人补体杀伤小鼠N9小胶质细胞及亚溶破模型的建立

Studies on Mechanism of Murine N9 Microglia Cell Induced Human Complement Activation and on the Construction of Its Sublytic Model

目的:探讨正常人血清(norm al hum an serum,NHS)补体杀伤小鼠N9小胶质细胞的机制,建立人补体攻膜复合物(sub lytic m embrane attack comp lex,sMAC)N9细胞亚溶破刺激模型。方法:用阻断补体活化途径的方法探讨N9细胞活化人补体系统的机制;采用微量补体反应性溶破法,以酵母多糖(Zymosan,Z)活化急性期病人血清制备补体优球蛋白C56,EDTA-NHS作为C7-C9的来源,组装N9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。结果:未致敏N9细胞可通过旁路途径直接活化人血清补体系统;当C56稀释度在1∶500以上,NHS(含1 mmol/L EDTA)稀释度为1∶20时,膜攻击复合物(m embrane attack comp lex,MAC)活性逐渐降低,细胞溶破减少,确定C56 1∶500,NHS 1∶20为N9细胞亚溶破补体量;激光共聚焦鉴定sMAC沉积于N9细胞表面;亚溶破剂量MAC刺激N9细胞后与对照组相比细胞脱落增加(P<0.05),而细胞活力正常。结论:探讨了小鼠N9细胞活化人补体的机制;通过建立N9细胞人补体sMAC亚溶破模型,证实sMAC刺激N9细胞后可降低细胞黏附力但不影响细胞活力;为sMAC对中枢神经系统小胶质细胞亚溶破刺激效应的深入研究提供了理论与实验基础。

Objective： To explore the mechanism of human serum complement induced murine N9 microglia cell lysis and to construct the complement non-lethal attack model on N9 cells. Methods： To study the mechanisms of human serum complement mediated N9 cell lysis, the complement activation was blocked. An activated complex of C56 was generated by activation of acute phase patient serum with Zymosam and C7-C9 came from EDTA-NHS. So as to assemble sublytic membrane attack complex （sMAC） on N9 cells by microassay of reactive hemolysis in vitro. The sublytic dose of sMAC was determined by CCK-8 assay, sMAC deposition was identified by laser confocal microscope. Activity of cast-off cells and the change of cell focal adhesion were determined by cast-off counting and Typan staining assay. Results： When C56 dilution was beyond 1 ： 500, NHS dilution （ including 1 mol/L EDTA） was 1： 20, the absorptance from CCK-8 assay was significantly higher than that when C56 dilution under 1 ： 500 （ P 〈 0.05） , and MAC ability and the lysis of N9 cells was decreased. The sublytic dose of MAC was determined as C56 1：500 and NHS 1：20（ 1 mol/L EDTA）. The sMAC deposition on the surface of N9 cells was proved by immunofluorescene method; The number of cast-off microglia cells which had normal cell abilities was greatly increased by sMAC compared to other controls （P 〈0.05）. Conehtsions： Our study results could help to elucidate the mechanism of N9 cell that activates human serum; through the experiments with model of sMAC on N9 cell, we show that the simulation of sMAC to N9 cell can reduce cell adhesion, but not cell activity; our study provides useful theoretical and experimental basis for further study on sublytic effects of sMAC on CNS microglia cells.