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Effect of X-linked inhibitor of apoptosis protein on imatinib resistance in human chronic myeloid leukemia cell line K562
韩晨阳;郭丽;盛泳佳;李文燕;杨毅;
摘要(Abstract):
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IC_(50)。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,IC_(50)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,IC_(50)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。
关键词(KeyWords): 伊马替尼;人慢性髓性白血病;X连锁凋亡抑制蛋白;敏感性
基金项目(Foundation): 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)
作者(Authors): 韩晨阳;郭丽;盛泳佳;李文燕;杨毅;
DOI: 10.19577/j.1007-4406.2019.05.002
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