人肝癌顺铂耐药细胞系SK-Hep1/CDDP的建立

Establishment of a cisplatin-induced multidrug resistance cell line SK-Hep1/DDP

背景与目的:多药耐药是肿瘤治疗的主要障碍,本研究旨在建立人肝癌多药耐药细胞株SK-Hep1/CDDP,并对其生物学特性及发生多药耐药的可能机制进行初步评价。方法:采用大剂量冲击,间歇诱导法获得人肝癌顺铂(Cisplatin,CDDP)多药耐药系SK-Hep1/CDDP;CCK-8法检测药物敏感性,计算半数抑制浓度(IC50)和耐药指数(RI);Western blot检测多药耐药基因(MDR1,ABCB1)、多药耐药相关蛋白1(MRP-1,ABCC1)、多药耐药相关蛋白2(MRP-2,ABCC2)、Bax蛋白的表达,以及加入MDR1抑制剂CsA对肝癌细胞MDR1蛋白的表达影响;流式细胞仪(FCM)检测细胞周期及凋亡率。结果:历时6个月建成SK-Hep1/CDDP细胞株,其对CDDP的耐药指数为13.76,并对盐酸阿霉素(doxorubicin,DOX)、氟尿嘧啶(5-Fluororacil,5-FU)等多种抗肿瘤药物交叉耐药;Westernblot发现SK-Hep1/CDDP与亲本细胞相比MDR1、MRP-1、MRP-2的表达明显升高(P<0.01),Bax蛋白表达明显降低(P<0.01),加入小剂量环孢素A对肝癌细胞MDR1蛋白的表达无影响(P>0.05);细胞周期分析发现相对于亲本SK-Hep1细胞[G1期为(59.83±3.28)%,S期为(27.91±2.16)%,G2/M期为(12.14±3.36)%],SK-Hep1/CDDP耐药细胞[G1期为(37.50±5.05)%,S期为(42.20±2.65)%,G2/M期(20.67±5.69)%]的G2/M,S期比例增加,G1期比例减少,两组相比差异均有统计学意义(P<0.01);流式细胞仪分析表明CDDP对耐药细胞SK-Hep1/CDDP的凋亡率明显减少,加入MDR1抑制剂干预后可明显增加CDDP对SK-Hep1/CDDP细胞的凋亡率。结论:成功建立MDR细胞株SK-Hep1/CDDP,其耐药机制可能与MDR1、MRP-1、MRP-2蛋白的表达增加,Bax蛋白表达降低及降低化疗药物诱导肿瘤细胞的凋亡作用相关。

Background and Objective： Multidrug resistance （MDR） is a major obstacle in the chemotherapy of cancer patients. The aim of this study was to establish a mutidrug-resistant cell line SK-Hep1/DDP and explore its molecular mechanism of the MDR. Methods： SK-Hep1/DDP cell line was induced by pulse treatment using a high concentration of cisplatin （DDP） in vitro. The chemoresistance indexes of cells were evaluated by CCK-8 assays. The protein of MDR1 （ABCB1）, MRP1 （ABCC1）, MRP2 （ABCC2） and Bax were detected by Western blotting, and the effect of MDR1 inhibitor cyclosporine A （CsA） on expression of MDR1 proteins in SK-Hep1 and SK- Hep1/DDP cell lines. Flow cytometry was performed to determine the distribution of the cell cycle and cell apoptosis ratio. Results： The SK-Hep1/ DDP cells were 13.76 times more resistant to DDP in comparison with SK- Hep1 cells, and SK-Hep1/DDP cells also exhibited cross-resistance to many other chemotherapeutic agents （for example adramycin and 5-fuorouracil）. MDR1, MRP1 and MRP2 protein expressions were significantly higher in the SK-Hepl/DDP than in the SK-Hep1 （P〈0.01）, but Bax was lower in the SK- Hep1/DDP than in the SK-Hepl （P〈0.01）. There was no obvious influence between SK-Hep1 and SK-Hep1/DDP cells in the expression of MDR1 by MDR1 inhibtor CsA （P〉 0.05）. The percentages of cells in G2/M and S phase were significantly increased in SK-Hep1/DDP in comparison with those in SK-Hep1 [（20.67±5.69）% vs. （12.14 ± 3.36）% ; （42.20±2.65）% vs. （27.91 ± 2.16）%; P〈 0.01]. Afterthe cells were exposed to 10 μg/mL DDP for 24 h, the cell apoptosis rate of SK-Hep1/DDP was decreased in comparison with SK-Hep1, but it was increased in those with pretreatment of MDR1 inhibitor CsA as compared with those without pretreatment. Conclusions： A reliable multidrug-resistant human hepatoma cell line SK- Hep1/DDP is successfully established. The MDR mechanisms of this cell lines are closely related to the over-expression of MDR1, MRP1 and MRP2, lower expression of Bax, and the attenuated cell apoptosis induced by chemotherapeutic agents.