三氧化二砷诱导NB4细胞凋亡相关基因表达改变的基因芯片研究

Studies with an oligonucleotide microarray on the changes of gene expression related with NB4 apoptosis induced by arsenic trioxide

目的利用基因芯片技术研究NB4细胞经三氧化二砷(As2O3)诱导凋亡后的凋亡相关基因表达变化。方法检索NCBI的Entrez以及HumanIPI数据库,通过染色体定位来排除冗余基因,共获得1384个凋亡相关基因。探针使用软件OligoArray2.0设计,并作Blast比对。设计基因特异寡核苷酸探针,合成后点样,制备寡核苷酸芯片。用2μmol/L的As2O3处理NB4细胞48h后,提取细胞总RNA,分别以Cy3,Cy5标记对照组及实验组,随后与含1384个凋亡相关基因的寡核苷酸芯片杂交,使用基因芯片扫描仪对杂交信号扫描,软件分析Cy3和Cy5两种荧光信号的强度和比值,找出经As2O3处理后出现差异表达的基因,并挑选其中差异表达最明显的4条基因,设计引物后与CNDA产物进行PCR扩增,琼脂糖凝胶电泳进行验证。结果NB4细胞经2μmol/L As2O3作用48h后共有4个基因表达上调,12个基因表达下调,且RT-PCR验证结果与基因芯片结果完全相符。结论As2O3可以诱导NB4细胞一系列基因表达的改变,这些涉及信号转导、转录调节、细胞周期、氧化应激、蛋白质的翻译合成及细胞分化等方面基因的差异表达,可能在NB4细胞凋亡中起着重要作用。

Objective Gene microarray technology was used to investigate the differential gene expression of apoptosis related genes in NB4 cells induced by arsenic trioxide. Methods The databases of Evntrez and Human IPI were searched with ＂apoptosis or apoptofic＂ as the key words, and 1384 apoptosis related genes were found after the redundant genes were eliminated by chromosomal localization. The probes of these genes were designed using OligoArray 2. 0, and then analyzed by BLAST. All the probes were immobilized on the glass slide, which were used as oligonucleofide microarray. PAter NB4 cells were treated with 2umol/L AseO3 for 48h, the total RNA were extracted, cDNAs of control group and test group were fluorescently labeled with Cy3 and Cy5, respectively, by RT-PCR. The fluorescent samples were hybridized with an oligonucleofide microarray containing 1384 apoptosis related genes to search for the differentially expressed genes in the cells with or without As203 treatment. The hybridization signals were scanned by oligonucleotide microarray, and then the fluorescent intensity of Cy3 and Cy5 and the ratio of two fluoresceins were analyzed using certain software. Then the differential expressed genes were analyzed after As2O3 treatment, in which the most distinctly differential expressed genes were chosen as targets, and through PCR amplification and gel electrophoresis the above genes were verified. Results There are 4 genes up-regulated and 12 genes downregulated in expression in NB4 cells after 48h treatment with 2umol/L AseOa, which were in accordance with the results of RT PCR and oligonucleotide microarray. Conclusion Differential gene expression in NB4 cells was induced by AseOa treatment. These differentially expressed genes, with relation to signal transduction, transcription regulation, cell cycles, oxidation response, protein translation and cell differentiation, may play an important role in NB4 cell apoptosis.