【摘要】 背景:蛋白激酶C家族有3组,经典型蛋白激酶C及非典型蛋白激酶C和新奇型蛋白激酶C。一些实验证实在低氧环境中,蛋白激酶C家族在脑缺血低氧预刺激中发挥了重要作用。目的:建立细胞缺氧模型,观察在细胞水平上低氧激活经典型蛋白激酶C各亚型(α,βⅠ,βⅡ,γ)膜转位现象的影响。设计:以细胞为对象,随机区组设计。单位:首都医科大学基础医学院神经生物学系。材料:实验于2004-05在首都医科大学神经生物学系细胞培养室完成。由本实验室培养的传代细胞,具有神经元特性的人神经母细胞瘤细胞系。方法:应用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和经典型蛋白激酶C蛋白印迹分析和免疫细胞化学等生化技术,观察培养的细胞经过0～24h不同时间段,低氧环境为体积分数0.01的氧气,体积分数0.05的二氧化碳,体积分数0.94的氮气,对人神经母细胞瘤细胞内经典型蛋白激酶C-α及βⅠ和βⅡ细胞膜转位水平的影响。主要观察指标:①观察持续低氧对人神经母细胞瘤细胞内经典型蛋白激酶C膜转位影响采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和经典型蛋白激酶C蛋白印迹分析检测。②观察持续低氧对人神经母细胞瘤细胞内经典型蛋白激酶C膜转位影响采用免疫细胞化学方法检测。结果:①经典型蛋白激酶C-α及βⅠ和βⅡ细胞膜转位水平均随持续性低氧时间的延长而明显增高(P<0.05),尤以经典型蛋白激酶C-βⅠ亚型在持续性低氧4h后的增高最为显著(P<0.001)。②无论在常氧还是在持续性低氧状态下,在人神经母细胞瘤细胞内均未检测到经典型蛋白激酶C-γ亚型的蛋白表达。应用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和经典型蛋白激酶C蛋白印迹分析和免疫细胞化学实验检测的结果均一致。结论:①在持续低氧的条件下存在于人神经母细胞瘤细胞内经典型蛋白激酶C-α及βⅠ和βⅡ亚型均被激活,发生膜转位变化。②经典型蛋白激酶C-γ亚型可能不存在于人神经母细胞瘤的细胞系中,或者这种细胞系某种特性已丧失。更多还原

【Abstract】 BACKGROUND:The protein kinase C(PKC) family consists of 3 groups of PKCs,namely the conventional PKC(cPKC),atypical PKC and novel PKC. Accumulating studies conducted in recent years have suggested that PKCs may play important roles in the development of cerebral ischemic/hypoxic preconditioning(I/HPC).OBJECTIVE:To observe membrane translocation of hypoxiaactivated cPKC isoforms(α,βI,βⅡand γ) at cellular levels in a cell hypoxia model.DESIGN:Randomized block design.SETTING:Department of Neurobiology,College of Basic Medical Sciences, Capital University of Medical Sciences.MATERIALS:The experiment was completed at the Neurobiological Cell Culture Laboratory of Capital University of Medical Sciences in May 2004. Human neuroblastoma cells with the properties of neurons were maintained and passaged in this laboratory.METHODS:The activation of cPKC isoforms under hypoxic condition and changes of cPKCα,βI,βⅡand γmembrane translocation(an indicator of PKCs activation) in SHSY5Y neuroblastoma cells in response to hypoxia (1%O2,5%CO2 and 94%N2) for 0 to 24 hours were observed using SDSPAGE, Western blotting and immunocytochemistry.MAIN OUTCOME MEASURES:Effect of sustained hypoxia on cPKC membrane translocation in human neuroblastoma cells was observed with SDSPAGE,cPKC Western blotting, and immunocytochemistry.RESULTS:cPKCα,βΙand βⅡmembrane translocation were increased significantly(P< 0.05) in a timedependent manner in response to hypoxic exposure, and the increase of cPKCβΙwas more evident(P< 0.001) after 4 hours of hypoxic exposure, whereas no cPKCγwas detected in SHSY5Y neuroblastoma cells either under normoxic or hypoxic condition.The results suggested that all cPKC isoforms,epically cPKCβΙ,could be activated by sustained hypoxia,and the absence of cPKCγin SHSY5Y neuroblastoma cells may be relevant to the loss of specific biological features of the cultured cells.CONCLUSION:Sustained hypoxia activates the isoforms of cPKCα,βI and βⅡin human neuroblastoma cells and induces their membrane translocation. cPKCγisoform may not exist in human neuroblastoma cells,or the cells has lost certain biological characteristics.更多还原