大鼠内毒素血症时外周血及肺泡内PMN死亡方式及呼吸爆发的检测

Apoptotic pattern of polymorphonuclear neutrophils in peripheral blood and alveolus and detection for respiratory burst in endotoxemia rats

目的 观察大鼠内毒素血症时肺组织中及外周血中性粒细胞 (PMN)凋亡、坏死及功能改变的差异。方法 Wistar大鼠 2 5只 ,腹腔注射内毒素 (LPS) (O55∶B5,5mg/kg)造成内毒素血症 ,分别在给予LPS前 (对照 )及给予LPS后 2、4、8、1 2h(每组 5只动物 )取血及支气管肺泡灌洗 ,密度梯度法分离PMN ,用流式细胞仪测定凋亡和坏死比例以及呼吸爆发功能的改变。结果 内毒素血症时 ,外周血和支气管肺泡灌洗液中PMN凋亡细胞比例相似。但与对照相比 ,外周血PMN坏死比例明显增加 ,呼吸爆发能力明显受抑 ,而支气管肺泡灌洗液PMN坏死比例显著减少 ,呼吸爆发能力显著增强。结论 在内毒素血症时 ,扣押于肺组织中的PMN在凋亡和坏死上表现出与外周血PMN不同的改变 ,其结果是组织中PMN存活PMN增加 ,并持续处于活化状态 ,这与PMN造成组织损伤有关。

Objective To investigate the differences of the peripheral polymorphonuclear neutrophils (PMN) and the PMN extravasated into pulmonary tissues in apoptosis, necrosis and respiratory burst following endotoxemia. Methods Twenty five Wistar rats were subjected to endotoxemia induced by intraperitoneal injection of LPS (O55∶B5, 5 mg/kg). Blood samples were collected and the bronchoalveoli were lavaged pre LPS and 2, 4, 8 and 12 h after LPS injection. After PMN was isolated with density gradient centrifugation, the apoptosis, necrosis of and respiratory burst of PMN were determined with flowcytometry. Results The apoptotic rate of PMN in peripheral blood was similar with that in bronchoalveoli lavage fluid (BLF). But compared with control, the PMN necrosis was increased and respiratory burst was suppressed significantly in peripheral blood after endotoxemia. While, for the PMN in BLF, the necrosis was decreased and respiratory burst was increased significantly. Conclusion Out results indicate that the PMN extravasted into the pulmonary tissue has different characters in apoptosis, necrosis and respiratory burst compared with peripheral PMN. The apoptosis of tissue PMN is delayed and the cytotoxicity increased. Thus, the retention of PMN in tissues and the abnormality in apoptotic pathway inevitably generate persistent activation of PMN and excessive release of toxic substances, which might result in tissue injury.