人体进行力竭运动时，会产生大量氧自由基，导致心肌细胞损伤和线粒体功能紊乱。因此，研究运动氧化应激致心肌线粒体功能调控对预防运动性心肌损伤非常重要。S100A1是一种钙结合蛋白，具有提高心肌收缩力，调节血管功能，刺激ATP合成酶活性等作用，与腺苷酸转运蛋白（ANT）结合，影响线粒体的功能等。但运动氧化应激致心肌损伤后S100A1是否通过ANT调控心肌线粒体功能尚不清楚。本课题拟通过建立运动氧化应激损伤动物模型和离体心肌细胞氧化应激损伤模型，研究S100A1和ANT蛋白在心肌组织中的表达和线粒体的功能变化；构建蛋白表达载体，表达纯化S100A1蛋白；采用外源干预和基因转染法研究：S100A1蛋白对氧化应激心肌线粒体功能的影响及调控，初步阐释其对氧化应激致心肌线粒体功能调控机制，为建立运动性心肌损伤防护新措施提供新思路。

Oxygen free radicals from human body during carrying out exhaustive exercise could result in functional disorder of mitochondria.Therefore it is important to study the functional control of mitochondria in cardiocytes under oxidative stress by exhaustive exercise for preventing cardiocyte from injury. S100A1 is a kind of calcium-binding protein.It can increase myocardial contractility, control vascular function and also stimulate activity of ATP synthetase. S100A1 can affect function of mitochondria through binding ANT( adenine nucleotide translocator,ANT) . Whether S100A1 control the function of mitochondria through interaction with ANT after cardiocytes injury by oxidative stress remains unclear.Our aim is to investigate the expression of S100A1 and ANT in cardiocytes and the change of mitochondria function by animal model under oxidative stress by exhaustive exercise and oxidative stress cardiocytes in vitro model. To express and purify human S100A1 protein, a pBV220-S100A1 expressing system was constructed and the purification technology of human S100A1 protein was established. We will reveal how S100A1 affect mitochondria function in oxidative stress cardiocytes by applying exogenous intervention and gene transfection, which maybe further support new concept for protecting cardiocytes from injury after exhaustive exercises.

人体力竭运动会产生大量氧自由基，导致心肌细胞损伤和线粒体功能紊乱。因此，研究运动氧化应激损伤心肌线粒体功能调控对预防运动性心肌损伤非常重要。S100A1蛋白可与腺苷酸转运蛋白（ANT）结合，影响线粒体功能。但S100A1是否通过ANT调控运动氧化应激损伤心肌线粒体功能未见报道。通过建立运动氧化应激损伤心肌动物模型，以及氧化应激损伤细胞模型，研究S100A1和ANT蛋白在损伤心肌中的表达和线粒体功能变化；采用S100A1干预和敲除S100A1基因，研究S100A1对氧化应激心肌线粒体功能的影响及调控。主要结果如下：一、S100A1和ANT蛋白在运动氧化应激损伤大鼠心肌组织中的表达变化Western-Blot结果表明，与对照组相比，损伤心肌组织S100A1和ANT蛋白表达水平降低。免疫组化显示，正常心肌组织S100A1和ANT蛋白均匀分布，而损伤心肌组织S100A1和ANT蛋白表达降低。二、S100A1蛋白表达载体构建及表达、纯化根据大肠杆菌的密码子偏好性，优化并合成了人源S100A1基因，将该序列酶切后连接pBV220载体并转化至E.coliDH5α，目的蛋白获得高效表达。采用离子交换层析法纯化，最终获得纯度为95%的目的蛋白。三、S100A1蛋白对氧化应激损伤心肌线粒体功能的影响及调控以1mM的H2O2干预12h，建立H9c2氧化应激损伤模型。结果表明，氧化应激损伤心肌S100A1、ANT、PGC-1α和Tfam的mRNA和蛋白表达降低，给予S100A1蛋白后，ANT、PGC-1α、Tfam的mRNA和蛋白表达升高。敲除S100A1基因后，H9c2细胞S100A1、ANT、PGC-1α和Tfam的基因和蛋白表达降低。综上，力竭运动可致大鼠心肌氧化应激损伤和线粒体功能紊乱，心肌S100A1和ANT蛋白表达降低。构建了S100A1蛋白表达载体，表达纯化获得了S100A1蛋白；H9c2细胞氧化应激损伤后，给予S100A1蛋白，H9c2细胞存活率升高，同时ANT、PGC-1α和Tfam基因和蛋白表达升高。敲除S100A1，H9c2细胞S100A1、ANT、PGC-1α和Tfam基因和蛋白表达降低。提示，S100A1蛋白对氧化应激损伤心肌线粒体功能影响及调控，可能是通过S100A1蛋白与靶标ANT相互作用，影响PGC-1α表达，同时伴有Tfam表达升高，进而促