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目的:比较两种分离方法原代培养所获小胶质细胞的的生物学差异。方法:分别采用振摇法和温和消化法从胶质细胞/神经元的混合培养体系中分离纯化小胶质细胞,通过1,1’-己二酸双酯-3,3,3’,3’-四甲基吲哚菁标记的低密度脂蛋白活体标记显示小胶质细胞的形态学变化,分别用Griess反应和酶联免疫吸附法测定培养小胶质细胞经脂多糖刺激后释放的一氧化氮(NO)和肿瘤坏死因子α(TNF-α)的浓度。结果:温和消化法小胶质细胞的得率是振摇法的5.7倍,且其获得小胶质细胞的纯度(99.9±0.1)%远高于后者。温和消化法所获的小胶质细胞转化为静息状态需要的时间较振摇法明显缩短;经脂多糖刺激后,振摇法所得的小胶质细胞释放NO和TNF-α的水平均较温和消化法者高。结论:温和消化法在小胶质细胞的得率、纯度以及与静息状态的相似性方面明显优于振摇法,因此应在与小胶质细胞有关的实验室研究工作中得到推广。
OBJECTIVE: To compare the biological differences of microglial cells obtained from primary culture by two separation methods. Methods: Microglia were isolated and purified from the mixed culture system of glial cells and neurons by shake method and mild digestion method, respectively. The microglial cells were separated by 1, 1 ’-adipate-3,3,3’, 3 ’- tetramethylindocyanine labeled low density lipoprotein (LDL) in vivo showed the morphological changes of microglia. The microglial cells were stimulated with lipopolysaccharide and then released by Griess reaction and enzyme - linked immunosorbent assay Nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) concentrations. Results: The yield of microglia in mild digestion method was 5.7 times that of shaking method, and the purity of microglia was 99.9 ± 0.1%. The time required for microglial cells to convert to resting state by mild digestion was significantly shorter than that of shaking method. After lipopolysaccharide stimulation, the levels of NO and TNF-α released from microglial cells by shaking method were significantly lower Gentle digestion is high. CONCLUSION: Mild digestion is better than shaking method in obtaining the microglia’s yield, purity and similarity with resting state. Therefore, it should be popularized in laboratory work related to microglia.