal of Pharmaceutical Analysis
