目的:探讨乳酸引起的酸度变化对外周全血吞噬细胞呼吸爆发与吞噬功能的影响及发生机制。方法:1)取20名健康男性外周静脉血10 ml,分10份建立乳酸体积量与血乳酸值的回归方程;2)另取10名健康男性外周静脉血20 ml分为对照组(Ctr组)、乳酸处理组(E组),各组再分10小组,按照第一步建立的回归方程加入不同体积的乳酸,同时Ctr组给予DPI处理,2组同时孵育10 min后各小组每份取100μl全血用流式细胞仪测定呼吸爆发和吞噬功能;3)分别取Ctr组、E组第6小组的血液分离中性粒细胞,利用免疫细胞化学与共聚焦技术进行NADPH氧化酶关键亚基gp91 phox与p47 phox的共定位实验。结果:1)低浓度(低于4.81 mmol/L)的乳酸抑制吞噬细胞的呼吸爆发和吞噬功能,中度浓度(4.81 mmol/L~8.14 mmol/L)的乳酸浓度可引起吞噬细胞呼吸爆发依赖乳酸浓度的增加而增加,高于此浓度可引起吞噬细胞呼吸爆发与急剧下降,而吞噬功能提前急剧下降;2)吞噬细胞NADPH氧化酶活性的改变与呼吸爆发的变化呈高度的相关性;3)乳酸浓度为8.14 mmol/L时中性粒细胞NADPH氧化酶关键亚基p47 phox移为至质膜与gp91 phox出现共定位。结论:乳酸引起的外周血酸度的变化可以通过调控NADPH氧化酶活性的改变影响其吞噬细胞呼吸爆发和吞噬功能。
Objective:To investigate the impact of acidity change caused by lactic acid on the phagocytes cell respiratory burst and the phagocytic function in peripheral whole blood and mechanism.Method:1) 10ml peripheral whole blood from 20 healthy male,was divided into 10 portions(1ml per portion) to establish the regression equation between concentration land the volume lactic acid.2) 20ml peripheral whole blood from another 10 healthy male,was divided into group Ctr and E,each group was further separated into 10 portions and added in the different volume of lactic acid according to the first step,the group Ctr was treated with DPI additionally.The blood of two groups was incubated for 10 minutes and then 100ul whole blood was taken from each portion to detect the function of respiratory burst and phagocytosis of neutrophils and mononuclear cell respectively by flow cytometery.3.The neutrophils from the sixth portion blood of group Ctr and E were taken and observed the co-localization of the gp91phox and p47phox in NADPH-oxidase using immunocytochemistry and confocal technology.Result:1) Low concentration(less than 4.81mmol / L) of lactic acid can inhibit the function of phagocytosis and respiratory burst of phagocytic cells,moderate concentration(4.81mmol / L~8.14mmol / L) of lactic acid can lead to the increase respiratory burst of phagocytic cells.The curve of respiratory burst can be declined sharply while above this concentration lactic acid and the curve of sharp decline of the function of phagocytosis was early than the curve of respiratory burst.2) It is a high degree of correlation between the activity NADPH oxidase and the respiratory burst in phagocytic cells.3) There is co-localization between the subunit p47phox and the gp91phox in NADPH-oxidase on the plasma membrane in neutrophils when the lactic acid concentration in blood is 8.14mmol / L.Conclusion:The acidity induced by lactic acid in peripheral blood can regulate the activity NADPH oxidase and lead to the change of the pha