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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。
Background: The protein kinase C family has three groups, the classic protein kinase C and atypical protein kinase C, and novel protein kinase C. Some experiments confirmed that the protein kinase C family plays an important role in hypoxic preconditioning of cerebral ischemia in hypoxic environment. OBJECTIVE: To establish a cell hypoxia model to observe the effect of hypoxia-activated membrane-bound translocation of classical protein kinase C subtypes (α, βⅠ, βⅡ, γ) at the cellular level. Design: Cell-based, randomized block design. SETTING: Department of Neurobiology, Capital Medical University. Materials: The experiment was performed at the Department of Neurobiology Cell Culture, Capital Medical University in 2004-05. Passage cells cultured in this laboratory have neuronal characteristics of human neuroblastoma cell line. Methods: Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and classical protein kinase C Western blot analysis and immunocytochemistry and other biochemical techniques were used to observe the cells cultured in different periods of 0 ~ 24h, hypoxic environment for the volume Oxygen with a fraction of 0.01, carbon dioxide with a volume fraction of 0.05, and nitrogen with a volume fraction of 0.94 influenced the translocation level of classical protein kinase C-α and βⅠ and βⅡ in human neuroblastoma cells. MAIN OUTCOME MEASURES: ①To observe the effect of continuous hypoxia on the translocation of classical protein kinase C in human neuroblastoma cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and classical protein kinase C Western blotting. ② To observe the effect of continuous hypoxia on the translocation of classical protein kinase C membrane in human neuroblastoma cells by immunocytochemistry. Results: ① The translocation levels of classical protein kinase C-α, βⅠ and βⅡ cells were significantly increased with the prolongation of hypoxia (P <0.05), especially in the classical protein kinase C-βⅠ subtype Hypoxia 4h after the most significant increase (P <0.001). ② No expression of classical protein kinase C-γ subtype was detected in human neuroblastoma cells in both normoxia and persistent hypoxia. The results of sodium dodecyl sulfate - polyacrylamide gel electrophoresis and classical protein kinase C Western blot analysis and immunocytochemistry experiments were consistent. CONCLUSIONS: ①At the end of hypoxia, the typical protein kinase C-α and the βⅠ and βⅡ isoforms are present in human neuroblastoma cells, and the changes of membrane translocation occur. ② The classical protein kinase C-γ subtype may not exist in human neuroblastoma cell lines, or some of the characteristics of this cell line has been lost.