大鼠小脑颗粒神经元凋亡相关的差异表达基因ARNT2的克隆

Cloning of Cerebellar Granule Neuron Apoptosis Related and Differentially Expressed Rat Gene ARNT2

【目的】研究凋亡与非凋亡大鼠小脑颗粒神经元(cerebellar granule neuron,CGN)细胞中基因表达的差异,克隆出与大鼠CGN凋亡相关的基因。【方法】用荧光差异显示聚合酶链反应(florescent differential display polymerase chain reaction,FDD PCR)从低钾诱导的大鼠CGN凋亡模型中筛选出差异表达序列标签(expressed sequence tag,EST),反向Northern blot杂交进一步验证后,用cDNA 5’末端快速扩增技术(rapid amplification of cDNA 5’Ends,5’RACE)克隆差异表达EST的目标基因,用RT-PCR及Western blot进一步验证目标基因mRNA 及蛋白质水平的表达差异。【结果】用FDD PCR筛选出5号差异表达EST,经反向Northern blot验证后,用5’RACE法成功地克隆到5号EST完整开放阅码框,序列同源性分析证实为大鼠基因ARNT2,RT-PCR及Western blot证实低钾诱导后ARNT2 mRNA与蛋白质水平的表达均显著地上调,统计学分析显示差异有显著性(P< 0.001)。【结论】ARNT2在低钾诱导的大鼠凋亡CGN中表达上调,提示ARNT2与CGN凋亡相关并在该过程中发挥重要作用。

[Objective]To explore the differences of gene expression between cerebellar granule neuron (CGN) of normal and apoptosis of rat so as to clone rat gene related with CGN apoptosis. [Methods] Fluorescent differential display PCR (FDD PCR) was used to screen differentially expressed sequence tag (EST) between CGN of normal and apoptosis induced by low K+. After verified by reverse Northern blot, rapid amplification of cDNA 5'ends (5' RACE)was applied to clone target gene of differentially expressed sequence tag;RT-PCR and Western blot was employed to further investigate the differential expression of target gene. [Results] EST 5 showed differentially expression after FDD PCR screening. Verified by reverse Northern blot, EST 5 was then successfully cloned by 5' RACE with an integrity open reading frame which completely homologous to that of ARNT2 of rat, both mRNA and protein expression levels of ARNT2 were verified up-regulated by low K+ significantly analyzed by statistic methods (P<0.001).[Conclusion] ARNT2 is up-regulated in apoptotic CGN of rat induced by low K+, suggesting that ARNT2 is related with CGN apoptosis and may play an important role in the process of CGN apoptosis.