Purification and Identification of the Bioactive Subs tance That Mediates Cardiac Response to the Ischemia Reperfusion Stresses and Development of Treatment

介导心脏对缺血再灌注应激反应的生物活性物质的纯化和鉴定以及治疗方法的开发

基本信息

批准号：
11470158
负责人：
SEKO Yoshinori
金额：
$ 9.54万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

1. Function of the bioactive substance that mediates cardiac response to hypoxia/reoxygenation : We showed that the conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation activated three MAPK family member kinases within 5 to 10 min and induced apoptosis of cardiac myocytes within 24 to 48 hrs. 2. Purification and identification of the bioactive substance released from cardiac myocytes in response to hypoxia/reoxygenation : (A) We showed that the activity for MAPK activation and apoptosis induction of conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation exists in the protein fraction with MW higher than 10kD.Then, we further purified the substance by chromatograpy such as isoelectric point, ion exchange, get filtration, and hydrophobic We finally analyzed the bands of SDS-PAGE by an amino acid sequencer or mass-spectrometry. We found that the substance consisted of several proteins and at least one novel protein. We are currently going on the process of affinity-purification with an antibody. 3. Autocrine mechanism of humoral factors involved in the activation of intracellular signaling in cardiac myocytes in response to hypoxia : (A) We found that VEGF plays a protective role in cardiac response to hypoxic stresses not only by suppressing apoptotic process but increases the adhesion between cardiac myocytes and extracellular matrix through activation of p125^<FAK> and paxillin in an autocrine fashion. (B) We found that similar autocrine mechanism mediated by VEGF is also involved in cardiac response to pulsatile mechanical stretch (relative hypoxia) and that there is further autocrine mechanism mediated by TGF-β upstream of that by VEGF.

1。介导心脏反应缺氧/氧化的生物活性物质的功能：我们表明，受到低氧/重氧的心脏心肌细胞的条件培养基激活了5至10分钟内的三个MAPK家族成员激酶，并在24至48 Hrs中诱导了心脏肌细胞的凋亡。 2。响应缺氧/氧化作用，从心肌细胞释放的生物活性物质的纯化和鉴定：（a）我们表明，MAPK激活和凋亡诱导从患有低氧/蛋白质的蛋白质级别的蛋白质疗程中的心肌细胞中有条件培养基的活性在MEW高于10kD的蛋白质障碍物中存在。然后，我们通过色谱法进一步纯化了该物质，例如等电点，离子交换，过滤和疏水性，我们最终通过氨基酸序列或质谱法分析了SDS-PAGE的频带。我们发现该物质由几种蛋白质和至少一种新型蛋白质组成。我们目前正在与抗体进行亲和力纯化的过程。 3。对缺氧反应的心肌细胞中细胞内信号传导激活的体液因素的自分泌机制：（a）我们发现，VEGF在心脏反应中对低氧应激的作用起着受保护的作用，不仅通过抑制凋亡过程，还可以通过抑制粘附性，还增加了通过paxix racix ractix in> pax inix <pax rive <pak ixpix^prak y ix的粘合剂。自分泌时尚。（b）我们发现，由VEGF介导的类似的自分泌机制也参与了对脉冲机械拉伸（相对缺氧）的心脏反应，并且存在由VEGF上游的TGF-β介导的进一步的自分泌机制。