目的: 通过观察乙酸铅[lead acetate, Pb(Ac)2]对革兰氏阴性菌内毒素脂多糖（lipopolysaccharide, LPS）介导大鼠原代小胶质细胞氧化应激反应的影响,探索中枢神经系统小胶质细胞对铅与内毒素产生的反应或后果。 方法: 随机将培养后的小胶质细胞分为阴性对照组（Control组）、 LPS组（100 ng/ml LPS）、不同浓度Pb (Ac)2组1（20、50、100 μM）、 LPS + Pb(Ac)2组[ 20、50、100 μM Pb(Ac)2 + 100 ng/ml LPS] 及不同浓度Pb(Ac)2组2（10、20 、50、100、200、500、1 000、2 000 μM）,置37℃、5 % CO2培养箱中孵育24 h或36 h,采用光学或免疫荧光显微镜观察或鉴定细胞,用MTT法检测铅作用后细胞存活率,用Griess Assay检测细胞分泌一氧化氮（nitric oxide, NO）情况、Western blot 检测诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）的表达水平。结果: LPS单用介导小胶质细胞24 h后,可使NO、iNOS水平上调（P < 0.05）;LPS与Pb(Ac)2共孵育后,NO水平下调（P < 0.05）,且iNOS与NO有一致性下调趋势;细胞生长状况随着铅浓度增加而出现损害现象,如细胞聚集、胞膜不完整、轮廓不清、粘合成团,核仁模糊不清;小胶质细胞存活率随Pb (Ac)2浓度的增加而下降。结论: LPS可导致原代小胶质细胞发生氧化应激反应,与Pb(Ac)2合用见氧化应激产物下调,似乎呈“保护”作用,但细胞形态学及细胞存活率呈现不良变化,与NO或iNOS的减少有“分离现象”。

Objective: To investigate the effect of lead acetate [Pb(Ac)2] on oxidative stress response mediated by lipopolysaccharide (LPS), bacterial endotoxin, in primary microglia derived from Sprague Dawley (SD) rats. To explore the reaction and consequence of microglia in central nervous system to lead and endotoxin. Methods: Microglia were randomly divided into negative group (Control group), LPS group (100 ng/mL LPS), Pb(Ac)2 groups 1 (20, 50, 100 μM), LPS + Pb(Ac)2 group [(20, 50, 100 μM Pb(Ac)2 + 100 ng/mL LPS] and Pb(Ac)2 group 2 (10, 20, 50, 100, 200, 500, 1 000, 2 000 μM) were incubated at 37℃ in 5% CO2 for 24 h or 36 h. The microglial cells were purified by shake-off in a orbit shaker, and identified by immunofluorescence staining microscopy. The cell survival rate was detected by MTT after Pb(Ac)2 was applied alone. The morphological changes of the cells were observed by microscopy. The nitric oxide (NO) or inducible nitric oxide synthase (iNOS) were respectively measured by Griess Assay or Western Blot. Results: NO or iNOS in LPS group were significantly up-regulated when compared to those in Control group (P < 0.05). Right after LPS was added to the microglia, 20, 50 or 100 µM of Pb(Ac)2 were added and then co-incubated with the cells for 24 h. NO level was significantly down-regulated (P < 0.05) in the supernatant. The expression of iNOS protein showed a consistently down-regulated trend with NO. To emphasize, the cells were found to be impaired with an increasing concentrations of Pb(Ac)2 at 20, 50 or 100 µM under the microscopy, which the celularl aggregation like adhesion clumps, broken cell membrane, and unclear nuclei were seen. Also, the survival rate of microglia was down-regulated while using increasing concentration of Pb(Ac)2 above. Conclusion: The primary microglia were activated such as the up-regulation of NO and iNOS by LPS alone. The oxidative-stress response were down-regulated after the microglia were exposed to Pb(Ac)2 with LPS in co-incubation. It showed the action as if “protectiove effect” from Pb(Ac)2 in the latter. Nevertheless, the morphological changes and the survival rate of the microglia were showed not in good condition. There was “segregation phenomenon” between the morphological changes and biochemical measure. However, the morphology of the microglia did not recover a normal feature when using Pb(Ac)2 alone. In a word, the reasons and mechanisms of “segregation phenomenon” still kept unclear. It suggested that the further studies be needed.