小胶质细胞激活研究中免疫组化和荧光技术的应用

Application of immunohistochemical and immunofluorescence techniques in the study of microglial activation

目的在体内和体外实验中应用免疫组化(immunohistochemistry, IHC)和免疫荧光(immunofluorescence, IF)技术检测神经炎症过程中小胶质细胞激活及炎症分子表达改变。方法应用IHC方法观察脂多糖(lipopolysaccharide,LPS)诱导SD大鼠脑内小胶质细胞离子钙接头蛋白分子1(ionized calciumbinding adaptor molecule 1, Iba-1)免疫标记的小胶质细胞改变;应用IF方法检测LPS诱导体外培养的小鼠小胶质细胞系BV2细胞吞噬异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记的荧光微球活性以及炎症细胞因子白细胞介素1 beta(interleukin 1beta, IL1beta)的表达变化。结果 IHC结果显示,2.5 mg/kg LPS可诱导大鼠皮层Iba-1阳性小胶质细胞形态改变;IF结果显示,10 ng/mL LPS组和100 ng/mL LPS组BV2细胞荧光微球吞噬率与对照组比较,差异均有统计学意义(P<0.05);100 ng/mL LPS处理后,BV2细胞的炎症细胞因子IL1beta表达上调。结论应用IHC和IF技术可证实LPS模型中小胶质细胞激活及炎症细胞分子表达改变。

Objective To evaluate the application of immunohistochemical (IHC) and immunofluorescence (IF) techniques in detecting various changes of microglial activation and inflammatory molecules expression in neuroinflammatory models both in vivo and in vitro . Methods Changes of microglial marker ionized calciumbinding adaptor molecule 1 (Iba-1)-immunolabeled microglial cells were observed by IHC staining in the brain of lipopolysaccharide (LPS)-treated Sprague-Dawley (SD) rats. Phagocytic activity to ingest fluorescein isothiocyanate (FITC)-labeled microspheres and the expression of inflammatory cytokine interleukin 1 beta (IL1beta) were determined by IF methods in LPS-treated BV2 cells, a murine microglial cell line. Results IHC staining showed 2.5 mg/kg LPS induced morphological changes of Iba-1-positive microglial cells in rat cortex. IF test found that the phagocytic rate of fluorescent microspheres was higher in 10 and 100 ng/mL LPS-treated BV2 cells compared with control cells ( P <0.05). The expression of IL1beta was upregulated in 100 ng/mL LPS-treated BV2 cells. Conclusions IHC and IF techniques can be applied to confirm microglial activation and inflammatory molecules expression in LPS models.