论文部分内容阅读
膜周蛋白PICK1(protein interacting C-kinase-1)参与多种膜受体与膜上蛋白的运输并影响细胞的功能。本研究旨在探索小胶质细胞PICK1与P2Y6受体之间的相互作用是否可改变P2Y6受体在细胞膜上的表达,以及对小胶质细胞吞噬功能的影响。采用小鼠脑内皮层原代培养的小胶质细胞进行免疫共沉淀实验揭示,与PICK1敲除小鼠比较,野生小鼠皮层小胶质细胞内存在PICK1-P2Y6受体相互作用。生物素化、密度梯度离心结合蛋白质印迹实验证明,PICK1基因敲除小鼠的小胶质细胞膜表面P2Y6受体表达水平降低。荧光胶珠吞噬实验结合免疫组织化学染色显示,PICK1基因敲除小鼠的小胶质细胞对UDP(刺激)引起的荧光胶珠吞噬作用减弱。蛋白质印迹实验显示,与野生型小鼠比较,PICK1基因敲除小鼠小胶质细胞中的Akt-308T磷酸化水平明显降低;使用Akt抑制剂API-2能有效抑制Akt在小胶质细胞内的(磷酸化)激活及UDP刺激引起的吞噬作用。上述结果表明,敲除PICK1能下调小胶质细胞膜上P2Y6受体的表达,并降低小胶质细胞的吞噬功能,且这一过程依赖Akt磷酸化修饰。总之,PICK1可促进P2Y6受体在细胞膜上的表达,是小胶质细胞吞噬功能的重要调节子;敲除PICK1可下调P2Y6膜受体表达,并降低小胶质细胞的吞噬功能。这一结果可加深对小胶质细胞的吞噬功能及机制的认识。
PICK1 (protein interacting C-kinase-1) is involved in the transport of multiple membrane receptors and membrane proteins and affects cell function. The aim of this study was to investigate whether the interaction between PICK1 and P2Y6 receptors in microglia could alter the expression of P2Y6 receptor on the cell membrane and its effect on microglial phagocytosis. Coimmunoprecipitation experiments using microglia primarily cultured in mouse brain cortex revealed PICK1-P2Y6 receptor interaction in the cortical microglial cells of wild-type mice as compared to PICK1 knockout mice. Biotinylation and density gradient centrifugation combined with Western blotting showed that the expression of P2Y6 receptor on the microglia membrane surface of PICK1 knockout mice was decreased. Fluorescent bead phagocytosis experiments combined with immunohistochemical staining showed that microglia in PICK1 knockout mice attenuated UDP-stimulated fluorescence bead phagocytosis. Western blotting showed that the phosphorylation level of Akt-308T in microglial cells of PICK1 knockout mice was significantly decreased compared with wild type mice; Akt inhibitor API-2 was able to effectively inhibit Akt in microglia (Phosphorylation) activation and UDP-stimulated phagocytosis. The above results indicate that knocking out PICK1 can down-regulate the expression of P2Y6 receptor on microglia and decrease the phagocytosis of microglia, and this process is dependent on Akt phosphorylation. In conclusion, PICK1 can promote P2Y6 receptor expression on the cell membrane and is an important regulator of microglial phagocytosis. Knockout of PICK1 can down-regulate the expression of P2Y6 membrane receptor and decrease the phagocytosis of microglia. This result can deepen the understanding of microglial phagocytic function and mechanism.