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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.介导心脏对缺氧/复氧反应的生物活性物质的功能：我们发现，缺氧/复氧心肌细胞的条件培养液在5~10分钟内激活了三个MAPK家族成员的激酶，并在24~48小时内诱导了心肌细胞的凋亡。2.心肌细胞缺氧/复氧释放活性物质的纯化与鉴定：(A)我们证明了缺氧/复氧心肌细胞条件培养液对MAPK的激活和诱导细胞凋亡的活性存在于分子量大于10kD的蛋白质组分中，然后通过等电点、离子交换、GET过滤和疏水性等层析进一步纯化该物质。我们发现该物质由几种蛋白质和至少一种新的蛋白质组成。我们目前正在进行与抗体的亲和纯化过程。3.体液因子参与低氧激活心肌细胞内信号转导的自分泌机制：(A)我们发现，血管内皮细胞生长因子在低氧应激心脏反应中发挥保护作用，不仅通过抑制细胞凋亡过程，而且通过自分泌方式激活p125^-lt、FAK&GT和paxlin，增加心肌细胞与细胞外基质之间的黏附。(2)我们发现血管内皮生长因子介导的自分泌机制也参与心脏对搏动性机械牵张(相对低氧)的反应，并且在血管内皮生长因子上游还有转化生长因子-β介导的进一步的自分泌机制。