多药耐药基因MDR1在Ph(+)急性淋巴细胞白血病伊马替尼耐药细胞株SUP-B15/RI耐药形成中的作用

Multidrug Resistant Gene MDR1 Contributes to Development of Imatinib-Resistance in Ph(+) Acute Lymphoblastic Leukemia Cell Line SUP-B15RI

目的研究多药耐药基因MDR1在Ph(+)急性淋巴细胞白血病〔Ph(+)ALL〕伊马替尼(IM)耐药细胞株SUP-B15/RI耐药机制中的作用。方法 RT-PCR技术检测敏感细胞株SUP-B15和IM耐药细胞株SUP-B15/RI MDR1mRNA表达,改良MTT法测定IM、柔红霉素(DNR)、长春新碱(VCR)、依托泊甙(VP-16)单药及联合维拉帕米作用于SUP-B15细胞和SUP-B15/RI细胞72h后增殖活性的改变,计算抑制率为50%时的药物浓度(IC50),DNR药物外排实验检测MDR1表达产物P-gp功能。结果 MDR1基因在SUP-B15/RI中的表达是敏感细胞SUP-B15的(2.02±0.04)倍(P<0.05)。IM、DNR、VCR、VP-16单药作用于SUP-B15/RI细胞的IC50值较SUP-B15细胞增高(P<0.05),相对耐药倍数(RF)分别为(20.52±2.34)、(10.33±1.88)、(9.78±1.27)、(3.84±0.69)倍。IM、DNR、VCR、VP-16与维拉帕米联合作用于SUP-B15/RI细胞后IC50值较单药时降低(P<0.05),耐药逆转倍数分别为(1.44±0.43)、(3.20±0.17)、(1.44±0.12)、(1.33±0.14)。P-gp蛋白功能在SUP-B15/RI细胞强于SUP-B15细胞,DNR泵出率分别为10.27%、3.43%。结论 MDR1基因在SUP-B15/RI细胞中表达增加,其表达产物P-gp功能增强,P-gp抑制剂维拉帕米可以部分逆转IM耐药。说明MDR1参与SUP-B15/RI细胞IM耐药机制的形成。

Objective To investigate the contribution of muhidrug-resistant gene MDR1 to development of imatinib-resistance in Ph（＋）acute lymphoblastic leukemia cell line SUP-BaS/RI. Methods RT-PCR was used to examine MDR1 mRNA levels, cytotoxic effects of imatinib （IM）, daunorubicin （DNR）, vincristine （VCR）, etoposide （VP-16） and the synergetic antiproliferation with P-gp inhibitor verapamil on sensitive SUP-B15 and SUP- B15/RI cell lines were detected by the MTT assay. The P-gp function was measured by flow cytometry. Results Increased expression of MDR1 gene in SUP-B15/RI than that of SUP-B15 cell line （P〈0.05） was observed when detected with RT-PCR. The IC50 values of SUP-B15/RI cell line inhibited by IM, DNR, VCR, VP-16 for 72 hours was higher than that of SUP-B15 （P〈0.05） and the resistant factor （RF） was （20. 52±9~. 34）,（10. 33±1. 88）, （9.78± 1. 27）, （3. 84±0. 69） respectively. The IC50 values of IM, DNR, VCR, VP-16 combined with P-gp inhibitor verapamil were decreased in SUP-B15/RI cells （P〈0.05）, reversal of drug resistance was （1.44± 0.43）, （3.20±0.17）,（1.44±0.12）,（1.33±0.14） respectively. The activity of P-gp in SUP-B15/RI measured by flow cytometry was higher than that of P-gp in SUP-B15/RI cell line. Conclusion The overexpression of MDR1 mRNA and higher activity of P gp is partially responsible for acquiring of imatinib resistance in SUP-B15/RI cell line. P-gp inhibitor verapamail can partially restored the sensitivity of the SUP-B15/RI cell line to anticancer agents.