急性低氧对红细胞的损伤及低氧习服的保护作用

发布时间：2018-08-14 13:36

【摘要】：目的：从红细胞变形性、红细胞的膜流动性、红细胞膜蛋白质表达、红细胞膜ATP酶活性及胞内Na~+和Ca~(2+)浓度方面探讨急性低氧对红细胞的损伤以及低氧习服的保护作用。方法：将健康雄性Wistar大鼠随机分为常氧对照组、急性低氧组和低氧习服组，每组10只。采用低压舱模拟高原低氧环境对大鼠分别进行急性低氧和间断低氧习服，麻醉后心脏采血，分别1．采用F-820全自动血细胞分析仪测定大鼠红细胞血液学指标，采用LG-B-190血细胞变形测试仪测定红细胞变形性；2．采用日立F-4000荧光分光光度计检测代表红细胞膜脂流动性的DPH荧光偏振度，采用U-2001紫外分光光度计测定红细胞膜胆固醇和总磷脂含量，采用薄层色谱分析法测定红细胞膜磷脂成分PC、PS、PE、SM的含量；3．采用双向电泳建立大鼠红细胞膜蛋白质双向电泳图谱，通过Image Master 2D Elite软件分析寻找出蛋白质差异点，对其进行质谱鉴定。4．采用红细胞膜ATP酶活性测定试剂盒测定红细胞膜ATP酶活性，采用红细胞内Na~+和Ca~(2+)浓度测定试剂盒测定红细胞内Na~+和Ca~(2+)浓度。结果：1．急性低氧大鼠红细胞总数、血红蛋白浓度增加，，红细胞变形性下降；红细胞膜脂流动性下降，红细胞膜胆固醇含量、总磷脂含量、胆固醇／总磷脂(C／P)比值下降，红细胞膜磷脂各成分含量发生了变化；建立了急性低氧大鼠红细胞膜蛋白质双向电泳图谱，并与常氧大鼠红细胞膜蛋白质双向电泳图谱比较，选取7个蛋白质点，其中4个在急性低氧后表达下降，其余3个表达改变不明显：红细胞Na~+-K~+-ATP酶和Ca~(2+)-Mg~(2+)-ATP酶活性下降，红细胞内Na~+浓度和Ca~(2+)浓度升高。2．低氧习服大鼠红细胞总数、红细胞比容、血红蛋白浓度更高，红细胞平均体积较急性低氧组和常氧对照组下降，红细胞变形性比急性低氧组提高，接近常氧对照组水平；红细胞膜脂流动性增强，红细胞膜胆固醇含量、总磷脂含量、胆固醇／总磷脂(C／P)比值升高，红细胞膜磷脂各成分含量发生了变化；建立了低氧习服大鼠红细胞膜蛋白质双向电泳图谱，并与急性低氧大鼠、常氧大鼠红细胞膜蛋白质双向电泳图谱比较，上述7个蛋白质点中4个在低氧习服后表达增加，3个表达下降。蛋白质差异点质谱技术鉴定结果分别为是补体结合蛋白、水通道蛋白、
[Abstract]:Objective: to investigate the protective effects of acute hypoxia on erythrocyte damage and hypoxia acclimation from the aspects of erythrocyte deformability, erythrocyte membrane fluidity, erythrocyte membrane protein expression, erythrocyte membrane ATP enzyme activity and intracellular Na ~ and Ca ~ (2) concentration. Methods: healthy male Wistar rats were randomly divided into normoxic control group, acute hypoxia group and hypoxic acclimatization group with 10 rats in each group. Rats were acclimated to acute hypoxia and intermittent hypoxia by simulated high altitude hypoxia environment in low pressure chamber. Blood was collected from the heart after anesthesia. The erythrocyte hematological indexes of rats were measured by F-820 automatic blood cell analyzer. Erythrocyte deformability was measured by LG-B-190 blood cell deformability test. The fluorescence polarization degree of DPH, which represents the fluidity of erythrocyte membrane lipid, was detected by Hitachi F-4000 fluorescence spectrophotometer. The contents of cholesterol and total phospholipid in erythrocyte membrane were determined by U-2001 ultraviolet spectrophotometer, and the content of phospholipid in erythrocyte membrane was determined by thin layer chromatography. Two-dimensional electrophoretic map of rat erythrocyte membrane protein was established by two-dimensional electrophoresis. The protein difference point was found by Image Master 2D Elite software, and identified by mass spectrometry. 4. The erythrocyte membrane ATP enzyme activity was determined by using the erythrocyte membrane ATP enzyme activity assay kit, and the erythrocyte membrane ATP enzyme activity was determined by using the red blood cell membrane ATP enzyme assay kit. The concentration of Na ~ and Ca ~ (2) in red blood cells was determined by the kit of Na ~ and Ca ~ (2) in erythrocytes. Results 1. The total number of erythrocytes, hemoglobin concentration, erythrocyte deformability decreased, erythrocyte membrane lipid fluidity decreased, erythrocyte membrane cholesterol content, total phospholipid content and cholesterol / total phospholipid ratio decreased in acute hypoxic rats. The contents of erythrocyte membrane phospholipid were changed, the two-dimensional electrophoresis map of erythrocyte membrane protein in acute hypoxia rats was established, and compared with that of normoxic rat erythrocyte membrane protein two-dimensional electrophoresis, 7 protein spots were selected. The expression of Na ~-K ~ -ATPase and Ca ~ (2) mg ~ (2) -ATPase in erythrocytes decreased, the Na ~ (2) and Ca ~ (2) -ATPase concentrations in erythrocytes increased. The specific volume of erythrocyte and hemoglobin concentration were higher, the mean volume of red blood cell was lower than that of acute hypoxia group and normoxic control group, the deformability of erythrocyte was higher than that of acute hypoxia group, which was close to the level of normoxic control group, and the fluidity of erythrocyte membrane lipid was enhanced. The contents of erythrocyte membrane cholesterol, total phospholipid and the ratio of cholesterol to total phospholipid (C / P) were increased, and the components of erythrocyte membrane phospholipid were changed. Compared with acute hypoxic rats and normoxic rats, the expression of erythrocyte membrane proteins increased in 4 of the 7 protein spots and decreased in 3 after hypoxia acclimation. Protein differential point mass spectrometry was used to identify complement binding protein and aquaporin.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R363

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