人可溶性DR5蛋白通过阻断TRAIL减轻心肌缺血-再灌注损伤的机制

Myocardial cell apoptosis is one of the main causes of cell death during ischemia/reperfusion progression. Previous studies suggested that TRAIL（Tumor necrosis factor-related apoptosis-inducing ligand）mediated apoptosis may play an important role in acute myocardial infarction(AMI). However, the molecular mechanisms have thus far remained unclear. Our previous studies observed the down-regulation of serumal TRAIL in AMI patients. Furthermore, TRAIL was also found up-regulated in rat myocardial cell after ischemia. Human soluble DR5 could obviously ameliorate myocardium apoptosis and reduce infarction size. In the present study, Wistar rats, H9C2(2-1) cell line and NRVMs are chose to generate ischemia/reperfusion or hypoxia/reoxygenation model. And then, the expression of TRAIL in serum, PBMCs and myocardial cells will be monitored at RNA and protein levels. The expression of DR5 along with down-stream procaspase-8, 3 and Bid will also be detected to confirm that TRAIL/DR5 apoptosis pathway is involved in the myocardial infarction progression. Finally, in vitro recombinant human soluble DR5 will be utilized to block the interaction of endogenous TRAIL with cell surface DR5, evaluating the protective effect of soluble DR5 in myocardial infarction by serum enzymology detection, TTC and TUNEL staining. The results will let us further understand the role of TRAIL/DR5 in myocardial infarction, which provides a new therapeutic strategy for myocardial ischemia /reperfusion injury.

抑制心肌细胞凋亡可有效缓解急性心肌梗死（AMI）及后续治疗引起的心脏缺血-再灌注损伤。我们前期研究证实TRAIL在AMI病人血清中显著降低，提示TRAIL介导的细胞凋亡与AMI密切相关；进一步在大鼠AMI模型中发现缺血区心肌组织TRAIL、死亡受体5（DR5）等表达上调，细胞凋亡增多。通过尾静脉注射人可溶性DR5（sDR5）蛋白可显著抑制模型大鼠心肌细胞凋亡，减少心肌梗死面积。本项目拟对缺血-再灌注大鼠和缺氧-复氧细胞模型中TRAIL、DR5及其下游通路活化情况和细胞色素C、ROS等的表达、释放情况进行详细考察，以期阐明TRAIL、DR5参与心脏缺血-再灌注损伤的分子机制；继而用人sDR5蛋白阻断TRAIL与细胞表面DR5的结合，分别从形态学、酶学、心功能及信号通路活化等方面综合评价sDR5对心肌细胞的保护作用。研究结果将为深入揭示心肌梗死发病机制提供实验基础，并为临床治疗开辟新思路。

防治缺血-再灌注损伤是心梗病人治疗过程中的难题，目前临床缺乏特异性治疗药物。细胞死亡和炎性反应是心梗后心肌损伤的重要形式。本项目研究发现，TRAIL与心梗后心肌损伤关系密切，利用sDR5阻断TRAIL 与其受体的结合可有效减少心肌梗死面积，这一效应归功于sDR5减少了TRAIL介导的心肌细胞凋亡和炎性损伤。为了使研究成果利于向临床转化，我们设计了sDR5-Fc抗体融合蛋白，并在CHO细胞中筛选得到高表达稳定细胞株，进而利用生物反应器放大生产。药效学结果表明，sDR5-Fc同样可明显减少大鼠心肌梗死面积。令人兴奋的是，在形态结构与人更接近的大型动物-猪心梗模型中，sDR5-Fc表现出更好的心脏保护效果。机制探讨发现，sDR5-Fc不仅可减轻心肌细胞凋亡，也可明显减少巨噬细胞向缺血区迁移，下调心脏组织炎症因子表达。本项目研究不仅丰富了对心脏缺血-再灌注损伤分子机制的认识，同时提供了良好的潜在靶点和候选药物。