人肝癌5-脱氧-5-氟尿苷耐药细胞模型的建立及耐药机制探讨

Establishment of a 5-DFUR-resistant human hepatocellular carcinoma cell model and preliminary study of the mechanisms of the drug resistance

目的建立耐药肝癌细胞模型BEL-7402/5-脱氧-5-氟尿苷(5'-DFUR)并研究其耐药特性。方法采用体外低浓度递增联合大剂量间断冲击的诱导方法建立耐药人肝癌细胞模型BEL-7402/5'-DFUR细胞株。用MTT法检测该耐药细胞模型的多药耐药性,以流式细胞术检测该耐药模型细胞周期中的分布、细胞表面多药耐药基因(mdr1)的表达产物P-糖蛋白(P-gp)、bcl-2及谷胱甘肽S-转移酶-π(GST-π)的表达等。结果(1)经MTT法检测BEL-7402/5'-DFUR细胞较亲本细胞的5'-DFUR半数抑制浓度(IC50)增大12.9倍,并对多种抗癌药物产生耐药性。(2)耐药细胞S期细胞减少,G1、G2期细胞增多。(3)该耐药模型细胞表面mdr1的表达产物P-gp、Bcl-2非常显著升高,而GST-π表达无明显变化。结论BEL-7402/5'-DFUR细胞是一个明确的多药耐药模型,具有耐药细胞的基本生物学特性,同亲本细胞相比,其IC50提高12.9倍。细胞内药物蓄积明显降低,细胞周期分布发生变化,其多药耐药性与P-gp、bcl-2的高表达有密切关系。

Objective To establish a multidrug-resistant hepatocellular carcinoma cell line BEL-7402/5-deoxy-5-fluorouridine （5＇-DFUR） and study the mechanisms of the drug resistance. Methods BEL-7402/5＇-DFUR cell line was induced by pulse therapy combined with continuous stepwise exposure to 5＇-DFUR in vitro. The multidrug resistance of BEL-7402/5＇-DFUR cell line to the antitumor agents was evaluated by MTT assay. The distribution of the cell cycle, the expressions of P-gp, bcl-2 and GST-π were detected by flow cytometry. Results The established BEL-7402/5＇-DFUR cell line was resistant to multiple antitumor agents, with IC50 of 5＇-DFUR 12.9 times higher than that of the parental cell line 7204. The BEL-7402/5＇-DFUR cells in S phase decreased while those in G1 and G2 phase increased, with significantly increased expressions of P-gp and bcl-2 but stable expression of GST-π. Conclusion Compared with its parent cell line BEL-7402, the multidrug resistant cell line BEL-7402/5＇-DFUR has a 12.9-fold increase in IC50 of 5＇-DFUR with decreased drug accumulation and altered cell cycle distribution. The multi-drug resistance of this cell line is closely related to the overexpression of P-gp and bcl-2.