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目的研究X连锁凋亡抑制蛋白(XIAP)在K562细胞对于伊马替尼耐药中的作用机制。方法采用首剂大剂量冲击和逐步增加剂量相结合的方法构建伊马替尼耐药的人髓性白血病耐药细胞K562/IMA。Western-Blot和RT-qPCR法检测耐药株中XIAP的表达水平。小干扰RNA沉默耐药株中XIAP的表达,将细胞分为对照组、siRNA-NC组以及siRNA-XIAP组。XIAP真核表达质粒转染普通K562细胞,构建XIAP过表达的K562细胞株,将细胞株分为对照组、空质粒组和实验组。CCK-8法检测各组细胞对于伊马替尼的敏感性,并计算半数抑制浓度IC50。流式细胞术检测各组细胞的凋亡率。Western-Blot法检测Bcl-2、Bax、Caspase-3和Caspase-9的表达水平,TUNEL染色观察细胞的凋亡水平。结果构建的耐药株对10μmol·L-1的伊马替尼耐药,在K562/IMA细胞株中XIAP表达水平显著增高。而XIAP沉默后,相比对照组,siRNA-XIAP组中细胞活力显著下调,凋亡率增高,IC50值下调,且凋亡蛋白Bax、Caspase-3和Caspase-9的表达上调,Bcl-2的表达下调。K562过表达XIAP后,IC50值增高,凋亡率下调,与对照组比较,实验组中凋亡蛋白Bax、Caspase-3和Caspase-9的表达下调,Bcl-2的表达上调。结论 XIAP在慢性髓性白血病对伊马替尼耐药中有着重要的作用,其表达水平增高可以降低细胞对于药物的敏感性,起到抗凋亡作用。
Abstract：AIM To investigate the mechanism of X-linked inhibitor of apoptosis(XIAP) in imatinib resistance of human chronic myeloid leukemia cell line K562. METHODS Imatinib-resistant human myeloid leukemia cell line K562/IMA was constructed by the combination of the first dose of high-dose shock and gradually increasing dose. Western-Blot and RT-qPCR were used to detect the expression level of XIAP in drug-resistant strains. Small interfering RNA silenced XIAP expression in drug-resistant strains and divided cells into control, siRNA-NC and siRNA-XIAP groups. The eukaryotic expression plasmid of XIAP was transfected into K562 cells and constructed the overexpressed K562 cell lines. The cell lines were divided into control group, empty plasmid group and experimental group respectively.CCK-8 method was used to detect the sensitivity of each group of cells to imatinib, and the half inhibitory concentration IC50 was calculated. The apoptosis rate of each group was detected by flow cytometry. The expressions of Bcl-2, Bax, caspase-3 and caspase-9 were detected by Western-Blot, and the apoptosis level was observed by TUNEL staining. RESULTS The constructed strain was resistant to imatinib at 10 μmol·L-1 and the expression of XIAP was significantly increased in K562/IMA cell lines. After XIAP silencing, compared with control group, siRNA-XIAP group significantly decreased cell viability, increased apoptosis rate, decreased IC50 value, and up-regulated the expression of apoptotic proteins Bax, caspase-3, caspase-9, and down-regulated the expression of Bcl-2. After K562 overexpression XIAP, the IC50 value increased and the apoptosis rate decreased.Compared with control, the expression of Bax, caspase-3 and caspase-9 were down-regulated and the expression of Bcl-2 was up-regulated in the experimental group. CONCLUSION XIAP plays an important role in the resistance of chronic myeloid leukemia to imatinib. The increased expression of XIAP can reduce the cell sensitivity to the drug and play an anti-apoptosis role.
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基本信息:
DOI：10.19577/j.1007-4406.2019.05.002
中图分类号:R733.72
引用信息:
[1]韩晨阳,郭丽,盛泳佳,等.X连锁凋亡抑制蛋白在人慢性髓性白血病细胞K562对于伊马替尼耐药中的作用[J].中国临床药学杂志,2019,28(05):326-332.DOI:10.19577/j.1007-4406.2019.05.002.
基金信息:
浙江省卫生厅面上项目(编号2018KY804); 嘉兴市科技计划项目(编号2018AY32009)
2019-09-25
2019-09-25