14-3-3η/PKCε信号通路介导的葛根素心肌保护作用

On the basis of summarizing predecessors' and our own work, we assume that: mechanism of puerarin against myocardial hypoxia / reoxygenation injury is at least partly connected with upregulation of 14-3-3η protein and activation of PKC epsilon in cytoplasm, and helping the latter to the migration of mitochondrial, finally achieves the series protective effect.To this end, the project intends primary cultured neonatal rat myocardial cells using adenovirus expression vector and RNAi technology, the preparation of the corresponding transgenic cells by in vitro acute hypoxia / reoxygenation injury model to investigate the relationship between myocardial protective effect of puerarin and 14-3-3η/PKCε expression levels; using laser confocal microscopy, immunofluorescence double staining method to clear the effects of puerarin on the interaction between 14-3-3η and PKCε and subcellular localization; combination of multiin items classic features detection, revealing the molecular mechanisms of myocardial protection mediated by puerarin-14-3-3η/PKCε. Finally, a comparative demonstration in the whole animal experiments. The purpose of this project is to further confirm andClarify the molecular mechanisms of the myocardial protective effect of puerarin, establishing experimental basis for scientific and rational interpretating health benefits of similar phytonutrients; provide strong theoretical basis for the development of Pueraria foods.

在总结前人和自身工作基础上，我们假设：葛根素抗心肌缺血/再灌注损伤作用机制至少部分与上调14-3-3η蛋白，激活胞浆PKCε，帮助后者向线粒体迁移，最终实现系列保护效应有关。为此，本课题拟用新生大鼠原代培养心肌细胞，运用腺病毒表达载体和RNAi技术，制备相应的转基因细胞，通过体外急性缺氧/复氧损伤模型，探讨葛根素心肌保护作用与14-3-3η/PKCε蛋白表达水平之间的关系；采用激光共聚焦显微镜、免疫荧光双染色等方法明确葛根素对14-3-3η与PKCε之间的相互作用和亚细胞定位影响；结合多项经典功能学检测，揭示葛根素在14-3-3η/PKCε介导的心肌保护作用分子机制；最后在整体动物实验上进行的比较论证。本项目旨在进一步确认并阐明葛根素心肌保护作用的分子机制，为科学、合理诠释类似植物营养素保健功效奠定实验基础。