Application study of a novel apoptogenic protein for the pathogenesis of atherosclerotic diseases

Apop是一种蛋白质，最初作为一种未知功能的因子从培养的动脉粥样硬化平滑肌细胞中分离出来。我们最近发现Apop的表达通过从线粒体释放细胞色素c诱导培养的血管平滑肌细胞（SMC）凋亡。细胞色素c从通透性转换孔（permeability transition pore，PTP）的释放被认为在缺血/再氧合诱导的心肌细胞凋亡中起重要作用，这有助于心肌梗死后球囊血管成形术后心力衰竭的发展。本研究旨在探讨Apop在缺氧/复氧诱导的心肌细胞死亡中的作用。从2至3日龄的Sprague-Dawley大鼠的心室制备新生心肌细胞，并维持在补充有10%胎牛血清的DMEM中。使用电穿孔器将阻止Apop表达的反义质粒或对照载体引入心肌细胞中。然后将细胞在缺氧条件（1%O2）下孵育 关于我们 ）48小时，然后再充氧2小时。MTT法检测细胞活力。抗凋亡的bcl-2家族蛋白，如bcl-2和bcl-xL不能阻止Apop诱导的细胞凋亡，而PTP的重要成分亲环素D的抑制剂可以阻止凋亡。这些结果表明Apop诱导的细胞凋亡不是通过bcl-2家族相关通道介导的，而可能需要线粒体膜上的PIP。将靶向Apop转录本的siRNA导入培养细胞中几乎完全消除Apop-GFP融合蛋白的绿色荧光，表明siRNA阻止Apop的表达。Western blot分析也证实了siRNA的抑制活性。在缺氧/复氧诱导的细胞死亡实验中，siRNA的引入抑制了caspase级联的激活和培养心肌细胞的死亡，而对照siRNA的引入既不影响caspase的活性也不影响存活率;抑制Apop的表达阻止了缺氧/复氧诱导的心肌细胞死亡，提示Apop蛋白可能在缺血再灌注损伤后心脏疾病的发生中发挥作用。少

Apop is a protein originally isolated as a factor of unlkown function from cultured atherosclerotic smooth muscle cells. We found recently that expression of Apop induces apoptosis of vascular smooth muscle cells (SMC) in culture by releasing cytochrome c from mitochondria. Release of cytochrome c from the permeability transition pore (PTP) is believed to play an important role in ischemia/reeoxygenation-induced cardiomyocyte apoptosis, which contributes the development of heart failure following balloon angioplasty after myocardial infarction. This study was performed to investigate the role of Apop in hypoxia/reoxygenation-induced death of cardiomyocytes. Neonatal cardiomyccybas were prepared from ventricles of 2- to 3-day-old Sprague-Dawley rats and maintained in DMEM supplemented with 10% fetal bovine serum. Antisense plasmid to prevent Apop expression or control vector was introduced into cardiomyocybes using an electroporator. Cells were then incubated in hypoxic condition (1% O2 … More ) for 48 hours followed by 2 hours of reoxygenation. Cell viability was assessed by MTT assay. Anti-apoptotic bcl-2 family proteins, such as bcl-2 and bcl-xL were unable to prevent Apop-induced apoptosis, while it was prevented by inhibitors of cyclophilin D, an important component of PTP. These findings indicate that the apoptosis induced by Apop is not mediated by bcl-2 family-related channels, but may require PIP on mitochondrial membrane. Introduction of siRNA targeted toward Apop transcript into cultured cells almost completely abolished the green fluorescence of Apop-GFP fusion protein, indicating that siRNA prevents the expression of Apop. The inhibitory activity of siRNA was also confirmed by Western blot analysis. In the experiments of hypoxia/reoxygenation-induced cell death, introduction of siRNA inhibited the activation of caspase-cascade and the death of cultured cardiomyccytes, whereas introduction of control siRNA affected neither caspase activity nor viability; Inhibition of Apop expression prevented hypoxia/reoxygenation-induced death of cardiomyocytes, suggesting that Apop protein may play roles in the development of cardiac diseases following ischemia-reperfusion injury. Less