硝普钠诱导K562细胞凋亡机制的研究  

Mechanism of Sodium Nitroprusside-Induced Apoptosis in K562 Cell Line

作　　者：周永列[1] 吕亚萍[2] 邱莲女[1] 王文松[1] 林惠君[1] 

机构地区：[1]浙江省人民医院中心实验室,杭州310014 [2]浙江工业大学药学院,杭州310014

出　　处：《中国实验血液学杂志》2005年第6期983-988,共6页Journal of Experimental Hematology

基　　金：浙江省医学科学研究基金资助项目;编号2002A0010

摘　　要：为了研究外源性一氧化氮供体硝普钠诱导K562细胞凋亡机制,将不同浓度的硝普钠与K562细胞在体外培养,同时设高铁氰化钾(PFC)对照组和空白对照。用AnnexinV/PI双标记、DNA片段原位末端标记法、DNA凝胶电泳、DNA含量及细胞周期分析等方法检测细胞凋亡;用Rh123/PI测定线粒体跨膜电位(ΔΨm);以双氢罗丹明123(dihydrorhodamin123,DRH)和2',7'二氯双氢荧光素二乙酸酯(2',7'dichlorodihydrofluoresceindiacetate,DCFHDA)为荧光探针,测定线粒体内和细胞胞质内过氧化物;用流式细胞术测定线粒体膜蛋白和bcl2、bax、bad、p53和Fas凋亡调控基因蛋白。结果表明:K562细胞经硝普钠作用后出现典型的细胞形态改变,DNA片段化,亚G1峰检出并显著增加,annexinV/PI和DNA片段原位末端标记表达增加,这些结果均证实NO能诱导K562细胞凋亡,大部分细胞阻滞于G0/G1期。硝普钠诱导K562细胞凋亡过程中,胞质和线粒体内活性氧自由基增加,线粒体跨膜电位降低,bcl2基因蛋白表达下调,同时bax、bad、p53、Fas和线粒体膜蛋白表达均上调。结论:NO诱导K562细胞凋亡是通过产生活性氧自由基,使p53基因表达增加,下调bcl2基因和使bax、bad基因表达上调,线粒体膜通渗性转运孔开放,ΔΨm降低来实现的,此外还存在Fas系统激活途径。To study the molecular mechanisms of nitric oxide donor sodium nitroprusside （SNP） -induced apoptosis in K562 human leukemia cell line, the different concentrations of SNP and different time of culture were used to treat K562 cell. At the same time, potassium ferricyamide （PFC） was used as control, blank was designed in experiment. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling（TUNEL） assay was used to quantify in situ cell apoptosis. Reactive oxygen species （ROS） in cells and mitochondrial transmembrance potential （△Ψm） were labeled by dihydrorhodamin 123 , 2′, 7′-dichlorodihydrofluorescein diacetate and rhodamin 123/PI . bcl-2 , bax, bad, p53 gene proteins and miotochondrial membrane protein were analysed by flow cytometry. The results showed that the K562 cell apoposis was comfirmed by typical cell morphology, DNA fragment, sub-G1 phase, TUNEL and annexin-V/PI labeling. A majority of K562 cells were arrested in G0/G1 phase. During the process of SNP-induced apoptosis in K562 cell, the mean fluorescence intensity of ROS in cells was significantly higher than those in blank and PFC control, while the △Ψm reduced . The expression of p53 ,bax,bad,Fas protein and mitochondrial membrane protein increased and bcl- 2 protein decreased after SNP treatment. It is concluded that SNP induces K562 cell apoptosis through increasing ROS in cells, expressing the p53, bax, bad, Fas protein and mitochondrial membrane protein and decreasing bcl-2 protein, opening the mitochondrial permeability transition pore and reducing △Ψm. Furthermore, the Fas was activated during the apoptosis process.

关 键 词：硝普钠 一氧化氮 K562细胞 细胞凋亡 线粒体膜电位 活性氧自由基 

分 类 号：R733.7[医药卫生—肿瘤] R979.1[医药卫生—临床医学]