细菌内毒素体外刺激小鼠腹腔巨噬细胞分泌一氧化氮实验模型的条件优化  

Optimization of a lipopolysaccharide-stimulated nitric oxide production model in mouse peritoneal macrophages

作　　者：黄利[1] 夏鸿[1] 伦玉宁[1] 余传林[1] 张群[1] 陈娜娜[1] 雷林生[1] 

机构地区：[1]南方医科大学药学院,广东广州510515

出　　处：《南方医科大学学报》2012年第11期1646-1650,共5页Journal of Southern Medical University

基　　金：广东省自然科学基金(S2011010003789);高等学校博士学科点专项科研基金(20114433110013)

摘　　要：目的优化细菌内毒素刺激小鼠腹腔巨噬细胞产生NO实验模型的条件。方法采用Hank＇s液灌洗小鼠腹腔，收集腹腔留居巨噬细胞，利用铜绿假单胞菌脂多糖（LPS）作为内毒素刺激巨噬细胞产生NO，以Griess法检测培养上清液中NO的浓度，考察细胞浓度、LPS浓度、LPS刺激时长、培养液体积等因素对巨噬细胞产生NO的影响，优化实验条件，并用已知药物验证模型的可靠性。结果本实验条件下，细胞浓度对NO的产生具有关键性影响，最适浓度为6×lO‘个细胞，ml，96孔板中每孔100μl；LPS浓度〈1 μg／ml时，NO的产生随LPS浓度的增加而增加（P〈0．001），LPS浓度〉1μg／ml时，NO的增加的幅度明显下降，当LPS浓度为10／μg／ml时，NO产生达到峰值；NO的产生随LPS刺激时间的延长，其含量相应增加，24与48h之间的增加幅度大于48h与72h之间的增加幅度（P〈O．05）；培养上清中NO的含量与培养液的体积有一定关系，100m时的NO含量最高。阿司匹林（1 mmol／L）、地塞米松（10laxlol／L）、环孢霉素A（10μmol／L）均能明显抑制LPS刺激小鼠腹腔巨噬细胞产生NO（P〈0．001）。结论巨噬细胞浓度、LPS浓度、LPS刺激时长是建立内毒素刺激小鼠腹腔巨噬细胞分泌一氧化氮实验模型的主要影响因素。推荐方案为：巨噬细胞浓度5×10‘个细胞，ml，100μl／孔；LPS浓度10μg/ml；LPs刺激时长24h或48h；培养液体积100～200μl。Objective To optimize the experimental model of nitric oxide （NO） production in mouse peritoneal macrophages in response to lipopolysaccharides （LPS） stimulation. Methods Mouse resident peritoneal macrophages were collected by lavaging the peritoneal cavity of mice with Hank＇s solution and stimulated with Pseudomonas aeruginosa LPS for NO production. NO concentration in the culture supematants was measured with Griess Reagent. The influences of cell density, LPS concentration, LPS stimulation duration and culture medium volume on NO production were investigated. Finally, the feasibility of the model was confirmed with specific anti-inflammatory drugs. Results The density of macrophages produced the most significant effect on NO production （P〈0.001）, and optimal results were obtained at the macrophage density of 6×106 cells/ml with a volume of 100 ~1 in each well in 96-well plate. At a LPS concentration below I μg/ml, NO production increased proportionally with the increment of LPS concentration （P〈0.001）, but the increment of NO production declined obviously at LPS concentrations beyond 1 μg/ml, and the peak NO production occurred at a LPS concentration of 10 μg/ml. NO production also increased significantly with the prolongation of LPS stimulation （P〈0.05）, and the increments were greater within 24-48 h than those in 48-72 h. NO content in the culture supernatant was associated with the medium volume, and the highest level occurred in a system volume of 100 gl. Aspirin （1 retool/L）, dexamethasone （10 μmol/L）, and cyclosporin A （10 μmol/L） all significantly inhibited LPS-stimulated production of NO in mouse resident peritoneal macrophages （P〈0.001）. Conclusions Macrophage density, LPS concentration, and the duration of LPS stimulation are the main factors affecting LPS-stimulated NO production in mouse resident peritoneal macrophages. The optimal results can be obtained with a macrophage density of 5 × 10^6 cells/ml （100 μl per well）, LPS concentration of

关 键 词：巨噬细胞 内毒素 脂多糖 一氧化氮 实验模型 

分 类 号：R96[医药卫生—药理学]