甲酰基肽受体1对BV-2细胞迁移的影响及机制的体外研究

Formyl peptide receptor 1 promotes migration in mouse microglial cell line BV-2

目的探讨甲酰基肽受体1(formyl peptide receptor 1,FPR1)对BV-2细胞迁移的影响及潜在机制。方法免疫荧光染色检测BV-2细胞FPR1的表达,Transwell小室实验分别检测浓度为1、10、50、100、500 nmol/L的FPR1特异性激动剂(formylmethionyl-leucyl-phenylalanine,f MLP)对细胞迁移的影响,划痕实验及Transwell小室实验验证f MLP促进细胞迁移的作用可以被FPR1特异性阻断剂BocMLF所抑制。Western blot检测激动剂组、阻断剂组和阻断剂+激动剂组BV-2细胞经干预后其FPR1蛋白和磷酸化细胞外信号调节激酶(phospho-extracellular regulate kinase,p-ERK)蛋白表达量的变化。结果经免疫荧光染色结果显示,BV-2细胞表达FPR1,主要位于细胞膜表面。随着f MLP浓度的升高,Transwell迁移实验结果显示BV-2细胞的迁移能力明显增加(P<0.05),呈现出明显的剂量依赖性。100 nmol/L及500 nmol/L激动剂组效果最明显,f MLP促进BV-2细胞迁移的最适浓度为100 nmol/L。划痕及Transwell小室实验结果显示5μmol/L的Boc-MLF可显著阻断上述现象(P<0.05)。f MLP可以上调BV-2细胞中FPR1的表达(P<0.05),并且显著活化胞内ERK信号通路,上调p-ERK蛋白的表达水平(P<0.05),Boc-MLF又可显著抑制上述改变(P<0.05)。结论 BV-2细胞表达FPR1,且FPR1在促进BV-2细胞迁移中具有重要作用。

Objective To determine the effect of formyl peptide receptor 1（ FPR1） on the migration of BV2 cells and investigate the underlying mechanism. Methods The expression of FPR1 receptor in BV-2cells was detected by immunofluorescence staining. Transwell assay was used to detect the effect of FPR1 receptor specific agonist,formylmethionyl-leucyl-phenylalanine（ f MLP）,at different concentrations（ 1,10,50,100 and 500 nmol / L） on the alterations of cell migration,while the scratch test and Transwell assay were used to verify the role of f MLP in the promotion of cell migration whether can be inhibited by FPR1 specific antagonist,Boc-MLF,or not. The protein expression of FPR1 was detected by Western blotting in the BV-2cells stimulated with different concentrations of f MLP. After stimulated with 100 nmol / L f MLP and 5 μmol / L Boc-MLF,the expression of phospho-extracellular regulate kinase（ p-ERK） in the cells was detected by Western blotting. Results Immunofluorescence staining indicated that FPR1 receptor was expressed in BV-2cells,mainly located in the cell membrane. With the increase of f MLP doses,Transwell assay showed that the migration of BV-2 cells was enhanced significantly（ P〈0. 05） in a dose-dependent fashion. The 100 and500 nmol / L f MLP showed most obvious effect,while the optimal f MLP concentration to promote BV-2 cell migration was 100 nmol / L. The scratch test and Transwell assay verified that those above effects could be inhibited by 5 μmol / L Boc-MLF（ P〈0. 05）. The specific FPR1 agonist,f MLP up-regulated the FPR1 receptor expression in BV-2 cells（ P〈0. 05）,activated ERK signaling pathway,and increased p-ERK protein expression significantly（ P〈0. 05）. The FPR1 specific antagonist Boc-MLF inhibited the above changes significantly（ P〈0. 05）. Conclusion FPR1 receptor is expressed in the BV-2 cells,and FPR1 plays an important role in the promotion of BV-2 cells migration.