青蒿琥酯逆转多发性骨髓瘤细胞株多药耐药的实验研究

Study on artesunate in reversing MDR of multiple myeloma cell lines

目的建立小鼠骨髓瘤耐阿霉素(ADM)细胞系SP2/0/ADM,研究青蒿琥酯(ART)对其的影响。方法 ADM低浓度加量持续诱导法建立耐药细胞株;罗丹明123(Rh123)排出实验观察SP2/0/ADM细胞对药物外排情况;采用四甲基偶氮唑蓝(MTT)实验分析SP2/0/ADM细胞株耐药谱和ART对SP2/0/ADM细胞的增殖抑制作用及耐药逆转作用;通过Western blot法检测10μg/ml ART作用24、48、72、96 h后,SP2/0/ADM细胞P-糖蛋白(P-gp)表达变化。结果MTT实验结果显示,SP2/0/ADM细胞不但对ADM产生了耐药性[耐药指数(RI)为22.6],对米托蒽醌、足叶已苷和甲氨蝶呤也产生了不同程度交叉耐药,其RI分别为4.1、2.8和5.3。Rh123排出实验结果显示SP2/0/ADM细胞荧光强度低,而SP2/0细胞中荧光强度高。随ART浓度增加,对SP2/0/ADM细胞增殖抑制作用增强,呈剂量依赖性(P<0.05),其20%抑制浓度(IC20)约为0.3μg/ml。选此浓度进行耐药逆转实验,结果显示ART可增强ADM对SP2/0/ADM细胞的毒性作用,其半数药物抑制浓度(IC50)由17.81μg/ml降至4.26μg/ml,耐药逆转倍数为4.15倍(P<0.05)。随ART作用时间延长,SP2/0/ADM细胞P-gp蛋白表达水平呈下降趋势。与对照组(6 568.25±156.93)比较,ART作用24、48、72、96 h组P-gp蛋白灰度值分别为(4 459.12±297.13)、(4 218.35±153.21)、(3 558.52±241.38)、(3 024.85±157.33),差异有统计学意义(P<0.05)。结论SP2/0/ADM细胞株是成功的骨髓瘤耐药细胞株;ART能逆转SP2/0/ADM细胞对ADM耐药;ART抑制P-gp蛋白表达是其重要作用机制。

Objective To establish the myeloma cell line SP2/0/ADM,which is resistant to adriamycin（ADM）,and study the effect of artesunate（ART） on them.Methods The myeloma cell line SP2/0/ADM resistant to ADM was established in vitro by exposing SP2/0 parent cells to increasing concentrations of ADM.The drug discharge capacity of SP2/0/ADM cells was assessed by the fluorescent dye rhodamine 123 efflux experiment.By MTT assay,resistance spectrum of SP2/0 /ADM cells was analyzed,and inhibiting proliferation and reversing multidrug resistance effect of ART on SP2/0/ADM cells were detected.Western blot was used to detect the expression of P-glycoprotein（P-gp） after 10 μg/ml ART treatment for 24 hours,48 hours,72 hours and 96 hours.Results SP2/0/ADM cells developed drug-resistance not only to ADM but also to mitoxantrone,etoposide and methotrexate.The resistance index was 22.6 for ADM,4.1 for mitoxantrone,2.8 for etoposide and 5.3 for methotrexate respectively.The fluorescent intensity of SP2/0/ADM cells was weaker than that in SP2/0 cells in the fluorescent dye rhodamine 123 efflux experiment.ART inhibited the proliferation of SP2/0/ADM cells in dose-dependent manners（P0.05）.Since IC20 value was approximately 0.3 μg/ml,resistance reversal experiments of 0.3 μg/ml ART on SP2/0/ADM cells were conducted.The IC50 values of ADM were 17.81 μg/ml without ART and 4.26 μg/ml with ART treatment,and the reversal index was 4.15（P0.05）.The expression of P-gp protein in SP2/0/ADM cells decreased gradually with the prolonged duration of ART.The gray value was 6 568.25±156.93 in cells without ART treatment,4 459.12±297.13（P=0.042） in cells treated with ART for 24 hours,4 218.35±153.21（P=0.039） for 48 hours,3 558.52±241.38（P=0.029） for 72 hours and 3 024.85±157.33（P=0.024）for 96 hours,respectively.The change in each group was statistically significant after ART treatment（P0.05）.Conclusion SP2/0/ADM cell line is a successful multidrug resistance cell line;ART could reverse the resistance of SP2/0/ADM cells to ADM by inhibiting the expression of P-gp protein.