冷应激前后大鼠心脏、肝脏及脾脏中α-烯醇化酶基因mRNA的转录水平

Transcription levels of α-enolase m RNA in heart, liver and spleen of rats before and after cold stress

目的研究冷应激前后大鼠心脏、肝脏、脾脏中α-烯醇化酶（α-enolase,ENO1）基因m RNA的转录水平。方法将大鼠随机分为正常对照组（24℃,正常饲喂7 d）和冷应激组（置4℃,应激12 h）,提取各组大鼠心脏、肝脏及脾脏细胞总RNA,反转录成c DNA,以其为模板,PCR扩增ENO1基因,应用生物信息学软件DNAstar进行同源性分析,实时荧光定量PCR法检测大鼠心脏、肝脏、脾脏中ENO1基因m RNA的转录水平。结果冷应激前后大鼠心脏、肝脏及脾脏总RNA的1%琼脂糖凝胶电泳可见28S、18S和5S 3条带,A260/A280值为1.8～2.0;ENO1基因经2%琼脂糖凝胶电泳分析可见107 bp的目的基因条带;ENO1基因序列与NCBI上公布序列的同源性为100%;冷应激组大鼠肝脏、脾脏组织中ENO1基因m RNA的转录水平明显高于正常对照组（P〈0.01）,正常对照组大鼠心脏组织中ENO1基因m RNA转录水平明显高于冷应激组（P〈0.05）。结论冷应激前后大鼠心脏、肝脏及脾脏中ENO1基因m RNA的转录水平均发生显著改变,本实验为动物应激状态评估及应激发生机制的研究提供了实验依据。

Objective To investigate the transcription levels of α-enolase（ENO1）gene in heart, liver and spleen of rats before and after cold stress. Methods Rats were randomly divided into normal control and cold stress groups. The rats in normal control group were raised at 24 ℃ for 7 d, while those in cold stress group at 4 ℃ for 12 h. Total RNAs were extracted from heart, liver and spleen of rats in various groups and reversely transcribed to c DNAs which were used as templates for amplification of ENO1 gene by PCR. The PCR product was analyzed for homology by using DNAstar software. The ENO1 m RNA transcription levels in heart, liver, spleen of rats were determined by real-time fluorescent quantitative PCR. Results Three RNA bands, 28 S, 18 S and 5S, were observed on 1% agarose gel electrophoretic profile of heart, liver and spleen of evaluation of status and rats before and after cold stress, of which A260/ A280 values were 1. 8 ～ 2. 0. Target gene band at a length of 107 bp was observed on 2% agarose gel electrophoretic profile of ENO1 gene. The homology of ENO1 gene sequence was 100% to that reported in NCBI. The transcription level of ENO1 m RNA in liver and spleen of rats in cold stress group were significantly higher than those in normal control group（P〈 0. 01）. However, the transcription of ENO1 m RNA in heart of rats in normal control group was significantly higher than that in cold stress group（P〈 0. 05）. Conclusion The transcription level of ENO1 m RNA in heart, liver and spleen of rats before and after cold stress changed significantly. It laid a foundation of study on mechanism of cold stress.