STUDY OF ADHESION MOLECULES, CYTOSKELETON AND SIGNAL TRANSDUCTION PATHWAY ON INJURY OF ENDOTHELIAL CELLS FOR ISOLATED PRESERVED LUNGS

我们发现中性粒细胞引起的肺损伤是由中性粒细胞与血管内皮细胞黏附后产生的黄嘌呤氧化酶(XO)产生的活性氧引起的。通常，XO不存在于中性粒细胞中，而存在于内皮细胞中。因此，我们推测这种肺损伤是否是由内皮细胞本身激活引起的。在大鼠离体肺灌流实验中，我们用蛋白激酶C拮抗剂研究了细胞内信号转导系统是否参与了中性粒细胞活化对肺血管内皮细胞的损伤。我们发现PKC拮抗剂以剂量依赖的方式改善中性粒细胞激活所致的肺血管损伤。此外，我们还发现不含中性粒细胞的PKC激动剂可增加肺血管通透性。另一方面，在细胞培养模型中，不可能测量细胞培养片的蛋白质渗透性，我们测量了反式…培养的肺血管内皮细胞膜有更多的内皮电阻(TER)。连续测定大鼠肺血管内皮细胞培养的细胞膜融合时的TER，并检测几种激动剂和拮抗剂对培养细胞膜的作用。PKC激动剂以剂量依赖的方式降低培养细胞膜的TERs，而PKC激动剂的载体DMSO则不能。这些发现支持大鼠离体肺实验的结果。我们还对肺血管内皮细胞内的肌动蛋白纤维进行了染色。肌动蛋白纤维在无刺激的情况下存在于胞浆的周边区，但被PKC激动剂解聚并在胞核周围移动。提示肺血管内皮细胞在一定刺激条件下被PKC解聚，使其形态发生改变，最终导致肺微血管通透性增加。较少

We showed that neutrophils-induced lung injury was induced by reactive oxygen species generated via xanthine oxidase (XO) following adhesion of neutrophils on the endothelial cells. Usually, XO does not exist in the neutrophils but endothelial cells. Therefore we speculate if this kind of lung injury is induced by activation of endothelial cells themselves. In the experiments of isolated perfused rat lungs, whether intracellular signal transduction systems were involved in the injury of the pulmonary vascular endothelial cells caused by activated neutrophils, we investigated using a protein kinase C antagonist. We showed that PKC antagonist ameliorated the pulmonary vascular injury caused by activated neutrophils in a dose dependent manner. Moreover, we showed that the pulmonary vascular permeability was increased by a PKC agonist without neutrophils. On the other hand, in cell culture models, it was impossible to measure protein permeability for cell culture sheets, we measured trans- … More endothelial electrical resistance (TER) across the cultured pulmonary vascular endothelial cell sheets. We continuously measured TER of the cultured cell sheets from rat pulmonary vascular endothelial cells when they were confluent and the tested the several kinds of agonists and antagonists to the cultured cell sheets. The TERs of the cultured cell sheets were decreased by PKC agonists by dose dependent manner, meanwhile DMSO, a vehicle of PKC agonist, failed. These findings supported the results from the experiment of isolated rat lungs. We also stained actin fibers in the pulmonary vascular endothelial cells. Actin fibers existed at the peripheral area of the cytosol without any stimulations, however, they were depolymerized and moved around nucleus by PKC agonist. These findings suggest that the pulmonary vascular endothelial cells were changed their morphology by depolymerizing actin fibers by PKC when they were activated by some stimulations, and finally pulmonary microvascular permeability was increased. Less