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  

The activation of microglia caused by lead and manganese co-exposure induces activation of astrocytes and decrease of glutamine synthetase activity

作　　者：官瑞丽 王涛[1] 陈景元[1] 骆文静[1] 刘明朝[1] 

机构地区：[1]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,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2016年第3期313-318,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81230063;81472942)

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Objective To establish microglia and astrocyte co-culture cell model and investigate the effects of lead and manganese alone or co-exposure on different glia cells and the effect of microglia activation on astrocytes＇ function. Methods MTT assay was performed to select the appropriate dose and time of lead and manganese exposure that didn＇t affect C6 cell growth. Then with BV2 microglia and C6 astrocytes,cel models were established through lead acetate and manganese chloride alone and co-exposure. The cel models were further divided into three types： direct stimulation,conditioned medium and co-culture. In the direct stimulation method,C6 cells were directly treated with complete medium or complete medium containing lead acetate and / or manganese chloride for 24 hours. In conditioned medium method,BV2 cells were cultured in the complete medium or complete medium containing lead acetate and / or manganese chloride for 24 hours,and then the supernatants were centrifuged and used to treat C6 cells for another 24 hours. In co-culture method,BV2 cells were seeded in semipermeable membrane inserts and the inserts were put in a normal 12-well plate; C6 cells were seeded in another12-well plate; complete medium or complete medium containing lead acetate and / or manganese chloride was added in the wells to culture BV2 cells for 24 hours; the culture medium was abandoned,the cells seeded in the inserts were gently rinsed with complete medium and then the inserts were put into the 12-well plate where C6 were seeded before; the BV2 cells and C6 cells were co-cultured for another 24 hours. The effects of lead and manganese alone or co-exposure on different glia cel s were analyzed through CR3 / CD11 b / OX42 and glial fibrillary acidic protein（ GFAP） expression detection by Western blotting;the effect of microglia activation on astrocyte glutamate-glutamine cycle loop was studied through glutamine synthetase（ GS）level detected by Western blotting. Results In direct stimulation method,10 μmol / L lead acetate

关 键 词：铅 锰 小胶质细胞 星形胶质细胞 谷氨酰胺合成酶 

分 类 号：Q256[生物学—细胞生物学] R994.6[医药卫生—毒理学]