摘要
目的:利用细胞凋亡和周期基因芯片研究As2O3作用前后NB4细胞基因表达谱的差异性,寻找As2O3诱导NB4细胞凋亡的相关基因并分析其可能机制。方法:流式细胞仪检测细胞凋亡率,抽提对照及诱导组细胞的mRNA,通过逆转录将As2O3处理前后的NB4细胞cDNA进行生物素标记,用含269个目的基因的细胞凋亡和周期基因芯片进行杂交,GEArray软件分析,筛选出As2O3诱导前后表达有差异的基因。芯片结果用荧光定量聚合酶链反应(realtimepolymerasechainreaction)进行验证。结果:筛选出As2O3作用前后表达有差异的基因共100条(占芯片基因总数的37.2%),其中97条(97/100,97%)基因表达上调,3条(3/100,3%)基因表达下调。表达上调的基因主要包括肿瘤坏死因子配体和受体家族、bcl2家族、半胱氨酸家族、DNA损伤检测和P53途径以及细胞分裂周期蛋白和激酶等基因。结论:As2O3主要通过上调促凋亡基因表达来诱导NB4细胞凋亡,其中TNFSF15、Apaf1、Caspase3和p16等基因可能参与As2O3诱导的NB4细胞凋亡,As2O3诱导NB4细胞凋亡的可能机制主要涉及TNF途径、线粒体途径、Caspase途径、细胞周期抑制途径和P53途径等。
Objective： To detect the change of gene expression in acute promyelocytic leukemia cell line NIM cells treated with arsenic trioxide （As2O3 ） by Human Apoptosis and Cell Cycle Gene Array and investigate the apoptosis-related genes and possibly involved mechanism. Methods： The apoptosis rate of NIM cell induced by As2O3 was detected by Annexin V FITC-PI double staining method and measured by flow cytometry （FCM）. Total RNA was isolated from NB4 cells which had been untreated or treated with As2O3 at the concentration of 4μmoL/L and reverse-transcribed into a cDNA probe labeled with Bio-16-dUTP. It was hybridized with a Human Apoptosis and Cell Cycle Gene Array containing up to 269 key genes involved in apoptosis, cell cycle and oxidative stress. The gene expression profile of the As203-treated NB4 cells was analyzed with GEArray Analyzer software. The microarray results were confirmed by real time polymerase chain reaction. Results： 4μmol/ L As2O3 treatment for 24h caused the expression alteration of 100 genes （37.2% of total genes on microarray） in NB4 cells related to cell apoptosis , cell cycle or stress ＆ toxicity. Among them, 97 （97% ,97/100） genes were upregulated, and 3 （3% ,3/100 ） genes were downregulated. The upregulated genes were mainly involved in TNF（ tumor necrosis factor,TNF） ligand and receptor superfamily, bcl-2 family, caspases family, DNA damage chechpoint/P53 and ATM pathway, inhibitor of apoptosis protein family （IAP）, cell division cycle proteins and kinases , cyclin, cyclin-dependent kinases and inhibitors, DNA replication, transcriptional control and others, oxidative ＆ metabolic stress and CIDE family. The downregulated genes mainly included TRAF2 of TRAF family, TNFSF10 of TNF ligand family and XRCC3 of DNA damage ＆ repair stress. Conclusion： The apoptosis of NB4 cells induced by As203 occured mainly by upregulating some proapoptotic genes expression. TNFSF15,Apaf-1, Caspase-3 and p16 genes might take part in the apoptosis process of NB4 cells induced by As203. The possible mechanism involved in TNF passway, mitochondrion passway, caspase passway, cell cycle inhibition and P53 passway.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2005年第11期7-11,共5页
China Biotechnology
基金
上海市科学技术委员会科研计划资助项目(045458032)
