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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: To screen for RNA molecules associated with early myocardial ischemia by suppression subtractive hybridization (SSH), providing the basis for related research and application. Methods Ten SD male rats were ligated with anterior descending coronary artery for 1 hour to make a model of myocardial ischemia. The RNA molecules of ischemic myocardium were used as the experimental group, and the non-ischemic RNA molecules were used as the control group. Clontech’s Suppression Subtraction Hybridization Kit was used for hybridization. The positive clones were screened by blue-white screening and confirmed by dot blot hybridization. The suppression subtractive hybridization library was constructed after myocardial ischemia in early rats, and the obtained sequences were compared with the Genbank database. Results The expressed sequence tags (ESTs) of 10 positive clones and 11 unknown genes were screened. According to the functional classification, they included metabolic enzymes, ribosomes and translation related genes, genes involved in myocardial remodeling Among them, cathepsin L1, basigin and other genes are the first reports associated with early myocardial ischemia. Conclusion The differentially expressed genes obtained by suppression subtractive hybridization can provide new target molecules for early myocardial ischemia.