抑制消减杂交技术筛选大鼠急性心肌缺血后差异基因

Screening of differential expressed genes of acute myocardial ischemia in rats using suppression subtractive hybridization

目的采用抑制消减杂交技术,筛选早期心肌缺血相关的RNA分子,为相关研究和应用提供依据。方法 10只SD雄性大鼠结扎冠状动脉前降支1h,制作心肌缺血模型。以缺血心肌的RNA分子为实验组,以非缺血区的RNA分子为对照组,用Clontech公司抑制消减杂交试剂盒进行杂交。经蓝白斑筛选阳性克隆测序并经过斑点杂交确认,构建早期大鼠心肌缺血后抑制消减杂交文库,所得序列和Genbank数据库比对。结果筛选到10个阳性克隆和11个未知基因的表达序列标签(expressed sequence tag,EST),按照功能分类,包括代谢相关的酶类基因,核糖体和翻译相关的基因,心肌重塑相关的基因等,其中组织蛋白酶L1,basigin等基因等是首次报道与早期心肌缺血相关。结论采用抑制消减杂交技术获得的差别表达基因可为早期心肌缺血提供新的目标分子。

Objective Suppression subtractive hybridization （SSH） technique has been adopted to screen the changes of RNA molecules after early myocardial ischemia （EMI） so thai more evidence could be provided for the further research and related application. Methods Ten Male SpragueDawley rats were used to make the in vivo EMI model by ligation of the left anterior descending artery for lh （LAD）. The RNA molecules from ischemia myocardium of EMI rats were used as the Tester and the control RNA from the non- ischemic counterparts as the Driver. The hybridization was conducted using a SSH kit from Clontech. After bluewhite reaction screening and dot-blot hybridization, the positive clones were sequenced and the SSH library for EMI rats was constructed. The gene identity was confirmed after comparison with Genhank dalabase. Results Ten genes and 11 Expressed Sequence Tags （ESTs） from unknown gene clones were revealed as the EMI-related genes, which were classified into metabolizing enzymes genes, ribosome and translation-related genes and myocardial remodeling-related genes based on their functions. It was the first time that cathepsin LI and basigin were reported as the EMI responding genes. Conclusion The EMI-related genes detected by SSH may provide new target molecules for early myocardial ischemia injury.