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目的:检测胰腺癌细胞膜上Na+非依赖的平衡型核苷转运载体(ENT)和Na+依赖性的集中型核苷转运载体(CNT),并对ENT进行定量检测。方法:将胰腺癌细胞株(Panc-1)在含潘生丁(100μmol/L)的培养基中分别温育8,15,60,120 min,收集细胞用乙腈裂解,荧光分光光度计测定细胞悬液中潘生丁的荧光强度,依据平行悬液中的细胞数量计算出单个细胞表面ENT的数量;再将Panc-1分别在含5-氟尿嘧啶(5-FU)和5-FU+潘生丁两种培养基中分别温育15,30,60,120,240 min,毛细管区带电泳法测定5-FU含量;根据平行悬液中的细胞数量和单细胞体积计算单细胞中5-FU的浓度,间接判定细胞膜上是否存在CNT载体。结果:Panc-1在含潘生丁的培养基温育8 min后即能检测到细胞的荧光强度,15 min达高峰20.2×10-19mol;借助潘生丁测得单个细胞表面有1.25×105个ENT。用潘生丁阻断ENT后,5-FU仍可被转运入胰腺癌细胞中,且细胞内的5-FU最高浓度达到(138.3±9.77)mg/L,明显高于培养基中5-FU的浓度(P=0.011)。结论:与潘生丁结合后,通过荧光法可以定量检测细胞表面的ENT;通过阻断ENT并测定细胞中5-FU浓度,可以判断胰腺癌细胞膜上存在CNT,从而对合理应用化疗药物,提高化疗效果提供依据。
OBJECTIVE: To detect Na + -independent balanced nucleoside transporter (ENT) and Na + -dependent concentrated nucleoside transporter (CNT) in pancreatic cancer cell membrane and quantitatively detect ENT. Methods: Panc-1 cells were incubated for 8, 15, 60 and 120 min in the medium containing dipyridamole (100 μmol / L). The cells were lysed with acetonitrile and determined by fluorescence spectrophotometer , And then calculate the number of ENT on the single cell surface according to the number of cells in the parallel suspension. Then, Panc-1 was separately incubated in 5-fluorouracil (5-FU) and 5-FU + The content of 5-FU was determined by capillary zone electrophoresis at 15, 30, 60, 120, 240 min. The concentration of 5-FU in single cells was calculated according to the number of cells in parallel suspension and single cell volume. Results: The fluorescence intensity of Panc-1 was detected after incubated with dipyridamole for 8 min, reaching a peak of 20.2 × 10-19 mol at 15 min. On the single cell surface, Panc-1 had 1.25 × 105 ENT. After dipyridamole was used to block ENT, 5-FU could still be transported into pancreatic cancer cells, and the highest intracellular concentration of 5-FU reached (138.3 ± 9.77) mg / L, which was significantly higher than that of 5-FU (P = 0.011). CONCLUSIONS: The binding of dipyridamole to ENT on the cell surface can be quantitatively detected by fluorescence spectroscopy. By blocking ENT and measuring the concentration of 5-FU in cells, the presence of CNTs on the membrane of pancreatic cancer cells can be judged, so that the chemotherapeutic drugs can be rationally used to improve the chemotherapy effect Provide evidence.