黑质内注射脂多糖对小胶质细胞MHCⅡ表达的影响

Effects of Microinjection of LPS into Substantia Nigra on the Expression of Microglia MHCⅡ

目的观察黑质内注入脂多糖(LPS)后对黑质小胶质细胞主要组织相容性复合物Ⅱ(MHCⅡ)表达的影响。方法采用立体定向技术向大鼠单侧黑质内注入LPS建立帕金森病动物模型,分别于注药后1、7、14、60 d时采用免疫组化法检测MHCⅡ阳性细胞;Western blot检测黑质小胶质细胞MHCⅡ蛋白的表达;双标荧光染色法检测p47phox(NADPH氧化酶标志物)和诱导型一氧化氮合酶(i NOS)在MHCⅡ阳性小胶质细胞的表达。结果注射LPS后1 d,注射侧黑质区始出现MHCⅡ阳性细胞,7 d时达高峰,14 d时减少,60 d时仅见少量的阳性细胞。Western blot检测结果也有类似的趋势。双标荧光染色法检测到p47phox和i NOS在MHCⅡ阳性小胶质细胞均有表达。结论黑质内单次注射LPS可激活黑质小胶质细胞并表达MHCⅡ;LPS可诱导MHCⅡ阳性小胶质细胞同时表达i NOS和NADPH氧化酶。

Objective To investigate the effect of lipopolysaccharide（LPS） microinjection into substantia nigra on the expression of major histocompatibility complex class Ⅱ（MHC Ⅱ ） in microglia on LPS-induced rat model of Parkinson＇s disease （PD）. Methods LPS was stereostatically injected into unilateral substantia nigra（SN） of rats. Immunohistochemistry and Western blot were used to detect midbrain MHC Ⅱ-positive microglia,and the expression of MHC Ⅱ protein, respectively. In order to determine the possible co-localization of inducible nitric oxide synthase（iNOS） and p47phox with microglia,double immunofluorescence was performed on nigral sections. Results MHC Ⅱ-positive microglia appeared at the first day following nigral LPS administration. The number of MHC Ⅱ -positive microglia reached the peak at 7th day and was significantly decreased at 60th day following nigral LPS administration. The levels of MHC Ⅱ detected by Western blot had a similar tendency. Double immunofluorescence for iNOS and p47phox with MHC Ⅱ-ir microglial marker OX6 showed a very close match between iNOS- ir, p47phox-ir and OX6-ir cells. Conclusion LPS microinjection into SN could induce the expression of MHC Ⅱ on microglia in SN, MHC Ⅱ-ir microglia could be also induced by LPS to express iNOS and p47phox in a rat model of PD.