细菌脂多糖诱导活性小胶质细胞对少突胶质细胞前体的毒性作用

Toxicity of lipopolysaccharide-induced activated microglia to oligodendrocyte precursor cells

背景:已有实验证实,脑白质病变如多发性硬化症和脑室周围白质软化等一些神经病变的发病机制,均涉及到局部小胶质细胞的激活。目的:探讨细菌脂多糖诱导活性小胶质细胞对少突胶质细胞前体的毒性作用。方法:取2d龄SD大鼠脑内小胶质细胞和少突胶质细胞前体共培养,分为共培养对照组和共培养脂多糖组。对共培养细胞经脂多糖100μg/L诱导48h,分别应用硝酸还原比色法检测一氧化氮含量,还原-比色法检测超氧阴离子含量,免疫细胞染色法检测过氧亚硝酸盐含量,Hochest33342/PI荧光染色法观察细胞死亡的形态学改变,以及流式细胞仪检测细胞成活率。结果与结论:与共培养对照组比较,脂多糖诱导导致共培养细胞内一氧化氮、超氧阴离子、过氧亚硝酸盐含量均明显增加,少突胶质细胞前体的凋亡率亦显著增加。通过体外观察,确认脂多糖诱导少突胶质细胞前体的死亡通路,是由于脂多糖诱导小胶质细胞激活,导致小胶质细胞表达一氧化氮合酶和活化NADPH氧化酶,致使其产物一氧化氮和超氧阴离子大量生成,两者进一步反应生成大量的毒性因子过氧亚硝酸盐,作用于少突胶质细胞前体,从而导致少突胶质细胞前体的死亡。

BACKGROUND：Studies have confirmed that localized activation of microglia,however,has been implicated in the pathogenesis of a number of neurological diseases and disorders,such as multiple sclerosis and periventricular leukomalacia（PVL）.OBJECTIVE：To explore the toxicity of lipopolysaccharide（LPS）-induced activated-microglia to oligodendrocyte precursor cells.METHODS：Co-cultured microglias and oligodendrocyte precursor cells obtained from two-day-old Sprague-Dawley rats were divided into the co-cultured control group and the co-cultured LPS group.After co-cultured cells were induced by LPS（100 μg/L） for 48 hours,the concentration of nitric oxide was measured by nitric acid-deoxidize colorimetry;the generated levels of（O2-） were determined by deoxidize colorimetry;the levels of peroxynitrite（ONOO-） were detected by immunocytochemistry.The morphologic changes of death oligodendrocyte precursor cells were observed using Hoechst 33342/propidium iodide staining and the survival rate of oligodendrocyte precursor cells was detected by flow cytometry.RESULTS AND CONCLUSION：Compared with co-cultured control group,the levels of nitric oxide,O2-and ONOO-increased significantly in co-cultured LPS group,and with a higher apoptotic rate of oligodendrocyte precursor cells.It is validated in vitro that the death pathway of LPS-induced activated-microglia to oligodendrocyte precursor cells involves in LPS-induced microglia activation,impels microglia to express inducible nitric oxide synthase and to activate NADPH oxidase,results in the overproduction of nitric oxide and O2-,which further forms ONOO-,a primary toxic factor to oligodendrocyte precursor cells,finally leads to oligodendrocyte precursor cells death.