原代小鼠小胶质细胞培养及尼古丁抑制肿瘤坏死因子α分泌的研究

目的 本研究通过摇床法分离纯化培养小鼠原代小胶质细胞，并研究尼古丁对小胶质细胞肿瘤坏死因子α（TNF-α ）的表达和分泌的影响。方法 从C57 BL/6新生鼠大脑中分离出皮层，切碎研磨后进行混合培养，摇床法分离纯化小胶质细胞并培养。CD11b、Iba1作为小胶质细胞特异性标记物用来检测、鉴定小胶质细胞，在激光共聚焦显微镜下观察、计数免疫荧光细胞化学染色后的阳性细胞。将纯化的小胶质细胞设置为三个组，分别为空白对照组，LPS组，LPS+NIC组。 LPS+NIC组先加入10 ug/ml LPS处理30 m

A Study of Primary Mouse Microglia Cell Culture and Nicotine Inhibition the Secretion of Tumor Necrosis Factor Alpha

Objective To study the effects of nicotine on expression and secretion of tumor necrosis factor alpha (TNF-α) in cultured mouse primary microglia by shaking table method.Methods The cortex of C57 BL/6 neonatal rats was isolated,chopped and ground, mixed culture was carried out, microglias were isolated and purified by shaking table method and cultured.CD11b and Iba1 were used as microglia cell specific markers to detect and identify microglia, and positive cells were observed and counted under laser confocal microscope after immunofluorescence cytochemical staining.The purified microglia were set into three groups:blank control group, LPS group and LPS+NIC group.The LPS+NIC group was treated with 10 ug/ml LPS for 30 min and 10 mol nicotine for 24 h.LPS group was treated with 10 g/ml LPS for 4 h.The blank control group received no treatment.QPCR was used to detect the expression of nicotine in microglial TNF-α, and elisa was used to detect the effect of nicotine on the secretion of TNF-α in microglial cells.Results The positive rate of microglia cells was over 95.54%.Immunofluorescence cytochemical method was used to identify microglia cell purity, microglia cell specific markers CD11b and Iba1, astroglia cell marker GFAP detection results, and ImageJ analysis of microglia positive rate＞95.54%, astroglia＜4%, other cells＜2%.The expression level of TNF-α in LPS+NIC group was significantly lower than that in LPS+NIC group (P＜0.05).TNF-α secretion was increased in microglia treated with LPS alone.However, in the LPS+NIC group, the secretion of TNF-α in microglia decreased, and there was a significant difference between the two groups (P＜0.05).Conclusion The primary microglia of mice with high purity were isolated and purified by differential adhesion. Compared with other methods, the microglia cells were less damaged and could be harvested many times with high cell yield.Nicotine can inhibit the secretion of TNF-α in primary microglial cells in mice, which may provide a new therapeutic method for the treatment of neurodegenerative diseases.