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目的观察磷脂爬行酶1(phospholipid scramblase 1,PLSCR1)在苦参碱(matrine,MAT)诱导维甲酸(all-trans retinoic acid,ATRA)耐药急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)细胞分化的表达,并探讨其与环磷酸腺苷(cyclic adenosine monophosphate,cAMP)/蛋白激酶A(protein kinase A,PKA)途径的相关性。方法以ATRA敏感的APL细胞系NB4以及ATRA耐药株NB4-R1为研究对象。通过NBT还原实验以及流式细胞仪(CD11 b)检测0.1 mmol/L MAT联合1μmol/L ATRA对两细胞株分化标记的影响;采用Western blot、实时荧光定量PCR技术测定细胞PML/RARα、PLSCR1蛋白及基因表达;同时应用PKA拮抗剂H89联合作用,观察细胞分化抗原及上述蛋白/基因表达的变化。结果 MAT联用ATRA能明显提高NB4-R1细胞NBT及CD1 1 b阳性率,并显著下调PML/RARα融合蛋白/基因的表达(P<0.05,P<0.01)。单用ATRA能使NB4细胞PLSCR1在蛋白及mRNA水平表达均得到明显增强(P<0.01),而在NB4-R1细胞内同样表达上调,但仅蛋白水平差异有统计学意义(P<0.01)。在联用MAT后,两细胞株PLSCR1蛋白表达进一步上升(P<0.01),并且NB4-R1细胞mRNA表达水平差异有有统计学意义(P<0.05)。上述作用均能被10μmol/L H89逆转(P<0.05,P<0.01)。结论 MAT联合ATRA能显著诱导NB4-R1细胞获得再分化,同时抑制PML/RARα融合基因/蛋白的表达,这可能与其上调PLSCR1表达有关。
Objective To investigate the effect of phospholipid scramblase 1 (PLSCR1) on all-trans retinoic acid (ATRA)-induced acute promyelocytic leukemia (APL) cells induced by matrine (MAT) The expression of differentiation and its correlation with cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway. Methods The ATRA-sensitive APL cell line NB4 and the ATRA-resistant line NB4-R1 were studied. The effects of 0.1 mmol/L MAT combined with 1 μmol/L ATRA on the differentiation markers of the two cell lines were detected by NBT reduction assay and flow cytometry (CD11 b); PML/RARα and PLSCR1 proteins were detected by Western blot and real-time fluorescent quantitative PCR. And gene expression; simultaneous application of PKA antagonist H89 combined effect, observed cell differentiation antigen and the above protein / gene expression changes. Results The combination of MAT with ATRA significantly increased the positive rate of NBT and CD1 1 b in NB4-R1 cells, and significantly down-regulated the expression of PML/RARα fusion protein/gene (P<0.05, P<0.01). The expression of PLSCR1 in protein and mRNA of NB4 cells was significantly increased by ATRA alone (P<0.01), but was also up-regulated in NB4-R1 cells, but only the difference in protein level was statistically significant (P<0.01). After combined use of MAT, the expression of PLSCR1 protein in the two cell lines increased further (P<0.01), and there was a statistically significant difference in mRNA expression levels of NB4-R1 cells (P<0.05). All these effects were reversed by 10 μmol/L H89 (P<0.05, P<0.01). Conclusion MAT combined with ATRA can significantly induce the differentiation of NB4-R1 cells and inhibit the expression of PML/RARα fusion gene/protein, which may be related to the up-regulation of PLSCR1 expression.