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化疗是急性髓系白血病的标准治疗手段,然而白血病细胞多药耐药的产生为白血病化疗的主要障碍.在耐阿霉素的K562细胞株(K562/ADR)中,MRP4表达较不耐药的K562细胞株明显增高.本研究试图运用慢病毒介导的siRNA沉默MRP4基因后研究能否增强K562/ADR对阿霉素的敏感性.首先设计并构建了5个针对MRP4基因的慢病毒介导的siRNA(lv-shRNAs-MRP4);随后用荧光显微镜观察lv-shRNA-MRP4感染K562/ADR细胞的感染效率;用real-time PCR及Western blot检测感染lv-shRNA-MRP4后K562/ADR细胞MRP4mRNA及蛋白表达;应用MTS法及流式细胞术检测感染lv-shRNAs-MRP4后K562/ADR细胞增殖活性及凋亡.荧光显微镜观察结果可见,K562/ADR细胞lv-shRNA-MRP4感染效率达到80%以上;相比较其他lv-shRNAs-MRP4,lv-shRNA1-MRP4对MRP4基因沉默效应最强;在K562/ADR细胞中lv-shRNA1-MRP4组mRNA及蛋白表达均较对照组明显受抑;联合lv-shRNA1-MRP4与阿霉素后K562/ADR细胞生长受抑且细胞凋亡增加.上述结果提示,MRP4基因可能参与了K562/ADR细胞株多药耐药机制,慢病毒介导的siRNA沉默MRP4基因可增强急性髓系白血病对阿霉素的敏感性.
Chemotherapy is the standard treatment of acute myeloid leukemia, however, the multidrug resistance of leukemia cells is a major obstacle to the chemotherapy of leukemia.MK462 cell line (K562 / ADR) is less resistant to MRP4 expression K562 cell line was significantly increased.This study attempts to use lentivirus-mediated silencing of MRP4 gene in siRNA can enhance the sensitivity of K562 / ADR to doxorubicin.First, we designed and constructed five lentivirus-mediated MRP4 gene (Lv-shRNAs-MRP4). The infection efficiency of lv-shRNA-MRP4 infected K562 / ADR cells was observed by fluorescence microscopy. The expression of MRP4 mRNA in K562 / ADR cells infected with lv-shRNA-MRP4 was detected by real-time PCR and Western blot MTS assay and flow cytometry were used to detect the proliferation and apoptosis of K562 / ADR cells infected with lv-shRNAs-MRP4.The results of fluorescence microscopy showed that the efficiency of lv-shRNA-MRP4 infection in K562 / ADR cells was 80% Compared with other lv-shRNAs-MRP4, lv-shRNA1-MRP4 strongest effect on MRP4 gene silencing; lv-shRNA1-MRP4 mRNA and protein expression in K562 / ADR cells were significantly inhibited compared with the control group; -shRNA1-MRP4 and doxorubicin following K562 / ADR cell growth Inhibition and increased apoptosis.The above results suggest that MRP4 may be involved in the multidrug resistance mechanism of K562 / ADR cell line, lentivirus-mediated silencing of MRP4 gene may enhance the sensitivity of doxorubicin to acute myeloid leukemia .