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

1.介导心脏对缺氧/复氧反应的生物活性物质的功能：我们表明，来自缺氧/复氧的心肌细胞的条件培养基在5至10分钟内激活三种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/复氧释放生物活性物质的纯化和鉴定：（A）我们发现，来自缺氧/复氧的心肌细胞的条件培养基的MAPK活化和凋亡诱导活性存在于分子量高于10 kD的蛋白质组分中。然后，我们通过色谱法如等电点、离子交换、过滤，最后用氨基酸序列分析仪或质谱仪对SDS-PAGE条带进行分析。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的亲和纯化过程。3.参与心肌细胞响应缺氧的细胞内信号转导激活的体液因子的自分泌机制：（A）我们发现VEGF在心脏响应缺氧应激中起保护作用，不仅通过抑制凋亡过程，而且通过以自分泌方式激活p125 β和桩蛋白增加心肌细胞与细胞外基质之间的粘附<FAK>。(B)我们发现，类似的自分泌机制介导的血管内皮生长因子也参与了心脏对脉动机械牵拉（相对缺氧）的反应，并有进一步的自分泌机制，由TGF-β介导的上游血管内皮生长因子。

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.