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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(sublyt-icmembraneattackcomplex,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.
OBJECTIVE: To establish a rat sub-lysis model of mouse microglia complement complex in vitro and to evaluate the function of sublyt-icmembraneattack complex (sMAC) on the sub-lysis of central nervous system microglia Effect.Methods The isolated and cultured mouse cerebral cortical microglial cells were isolated and cultured with zymosan to activate the serum of patients with acute phase preparation of complement euglobulin C56, fresh normal human serum (NHS) as C7 ~ C9 source, in vitro assembly of small mouse glue The sMAC sub-dissolutive model of SCC was determined by colorimetric assay of CCK-8, and the sMAC deposition was determined by confocal laser scanning microscopy (LSCM). The change of cell adhesion and shedding were determined by cell counting and trypan blue exclusion Cell viability.ELISA was used to determine the effect of sMAC on the secretion of NO and TNF-α in microglia.Results The volume of microglia subsolidosis was determined by C561:480 and NHS 1:20. LSCM showed that sMAC was deposited on microglia (P <0.05), while the viability was normal.The amount of NO and TNF-α secreted by sMAC increased significantly (P <0.05) at 12h after stimulation compared with the control group, but not at the same time Phase observation sMAC stimulation The secretion of TNF-αin microglia was significantly higher than that of inactive sMAC (P0.05) .Conclusion The sMAC stimulated the secretion of NO and TNF-α after microglial stimulation, and could reduce the adhesion of microglial cells without affecting Cell viability, suggesting that sMAC has an inflammatory stimulatory effect on microglia.