急性淋巴细胞白血病阿糖胞苷耐药细胞株的构建及机制研究

Establishment of Cytarabine-resistant Acute Lymphoblastic Leukemia Cell Lines and Its Resistance Mechanism

目的:构建耐阿糖胞苷(Ara-C)的急性淋巴细胞白血病(ALL)耐药细胞株,并探讨可能耐药机制。方法:低浓度Ara-C持续诱导培养Jurkat和Nalm-6细胞,构建Jurkat/Ara-C和Nalm-6/Ara-C耐药细胞株。CCK-8法检测细胞增殖活力,流式细胞术检测细胞周期分布,实时荧光定量PCR检测多药耐药基因和Ara-C代谢酶m RNA表达水平,免疫印迹法检测细胞周期蛋白表达水平。结果:成功构建了Jurkat/Ara-C和Nalm-6/Ara-C耐药细胞株,耐药指数分别为1 973.908±161.163和7 231.643±1 190.624,耐药细胞株对阿霉素等常用化疗药物无交叉耐药。流式细胞术检测结果显示,Jurkat/Ara-C细胞G_(0)/G_(1)期比例增加(P<0.05),G_(2)/M期减少(P<0.05),而Nalm-6/Ara-C细胞周期分布较Nalm-6细胞无明显改变。实时荧光定量PCR检测发现脱氧胞苷激酶和胞苷脱氨酶在耐药细胞中低表达(P<0.05),MRP在Nalm-6/Ara-C细胞中高表达(P<0.05),MDR1、LRP和BCRP未见明显增加。蛋白免疫印迹检测发现Cyclin B1在耐药细胞中明显低表达(P<0.05),Cyclin D1未见明显改变。结论:低浓度持续诱导法成功构建了ALL耐Ara-C细胞株,其耐药机制可能与脱氧胞苷激酶和Cyclin B1的缺乏有关。

Objective:To establish cytarabine-resistant acute lymphoblastic leukemia(ALL)cell lines and investigate its possible resistant mechanism.Methods:Low-concentration cytarabine(Ara-C)continuously induced and cultured Jurkat and Nalm-6 cells to construct cytarabine-resistant cell lines Jurkat/Ara-C and Nalm-6/Ara-C.The cell viability was detected by CCK-8 assay,and the distribution of cell cycle was detected by flow cytometry.Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of multidrug resistant gene and Ara-C metabolic enzymes.The expression levels of cyclin were detected by Western blot.Results:Jurkat/Ara-C and Nalm-6/Ara-C drugresistant cell lines were successfully established,the resistance index of which was 1973.908±161.163 and 7231.643±1190.624,respectively.Drug-resistant cell lines had no cross-resistance to commonly used chemotherapeutic drugs,such as doxorubicin.Flow cytometry showed that the ratio of G0/G1 phase in Jurkat/Ara-C cells increased but G2/M phase decreased(P<0.05),while the cell cycle distribution of Nalm-6/Ara-C cells did not change in comparison with Nalm-6 cells.The results of real-time quantitative PCR showed that the expression of deoxycytidine kinase(DCK)and cytidine deaminase(CDA)were significantly down-regulated in drug-resistant cells(P<0.05),MRP was up-regulated in Nalm-6/Ara-C cells(P<0.05),while MDR1,LRP and BCRP did not increase in comparison with parental cells.Western blot analysis revealed that cyclinB1 was significantly under-expressed in drug-resistant cells(P<0.05),while cyclinDl did not change,when compared with parental cells.Conclusion:Cytarabine-resistant ALL cell lines are successfully established by using low concentration continuous induction method,and its drug-resistant mechanism may be related to the deficiencies of DCK and cyclinB 1.