摘要
目的研究亚砷酸对白血病多药耐药细胞株K562/A02细胞的作用及对多药耐药基因1(mdr1)、P糖蛋白(P-gp)和血管内皮生长因子(VEGF)表达的影响,探讨亚砷酸逆转白血病多药耐药(MDR)的作用机制。方法将K562/A02细胞与不同浓度的亚砷酸(0、0.5、2.0、5.0μmol/L)共同孵育,分别在24、48、72h时,用Wrights染色法观察K562/A02细胞形态,流式细胞术(FCM)检测凋亡细胞,MTT法检测细胞增殖活性,RT-PCR法检测mdr1mRNA的表达,免疫组织化学法观察P-gp表达,并用ELISA法检测VEGF的浓度。结果随着亚砷酸作用浓度和时间的增加,K562/A02细胞的数目减少,并出现凋亡形态学改变,FCM显示随着作用浓度和时间的增加,细胞凋亡率逐渐上升;从作用48h组开始,mdr1mRNA及P-gp的表达出现显著降低(P<0.05),mdr1mRNA条带颜色较空白对照组明显变浅,mdrl/GAPDH灰度比值下降,此时,P-gp的灰度值上升,且随亚砷酸作用浓度和时间的增加,mdr1mRNA条带颜色进一步变浅,表达水平进一步下降;ELISA法检测表明从0.5μmol/L作用24h组开始,VEGF浓度即下降至405.02pg/mL(P<0.05),相同作用时间下以5.0μmol/L组较其它浓度组下调VEGF的作用最为显著。结论亚砷酸呈药物浓度-时间依赖性抑制K562/A02细胞增殖,诱导其凋亡,下调VEGF表达,有效抑制了mdrlmRNA的产生和P-gp的合成。
Objective To study the expression of multidurg resistance gene（mdr1） 、P-glycoprotein（P-gp） and vascular endothelial growth factor（VEGF） during apoptosis induced by Arsenious acid and explore the mechanism of reversal of apoptosis resistance by Arsenic trioxide in multidurg-resistant human leukemia K562/ A02 cell.Methods The K562/A02 cell was cultured in different concentration of Arsenious acid（0,0.5,2.0,5.0 μmol/L） and harvested in 24 h,48 h,72 h.The cell morphology was assayed by inverted microscope and Wright＇s stain.The cell apoptosis was examined by flow cytometer（FCM） .The cell proliferating activity was assessed by MTT assay.The expression of mdr1 mRNA was examined with reverse ranscription-polymerase chain raction（RT-PCR） .The P-gp was examined by immunohistochemistry assay.The VEGF levels were measured with enzyme-linked immunosorbent assay（ELISA） in cultural supernatants of K562/A02 cell.Results Arsenious acid could decrease the number of K562/A02 cell and increase the number of K562/A02 cell debris.The cell morphology showed typical morphological changes of apoptosis in K562/A02 cells with the apoptosis-induction of Arsenious acid,such as chromatin condensation,nuclear fragmentation and so on.It was showed apoptosis rate of K562/A02 cells was obviously increased in a dose-and time-dependent,respectively.Constitutive expression of mdr1 mRNA、P-gp and VEGF were detected by RT-PCR,immunohistochemistry assay and ELISA respectively.After treatmeat with Arsenious acid for 48h,the expression of mdr1 mRNA and P-gp showed significent decrease（P0.05） .K562/A02 cells exhibited decreased mdr1 mRNA and P-gp expression,grey scale ratio and grey scale numerus were respectively（P0.05） ,the expression of mdr1 mRNA and P-gp in K562/A02 cells decreased futher（P0.05） .After treated with the same time,when incubated with 5.0μmol/l Arsenious acid,the expression of VEGF was the least.Conclusion Arsenious acid can inhibit the proliferation of multidrug-resistant K562/A02 cell and induce the cell to undergo apoptosis in a dose-and time-dependent manner.Arsenious acid can inhibit the expression of VEGF,and inhibit mdr1 mRNA and P-gp expression in the cells.
出处
《中南医学科学杂志》
CAS
2011年第4期385-389,415,共6页
Medical Science Journal of Central South China
