【摘要】：目的：從紅細胞變形性、紅細胞的膜流動性、紅細胞膜蛋白質(zhì)表達、紅細胞膜ATP酶活性及胞內(nèi)Na~+和Ca~(2+)濃度方面探討急性低氧對紅細胞的損傷以及低氧習服的保護作用。 方法：將健康雄性Wistar大鼠隨機分為常氧對照組、急性低氧組和低氧習服組，每組10只。采用低壓艙模擬高原低氧環(huán)境對大鼠分別進行急性低氧和間斷低氧習服，麻醉后心臟采血，分別1．采用F-820全自動血細胞分析儀測定大鼠紅細胞血液學指標，采用LG-B-190血細胞變形測試儀測定紅細胞變形性；2．采用日立F-4000熒光分光光度計檢測代表紅細胞膜脂流動性的DPH熒光偏振度，采用U-2001紫外分光光度計測定紅細胞膜膽固醇和總磷脂含量，采用薄層色譜分析法測定紅細胞膜磷脂成分PC、PS、PE、SM的含量；3．采用雙向電泳建立大鼠紅細胞膜蛋白質(zhì)雙向電泳圖譜，通過Image Master 2D Elite軟件分析尋找出蛋白質(zhì)差異點，對其進行質(zhì)譜鑒定。4．采用紅細胞膜ATP酶活性測定試劑盒測定紅細胞膜ATP酶活性，采用紅細胞內(nèi)Na~+和Ca~(2+)濃度測定試劑盒測定紅細胞內(nèi)Na~+和Ca~(2+)濃度。 結(jié)果：1．急性低氧大鼠紅細胞總數(shù)、血紅蛋白濃度增加，，紅細胞變形性下降；紅細胞膜脂流動性下降，紅細胞膜膽固醇含量、總磷脂含量、膽固醇／總磷脂(C／P)比值下降，紅細胞膜磷脂各成分含量發(fā)生了變化；建立了急性低氧大鼠紅細胞膜蛋白質(zhì)雙向電泳圖譜，并與常氧大鼠紅細胞膜蛋白質(zhì)雙向電泳圖譜比較，選取7個蛋白質(zhì)點，其中4個在急性低氧后表達下降，其余3個表達改變不明顯：紅細胞Na~+-K~+-ATP酶和Ca~(2+)-Mg~(2+)-ATP酶活性下降，紅細胞內(nèi)Na~+濃度和Ca~(2+)濃度升高。2．低氧習服大鼠紅細胞總數(shù)、紅細胞比容、血紅蛋白濃度更高，紅細胞平均體積較急性低氧組和常氧對照組下降，紅細胞變形性比急性低氧組提高，接近常氧對照組水平；紅細胞膜脂流動性增強，紅細胞膜膽固醇含量、總磷脂含量、膽固醇／總磷脂(C／P)比值升高，紅細胞膜磷脂各成分含量發(fā)生了變化；建立了低氧習服大鼠紅細胞膜蛋白質(zhì)雙向電泳圖譜，并與急性低氧大鼠、常氧大鼠紅細胞膜蛋白質(zhì)雙向電泳圖譜比較，上述7個蛋白質(zhì)點中4個在低氧習服后表達增加，3個表達下降。蛋白質(zhì)差異點質(zhì)譜技術鑒定結(jié)果分別為是補體結(jié)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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R363

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