摘要
目的:确定蛋白免疫印迹检测大鼠脑组织中P-糖蛋白的最佳实验条件。方法:将成年雄性Wistar大鼠随机分为正常组和模型组。模型组分八个时间点,采用反复夹闭颈总动脉辅以硝普钠降压的方法制备脑缺血再灌注模型。正常组直接断头取脑分别进行组织匀浆和提取微血管,而模型组断头取脑均提取微血管,利用蛋白免疫印迹检测大鼠脑内P-糖蛋白的表达水平。结果:最佳实验条件是:提取微血管,用裂解液C冰上裂解30min,然后40000g离心30min留上清,蛋白定量后在5*上样缓冲液中25℃处理30min。而模型组各时间点P-糖蛋白的表达量随时间的延长逐渐增多。结论:利用提取脑微血管的方法在上述实验条件下进行P-糖蛋白的检测方便、可行,明显优于脑组织匀浆;并且在脑缺血再灌注这种病理状态下脑组织内P-糖蛋白的表达呈现一定的时序特征。
Objective： To determine the optimum experiment condition of detecting P-glycoprotein with Western blotting. Methods： Adult male Wistar rats were randomly divided into normal group and model group with different time points, including 2h, 6h, 24h, 2d, 3d, 4d, 7d and 10d. The model rat of cerebral ischemia and reperfusion was induced by repeated common carotid artery occlusion by assisting depressurizadon with Sodium Nitroprusside-glucose solution. The normal group were direcdy decapitated and conducted tissue homogenate and capillaries isolation respectively. At the same time all rats of model group were also decapitated, when reaching the time point of ischemia and reperfusion, taken out of brain tissues, and were isolated capillaries from rat brains by means of strainer mesh. Western blotting was used to detect the level of P-glycoprotein in cerebral homogenate and isolated capillaries. Results： The optimum experiment condition was isolating capillaries, splitting for 30min in ice with solution C, centrifuging at 40000g for 30min, measuring supernatant proteins, and solubilizing 30min at 25℃ in 5＊ loading buffer. The level of this protein changed respectively to different degrees among the model group of different time points, and along with time the expression of P-glycoprotein gradually increased. Conclusions： It is more convenient and feasible using isolation of brain capillaries to detect P-glycoprotein under the above experiment condition than cerebral homogenate. And the expression of P- glycoprotein in brain tissue shows differential characteristic of time series in the pathologic status of cerebral ischemia and reperfusion.
出处
《现代生物医学进展》
CAS
2007年第3期329-331,共3页
Progress in Modern Biomedicine
基金
国家自然科学基金面上项目课题(30300469)
北京市科技新星计划(2006A48)
关键词
P-糖蛋白
蛋白免疫印迹
检测方法
脑缺血再灌注
微血管
P-glycoprotein
Western blotting
Detection method
Cerebral ischemia and reperfusion
Capillaries
