探讨一种新型Wnt替代蛋白激活脑血管Wnt信号通路对血脑屏障的保护作用

摘要

目的 探讨一种新型Wnt替代蛋白( Wnt-S)对人脑血管内皮细胞中Wnt/β-catenin信号通路的作用,可能提供一种保护血脑屏障的新途径.方法 用分子克隆技术构建Wnt-S in pcDNA3. 1( +)质粒,在HEK293F细胞中瞬时转染该质粒,表达纯化Wnt-S蛋白;Western blot检测含5x His-tag的Wnt-S蛋白;免疫荧光染色检测人源脑血管内皮细胞(hCMEC/D3)标记物CD31、VE-cadherin、GLUT1和MFSD2A;实时荧光定量PCR检测HEK293和hCMEC/D3细胞的Wnt蛋白受体FZD1-10和LRP5/6的表达情况,检测加药处理后HEK293和hCMEC/D3细胞中Wnt/β-catenin信号通路下游靶基因AXIN2的表达情况.结果 核酸电泳结果显示Wnt-S质粒构建完成;Western blot结果显示检测到了Wnt-S蛋白;HEK293中FZD1-7和LRP6高表达,hCMEC/D3中FZD2,4,6和LRP6高表达, FZD1,3低表达;实时荧光定量PCR结果显示,加Wnt-S蛋白处理后,HEK293和hCMEC/D3细胞中AXIN2基因表达水平显著上调(P<0. 001),说明Wnt-S蛋白有生物活性,并且激活了Wnt/β-catenin信号通路.结论 Wnt-S蛋白具有与天然Wnt3a蛋白类似的生物功能,能够激活人脑血管内皮细胞中的经典Wnt信号通路,以保护血脑屏障.%Objective To investigate the effect of a novel Wnt surrogate protein (Wnt-S) on the Wnt/β-catenin sig-naling pathway in human brain microvascular endothelial cells,and may provide a new strategy for the protection of blood-brain barrier (BBB). Methods Wnt-S in pcDNA3. 1( +) plasmid was constructed by molecular cloning technology,and was transfected into HEK293F cells to express and purify Wnt-S protein;Western blot tested Wnt-S with a 5x His-tag;Hu-man brain endothelial cells (hCMEC/D3) marker CD31,VE-cadherin,GLUT1 and MFSD2A were detected by immunofluo-rescence staining;Quantitative real-time PCR was performed to detect the expression of Wnt receptors FZD1-10 and LRP5/6 in HEK293 cells and hCMEC/D3 cells,and the expressions of target gene AXIN2 downstream of the Wnt/β-catenin signa-ling pathway in HEK293 cells and hCMEC/D3 cells after Wnt-S treatment. Results Nucleic acid electrophoresis results showed that Wnt-S plasmid construction was completed;Western blot’s results showed that Wnt-S protein was secreted and detected;FZD1-7 and LRP6 were highly expressed in HEK293 cells, FZD2,4,6 and LRP6 were highly expressed,and FZD1,3 were poorly expressed in hCMEC/D3;Quantitative real-time PCR results showed that AXIN2expression level was significantly up-regulated in HEK293 and hCMEC/D3 cells after addition of Wnt-S protein ( P<0. 001),indicating that Wnt-S protein had biological activity and activated the Wnt/β-catenin signaling pathway. Conclusion Similar to natural Wnt3a protein,Wnt-S protein has a biological function and can activate the canonical Wnt signaling pathway in human cere-brovascular endothelial cells,and thus may protect the BBB.