Tau蛋白聚集体促进小胶质细胞活化的机制研究

Tau aggregates promote microglia activation

目的探讨Tau蛋白聚集体对小胶质细胞活化的影响及机制。方法纯化重组的Tau蛋白并制备Tau聚集体,处理小鼠小胶质细胞(BV2 microglia of mice,BV2),Western blot检测半乳糖凝集素(Galectin,Gal)-9的表达水平,酶联免疫吸附测定(Enzyme linked immunosorbent assay,ELISA)法检测细胞培养基中Gal-9蛋白的水平;体外表达纯化Gal-9蛋白,处理BV2,Western blot检测细胞中白细胞分化抗原68(Cluster of differentiation 68,CD68)和电离钙结合适配器分子1(Lonized calcium-binding adapter molecule 1,IBA1)蛋白的表达水平;免疫荧光检测CD68的表达水平;ELISA检测细胞培养基中炎症因子肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)的水平,逆转录-聚合酶链反应(Reverse transcription-Polymerase chain reaction,RT-PCR)检测细胞中TNF-α的转录水平;Tau蛋白聚集体与短发夹RNA(Short hairpin RNA,ShRNA)-Gal-9同时处理BV2,观察阻断Gal-9的作用对小胶质细胞活化的影响。结果与对照组比较,Tau蛋白聚集体处理BV2引起Gal-9蛋白表达增加,并释放到细胞外;Gal-9蛋白处理BV2导致其活化,CD68和IBA1表达增加;促炎性细胞因子TNF-α表达增加;阻断Gal-9的作用可减轻Tau聚集体诱导的小胶质细胞活化。结论Tau聚集体促进BV2表达Gal-9蛋白,后者诱导小胶质细胞激活和促炎性细胞因子释放。

ObjectiveTo investigate the effect of tau aggregates on microglial activation.Methods BV2 cells were exposed to recombinant tau aggregates.The levels of galectin-9(Gal-9)were detected using Western blot analysis.The levels of Gal-9 in the culture medium were detected by ELISA.Purified Gal-9 was used to treat BV2 cells.The levels of CD68 and IBA1 were analyzed by Western blots.Immunofluorescence was used to detect the expression of CD68.The level of tumor necrosis factor-α(TNF-α)was determined by ELISA.Reverse transcription-Polymerase chain reaction(RT-PCR)was used to assess the mRNA levels of TNF-α.The role of Gal-9 in the activation of microglia was observed by treating BV2 cells with Tau aggregates and ShRNA-Gal-9.Results Compared with the PBS control,Tau PFFs elevated Gal-9 levels in BV2 cells.The levels of Gal-9 in the medium were also increased after treatment with Tau PFFs.Compared with control BV2 cells,the levels of CD68 and IBA1 in BV2 cells were increased in cells exposed to Gal-9,indicating that Gal-9 induces microglial activation.The protein and mRNA levels of the pro-inflammatory factor TNF-αwere increased after Gal-9 treatment.Blocking the expression of Gal-9 attenuated the activation of microglia induced by Tau aggregates.Conclusion Tau aggregates promote Gal-9 expression in BV2 cells,which in turn induces microglial activation.