小胶质细胞人补体攻膜复合物亚溶破模型制作及功能鉴定_医学论文.doc免费

小胶质细胞人补体攻膜复合物亚溶破模型制作及功能鉴定_医学论文 小胶质细胞人补体攻膜复合物亚溶破模型制作及功能鉴定_医学论文 作者：罗娜，白云，周静然，宋敏，王艳艳，杨晓亚，许雪青，段文元，熊加祥 【关键词】 C56 　　Preparation and functional identification of sublytic complement membrane attack complex on cultured mouse microglia cells 　　【Abstract】 AIM: To establish sublytic membrane attack complex (sMAC) models and identify the sublytic effects on mouse microglia cells in vitro. METHODS: An activated complex of C56 was generated by treatment of acute phase serum and C7-C9 came from fresh normal human serum to assemble sMAC on cultured microglia cells in vitro. The sublytic dose of sMAC was determined by CCK8 assay. SMAC deposition was identified by laser confocal microscope (LSCM). The activity of castoff cells and the change of cell focal adhesion were determined by castoff counting and Trypan blue staining assay. ELISA was used to detect the change of NO and TNFα secretion by sMAC. RESULTS: The sublytic dose of MAC was determined as C56 1∶480 and NHS 1∶20. SMAC deposition on microglia cells was seen under a LSCM The number of castoff microglia cells which had normal cell abilities was greatly increased by sMAC compared to controls (0.05), SMAC increased NO and TNFα secretion in microglia cells after 12 h stimulation (0.05 vs controls). TNFα secretion was significantly increased by activated sMAC compared to inactivated sMAC at different time points (0.05). CONCLUSION: NO and TNFα secretion are increased by sMAC stimulation. SMAC can reduce cell adhension, but not affect cell activity, which suggests that sMAC may have proinflammatory effects on microglia cells. 　　【Keywords】 C56 sublytic MAC microglia microscopy, confocal cell ability nitric oxide tumor necrosis factor 　　【摘要】　目的： 体外建立小鼠小胶质细胞补体攻膜复合物亚溶破模型，并进行功能鉴定, 探讨补体攻膜复合物( sublytic membrane attack complex, sMAC ) 对中枢神经系统小胶质细胞的亚溶破刺激效应. 方法: 分离培养新生小鼠大脑皮层小胶质细胞, 用酵母多糖激活急性期患者血清制备补体优球蛋白 C56，以新鲜正常人血清 ( NHS ) 作为 C7～C9 来源，体外组装小鼠小胶质细胞 sMAC 亚溶破模型，CCK8 比色实验确定 sMAC 亚溶破剂量，激光共聚焦显微镜 ( LSCM )鉴定 sMAC 沉积.脱落细胞计数及台盼蓝拒染法分别判定细胞黏附力变化及脱落细胞活力.