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

作　　者：王羽茜[1] 张思然 陈璐[1,3] 李成檀 郑威[1] 王琼珠[1] 赵建波 王艳芳[1] 张丽慧[1] WANG Yu-xi;ZHANG Si-ran;CHEN Lu;LI Cheng-tan;ZHENG Wei;WANG Qiong-zhu;ZHAO Jian-bo;WANG Yan-fang;ZHANG Li-hui(Hangzhou Key Laboratory of Medical Neurobiology,School of Medicine,Hangzhou Normal University,Hangzhou 310036;Department of Neurology,Zhejiang Hospita,Hangzhou 310013;Changzhou Hygiene Vocational Technology College,Changzhou 213002,China)

机构地区：[1]杭州师范大学医学院医学神经生物学市级重点实验室,浙江杭州310036 [2]浙江医院神经内科,浙江杭州310013 [3]常州卫生高等职业技术学校,江苏常州213002

出　　处：《健康研究》2019年第2期165-169,共5页Health Research

基　　金：国家自然科学基金项目(81671188);浙江省自然科学基金项目(LY12H31010)

摘　　要：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.

关 键 词：脑 小胶质细胞 免疫组化 免疫荧光 

分 类 号：R965.2[医药卫生—药理学]