权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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. 生物活性物质介导心脏对缺氧/再氧化反应的功能：我们发现缺氧/再氧化心肌细胞的条件培养基在5至10分钟内激活了三种MAPK家族成员激酶，并在24至48小时内诱导心肌细胞凋亡。2. 心肌细胞缺氧/再氧化释放的生物活性物质的纯化和鉴定：(A)我们发现缺氧/再氧化心肌细胞条件培养基中的MAPK活化和诱导凋亡活性存在于分子量大于10kD的蛋白片段中。然后通过等电点、离子交换、过滤、疏水等色谱层析对物质进行纯化，最后通过氨基酸测序仪或质谱分析SDS-PAGE的谱带。我们发现这种物质由几种蛋白质和至少一种新蛋白质组成。我们目前正在进行抗体亲和纯化的过程。3. (A)我们发现VEGF不仅通过抑制凋亡过程，而且通过自分泌方式激活p125^<FAK>和paxillin，增加心肌细胞与细胞外基质之间的粘附，从而在心肌细胞对缺氧应激的反应中起保护作用。(B)我们发现类似的由VEGF介导的自分泌机制也参与心脏对搏动性机械拉伸（相对缺氧）的反应，并且在VEGF介导的自分泌机制的上游还有由TGF-β介导的自分泌机制。