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目的探讨模拟高原低氧对大鼠肝脏中CYP2E1活性和蛋白表达的影响。方法将12只健康雄性Wistar大鼠随机分成对照组(1500m)和低氧组(4100 m)。低氧组置于高原模拟氧舱,3d后解剖,取大鼠肝脏、提取肝微粒体,用于CPY2E1的分析。采用Thermo AcclaimTM120C18柱(3 mm×150 mm,3μm),流动相:甲醇-水(V∶V)60∶40,流速:1.0ml·min-1,柱温:25℃,检测波长:280nm,进样体积:20μl,建立测定氯唑沙宗浓度的HPLC方法,Western blot方法检测大鼠肝脏中CYP2E1的蛋白表达。结果活性测定结果显示,对照组氯唑沙宗在孵育体系中的平均反应速度为0.1384±0.0148μg·min-1,其酶动力学参数:最大反应速度(Vmax)为2.06μg·min-1,米氏常数(Km)为116.38±3.46μg·ml-1。低氧组氯唑沙宗平均反应速度为0.0822±0.0071μg·min-1,Vmax为2.60μg·min-1,Km为142.88±5.66μg·ml-1。Western blot结果显示,对照组CYP2E1的蛋白表达量是低氧组的9.88倍。结论与对照相比,低氧组大鼠的CYP2E1活性降低,蛋白表达量降低。
Objective To investigate the effects of simulated plateau hypoxia on CYP2E1 activity and protein expression in rat liver. Methods Twelve healthy male Wistar rats were randomly divided into control group (1500m) and hypoxia group (4100m). The hypoxia group was placed in the simulated oxygen chamber in the plateau, and after 3 days, the liver was taken and the liver microsomes were extracted for the analysis of CPY2E1. The mobile phase consisted of methanol-water (V: V) 60:40, flow rate 1.0ml · min-1, column temperature 25 ℃, detection wavelength 280nm, Sample volume: 20μl, established HPLC method for the determination of chlorzoxazone concentration, Western blot method to detect the protein expression of rat liver CYP2E1. Results The results of activity assay showed that the average reaction rate of chlorzoxazone in the control group was 0.1384 ± 0.0148μg · min-1, and its kinetic parameters were as follows: the maximum reaction velocity (Vmax) was 2.06μg · min-1, The Michaelis constant (Km) was 116.38 ± 3.46 μg · ml-1. The average response rate of chlorzoxazone in hypoxia group was 0.0822 ± 0.0071μg · min-1, Vmax was 2.60μg · min-1, and Km was 142.88 ± 5.66μg · ml-1. Western blot results showed that the protein expression of CYP2E1 in the control group was 9.88 times that of the hypoxia group. Conclusion Compared with control, the activity of CYP2E1 in hypoxia rats decreased and the protein expression decreased.