铅锰联合暴露通过诱导小胶质细胞活化引起星形胶质细胞活化及谷氨酰胺合成酶的活性降低

发布时间：2018-06-27 03:35

本文选题：铅 + 锰　；参考：《细胞与分子免疫学杂志》2016年03期

【摘要】：目的以小胶质细胞与星形胶质细胞建立共培养模型,研究铅、锰单独及联合暴露下不同类型胶质细胞的反应及小胶质细胞活化对星形胶质细胞功能的影响。方法先通过MTT法筛选对C6星形胶质细胞生长无影响的铅和锰的暴露剂量和作用时间,再选取BV2小胶质细胞和C6细胞以铅、锰单独及联合染毒建立细胞模型。分为直接刺激法、条件培养基法和共培养法3种细胞模型。直接刺激法采用完全培养基或含醋酸铅、氯化锰培养基直接作用于C6细胞24 h;条件培养基法采用完全培养基或含铅、锰培养基作用于BV2细胞24 h,取上清经离心后作用于C6细胞24 h;共培养法将BV2细胞接种于TranswellTM共培养模型上层小室内,再将上层小室置入空白12孔板内,将C6细胞接种于另一12孔板内,以完全培养基或含铅、锰培养基作用接种于TranswellTM共培养模型上层小室内的BV2细胞,24 h后弃去培养基,以完全培养基轻柔洗涤后,将含BV2细胞的上层小室置入接种C6细胞的12孔板内继续培养24 h。采用Western blot法检测补体受体3(CR3/CD11b/OX42)、胶质纤维酸性蛋白(GFAP)水平,确定铅、锰单独及联合暴露下对不同类型胶质细胞的影响;检测谷氨酰胺合成酶(GS)水平确定小胶质细胞的活化对星形胶质细胞谷氨酸-谷氨酰胺循环环路的影响。结果直接刺激模型中,10μmol/L铅、100μmol/L锰作用星形胶质细胞24 h,铅、锰单独及联合暴露不能引起星形胶质细胞显著活化及其GS表达的改变;条件培养基模型中,10μmol/L铅、100μmol/L锰作用于BV2小胶质细胞24 h,BV2小胶质细胞OX42表达水平显著升高,将其培养基作用于C6星形胶质细胞24 h后,C6细胞GFAP表达显著性升高,GS表达显著性降低;共培养模型中,10μmol/L铅、100μmol/L锰作用于BV2小胶质细胞24 h,BV2小胶质细胞OX42表达水平显著升高,将其洗涤后与C6星形胶质细胞共培养24 h后,星形胶质细胞GFAP表达显著性升高,GS表达显著性降低。结论低剂量铅、锰单独及联合暴露能够引起体外培养的小胶质细胞活化,而活化的小胶质细胞能够诱导星形胶质细胞活性增强及GS表达水平降低,且铅锰联合暴露比单独暴露效应更为显著。
[Abstract]:Objective to establish a co-culture model of microglia and astrocytes to study the effects of different types of glial cells exposed to lead and manganese alone or in combination and the effects of microglial activation on the function of astrocytes. Methods the exposure dose and time of exposure to lead and manganese which had no effect on the growth of C6 astrocytes were screened by MTT assay. Then BV2 microglial cells and C6 cells were selected to be exposed to lead and manganese alone or in combination to establish the cell model. It was divided into three cell models: direct stimulation method, conditioned medium method and co-culture method. Direct stimulation was carried out on C6 cells in full medium or lead acetate, manganese chloride medium was directly applied to C6 cells for 24 h, and conditional medium method was used in complete medium or lead. BV2 cells were treated with manganese medium for 24 h, the supernatants were centrifuged for 24 h, BV2 cells were co-cultured in the upper chamber of Transwell TM co-culture model, and then the upper chamber was placed into a blank 12-well plate. C6 cells were inoculated in another 12-well plate. BV2 cells in the upper chamber of Transwell TM co-culture model were inoculated in full medium or lead and manganese medium for 24 hours. The upper chamber containing BV2 cells was cultured in a 12-well plate inoculated with C6 cells for 24 h. The levels of complement receptor 3 (CR3 / CD11b / OX42) and glial fibrillary acidic protein (GFAP) were determined by Western blot assay to determine the effects of lead and manganese exposure alone or in combination on different types of glial cells. The level of glutamine synthase (GS) was measured to determine the effect of microglia activation on the glutamate-glutamine loop in astrocytes. Results in the direct stimulation model, 10 渭 mol / L lead and 100 渭 mol / L manganese could not induce the activation of astrocytes and the changes of GS expression in astrocytes after exposure to lead and manganese alone or in combination for 24 h. In the conditioned medium model, the expression of OX42 in BV2 microglia cells was significantly increased by 100 渭 mol / L mn (10 渭 mol / L) on BV2 microglia cells, and the expression of GFAP in C6 astrocytes was significantly increased after 24 hours of treatment with the medium and the expression of GS in BV2 microglial cells was significantly decreased. The expression of GFAP in BV2 microglial cells was significantly decreased after treated with 10 渭 mol / L lead 100 渭 mol / L manganese for 24 h. In the co-culture model, 10 渭 mol 路L ~ (-1) lead and 100 渭 mol 路L ~ (-1) manganese increased the expression of OX42 in BV2 microglia for 24 h, and then co-cultured with C6 astrocytes for 24 h after washing. The expression of GFAP in astrocytes increased significantly and the expression of GS decreased significantly. Conclusion low dose lead and manganese exposure alone and in combination can induce activation of microglia in vitro, while activated microglia can induce increased activity of astrocytes and decrease of GS expression. The combined exposure of lead and manganese was more significant than that of single exposure.
【作者单位】：第四军医大学军事预防医学系军队劳动与环境卫生学教研室;
【基金】：国家自然科学基金(81230063,81472942)
【分类号】：R114

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