The effect of cytokines on leukocyte kinetics in rat pulmonary microcirculation.

尽管有报道称细胞因子和白细胞在急性肺损伤中起主要作用，但细胞因子对肺微循环中白细胞动力学的影响尚不完全清楚。为了研究白细胞在大鼠急性肺损伤模型中的作用，我们建立了两种新的方法来检测大鼠和兔肺微循环中的过氧化氢。我们将荧光染料2‘，7’-二氯二乙酸酯(DCFH-DA)应用于活体动物，该荧光染料已被用来通过流式细胞术测量分离的中性粒细胞产生的过氧化氢。我们还将Ce技术应用于活体动物，该技术已被用于通过电子显微镜检测分离的中性粒细胞产生的过氧化氢。以5 mg/kg/h的速度持续滴注内毒素复制大鼠急性肺损伤模型。利用这些技术，我们检测和定量了中性粒细胞与肺毛细血管内皮细胞黏附的过氧化氢的产生。我们还发现，这种过氧化氢扩散到内皮细胞中。用兔抗大鼠肿瘤坏死因子α多克隆抗体对注入内毒素的大鼠肺组织进行免疫组化染色。大鼠肺泡壁呈阳性反应。用双抗体夹心法测定内毒素肺组织中肿瘤坏死因子α(TNFa)的含量。注入内毒素的大鼠肺组织中肿瘤坏死因子α的含量是对照大鼠肺组织的5倍。在这个项目中，我们展示了中性粒细胞和肿瘤坏死因子α在急性肺损伤动物模型肺微循环中的作用。

Although, it was reported that cytokines and the leukocyte play major roles in acute lung injury, the effect of cytokines on the leukocyte kinetics in the pulmonary microcirculation is not fully understood. To investigate the role of leukocytes in the rat acute lung injury model, we developed two novel methods to detect hydrogen peroxide in the rat and rabbit pulmonary microcirculation. We applied the fluorescent dye, 2', 7'-dichlorofluorescin diacetate (DCFH-DA), which has been used to measure hydrogen peroxide generated from isolated neutrophils by the flowcytometry, to a live animal. We also applied the cerium technique, which has been used to detect hydrogen peroxide generated from an isolated neutrophil by the electron microscopy, to a live animal. The acute lung injury rat model was made by a continuous infusion of endotoxin at a rate of 5 mg/kg/hr. Using these techniques, we detected and quantified the hydrogen peroxide production in the neutrophil adhered to the pulmonary capillary endothelium. We also found that this hydrogen peroxide diffuses into the endothelial cell. The rat lung infused with endotoxin was immunostained with the rabbit polyclonal antibody against rat TNFalpha. The alveolar wall of the rat was positively stained with that. We also measured TNFa contents in the rat lung infused with endotoxin by ELISA method. The contents of TNFalpha in the rat lung infused with endotoxin was five times higher than that in the control rat lung. In this project, we demonstrated roles of neutrophil and TNFalpha in the pulmonary microcirculation of the acute lung injury animal model.