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  

Preparation and functional identification of sublytic complement membrane attack complex on cultured mouse microglia cells

作　　者：罗娜[1] 白云[1] 周静然[2] 宋敏[1] 王艳艳[1] 杨晓亚[1] 许雪青[1] 段文元[1] 熊加祥[1] 

机构地区：[1]第三军医大学医学遗传学教研室,重庆400038 [2]第三军医大学免疫学教研室,重庆400038

出　　处：《第四军医大学学报》2006年第10期872-874,共3页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金海外青年学者合作研究基金(30228018)

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，CCK-8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．ELISA法测定sMAC对小胶质细胞NO和TNF—α分泌量的影响．结果：确定C561：480，NHS1：20为小胶质细胞亚溶破补体量；LSCM显示sMAC沉积于小胶质细胞表面；sMAC刺激小胶质细胞后与对照组相比细胞脱落增加（P〈0．05），而活力正常．刺激后12hNO及TNF-α分泌量比对照组显著增加（P〈0．05），不同时相观察sMAC刺激后小胶质细胞TNF—α分泌量均显著高于失活sMAC（P〈0．05）．结论：sMAC刺激小胶质细胞后分泌NO及TNF—α增多，并且可降低小胶质细胞黏附力而不影响细胞活力，推测sMAC对小胶质细胞具有炎性刺激效应．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 CCK-8 assay. SMAC deposition was identified by laser confocal microscope （LSCM）. The activity of cast-off cells and the change of cell focal adhesion were determined by cast-off counting and Trypan blue staining assay. ELISA was used to detect the change of NO and TNF-α 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 cast-off microglia cells which had normal cell abilities was greatly increased by sMAC compared to controls （ P 〈 0.05 ）, SMAC increased NO and TNF-α secretion in microglia cells after 12 h stimulation （ P 〈 0.05 vs controls）. TNF-α secretion was significantly increased by activated sMAC compared to inactivated sMAC at different time points （P 〈0.05 ）. CONCLUSION： NO and TNF-α secretion are increased by sMAC stimulation. SMAC can reduce cell adhension, but not affect cell activity, which suggests that sMAC may have prointlammatory effects on microglia cells.

关 键 词：C56 补体攻膜复合物 小神经胶质细胞 显微镜检查 共焦 细胞活力 一氧化氮 肿瘤坏死因子 

分 类 号：R392.11[医药卫生—免疫学]