苦参碱对急性早幼粒细胞白血病细胞维甲酸耐药的逆转作用研究

目的 探讨苦参碱逆转急性早幼粒细胞白血病(APL)全反式维甲酸(ATRA)耐药的作用及可能的机制.方法 以ATRA敏感的APL细胞系NB4及其ATRA耐药株NB4-R1、NIM-R2作为研究对象.用MTT法明确苦参碱低毒性剂量,进一步计算ATRA联合100μmoL/L苦参碱作用前后细胞ATRA IC50值,明确苦参碱的逆转耐药倍数;用NBT还原实验分析不同浓度(10、8、6、4、2、1、mmol/L,100、10、1μmol/L)苦参碱联用1μmol/L ATRA对耐药细胞分化能力的影响,并观察细胞形态变化;Annexin V/PI染色流式细胞术检测不同浓度苦参碱联用1 μmol/L ATRA作用下细胞凋亡率.结果 ①苦参碱对NB4、NB4-R1、NB4-R2细胞的增殖抑制作用随浓度增加而增强,IC50值分别为(0.661±0.035)、(0.673±0.132)、(0.329±0.020)mmol/L;②联用100 μmol/L苦参碱能显著逆转NB4-R1细胞的耐药性(逆转耐药倍数为4.96±1.15),但不能增强NB4-R2细胞对ATRA的敏感性,甚至增强其耐药性(逆转耐药倍数为0.66±0.17);③苦参碱与1 μmol/L ATRA联用时,NB4、NB4-R1细胞的分化能力随苦参碱作用浓度的增大而增强,且在苦参碱为100 μmol/L时达到峰值(P<0.05),但对NB4-R2细胞无明显影响;④1 μmol/L ATRA联合苦参碱能显著提高NB4、NB4-R1细胞凋亡率(P<0.05和P<0.01),但对NB4-R2细胞同样无明显作用.结论 苦参碱能有效逆转NB4-R1细胞的ATRA耐药,可能与其协同ATRA诱导分化及促进细胞凋亡有关.苦参碱与ATRA联用非但不能缓解NB4-R2细胞的ATRA耐药,甚至可能加重耐药、阻断耐药细胞的凋亡过程.

Study on matrine alleviating retinoic acid resistance in acute promyelocytic leukemia

Objective To explore the role of matrine ( MAT) alleviating all-trans retinoic acid (ATRA) resistance in acute promyelocytic leukemia (APL) and its mechanism. Methods ATRA sensitive strain of APL (NB4) and resistant strain ( NB4-R1, NB4-R2) were used in this study. The low toxic dosage of MAT was established by MTT test, and ATRA IC50 of the cell strains ( cultured with or without 0.1 mmol/L MAT) were obtained to confirm the reversal index (RI); the influence of MAT (10, 8, 6, 4,2, 1, 0. 1, 0. 01, 0. 001 mmol/L) combine with 1 μmol/L ATRA on the differentiation of the three cell strains were observed by nitro blue tetrazolium chioride (NBT) test and morphologic changes. The apoptosis rate of cells treated with different concentration of MAT combined with 1 μmol/L ATRA was tested by flow cytometry with Annexin V/PI staining. Results ①The toxicity of MAT to NB4, NB4-R1, and NB4-R2 cells was increased with the concentration, IC50 value was (0.661 ±0.035) mmol/L, (0.673 ±0. 132) mmol/L and (0.329 ±0.020) mmol/L, respectively; ②After treated with 0. 1 mmol/L MAT, the ATRA resistance factor of NB4-R1 decreased markedly (RI =4.96 ± 1. 15) , but did not of NB4-R2( RI =0.66 ±0. 17) ; ③The differentiation capacity of NB4 and NB4-R1 was enhanced with increase of MAT, and peaked at 0. 1 mmol/L (P <0.05) , but did not of NB4-R2; ④ After treated with MAT, the ATRA( 1 μmol/L) induced apoptosis rate in NB4 and NB4-R1 increased significantly ( P < 0. 05 and P < 0. 01, respectively).Conclusion MAT can reverse the ATRA resistance of NB4-R1, which may relate to the effect of MAT on differentiation and apoptosis. Treatment with MAT plus ATRA may exaggerate the cells resistance potency.