【摘要】 目的:构建重组鼠骨形态发生蛋白7(BMP-7)的真核表达载体,并观察其在原代培养乳鼠心肌细胞中的表达情况。方法:将RT-PCR法获得的重组鼠BMP-7cDNA克隆入真核表达载体pcDNA3.1中,在FuGENE6.0介导下转染差速贴壁法体外培养的乳鼠心肌细胞,72h后分别应用RT-PCR、免疫细胞化学法和Western blotting检测mRNA和蛋白水平的表达情况。结果:经过PCR及酶切鉴定证明获得了真核表达质粒pcDNA3.1-BMP-7;通过RT-PCR、免疫细胞化学法和Western blotting证明pcDNA3.1-BMP-7能有效转染心肌细胞并表达BMP-7蛋白。结论:应用pcDNA3.1载体系统可有效转染重组鼠BMP-7,为应用于抗缺血再灌注损伤的研究创造了条件。更多还原

【Abstract】 Objective:To construct an eucaryotic expression plasmid carrying recombinant rat bone morphogenetic protein-7(BMP-7)gene and its expression in rat cardiomyocytes.Methods:Recombinant rat BMP-7 was cloned into eucaryotic expression vector pcDNA3.1.At the same time,cardiomyocytes were isolated and cultured in vitro.The plasmid carrying BMP-7 was transfected into cardiomyocytes with FuGENE 6.0 reagent,and BMP-7 mRNA and protein expressions were detected by reverse transcription polymerase chain reaction,immunohistochemical method,and Western blotting 72 hours after the transfection.Results:The eucaryotic expression vector pcDNA3.1 carrying BMP-7 was obtained,and it could be transfected into cardiomyocytes and express BMP-7 protein.Conclusion:The vector pcDNA3.1 carrying recombinant rat BMP-7 can be successfully constructed,and its effective expression in cardiomyocytes may provide basis for BMP-7 gene therapy of ischemia-reperfusion injury.更多还原