AKT/eNOS信号途径在脑源性神经营养因子诱导内皮细胞血管新生中的作用

目的探讨脑源性神经营养因子（BDNF）促进内皮细胞血管新生的机制及其参与的信号通路，为抗多发性骨髓瘤血管生成的研究提供新的实验依据。方法以人脐静脉内皮细胞（HUVEC）为对象，采用Western blot印迹法检测细胞内磷酸化丝／苏氨酸激酶（AKT）、内皮细胞一氧化氮合酶（eNOS）蛋白质的表达；采用Transwell小室迁移实验、小管形成实验评价体外内皮细胞血管新生的能力，小鼠体内Matrigel plug实验评估体内内皮细胞血管新生的能力，采用硝酸还原酶法检测HUVEC上清中内皮细胞源性一氧化氮（eNO）的含量，FITC-Annexin V／PI双染色流式细胞术分析细胞凋亡。结果BDNF以时间一浓度依赖性的方式激活P13K／AKT／eNOS信号通路，应用PI3K激酶抑制剂Ly294002可以明显阻断BDNF刺激后内皮细胞eNO的生成。在体外，BDNF诱导的细胞迁移和小管形成效应均分别能被Ly294002和L-NAME（NOS抑制剂）阻断；而BDNF的抑制凋亡效应与L-NAME无关，仅受Ly294002影响；在体内，经L-NAME喂养的小鼠皮下Matrigel栓中的新生血管数量与未经L-NAME喂养的小鼠相比显著减少。结论BDNF通过AKT／eNOS途径活化介导血管薪生，这可能成为治疗多发性骨髓瘤血管新生的新靶点。

Involvement of AKT/eNOS in brain derived neurotrophic factor-induced angiogenesis

Objective To investigate the signaling pathways involved in brain derived neurotrophic factor(BDNF)-induced angiogenesis and to provide a novel pathway to auti-angiogenesis in muhiple myelo- ma.Methods The phosphorylation of AKT and endothelial NO synthase(eNOS)in human umbilical venous epithelial cells(HUVEC)were detected by Western blot.The angiogenic activity in vitro was evaluated by tr- answell migration assay and tube formation assay.BDNF-induced in vivo angiogenic activity was evaluated by Matrigel plug assay.The concentration of NO was detected bv nitric acid deoxidizase assay.Cell apoptosis was detected by FITC-Annexin V/PI double staining and flow cytometry.Results BDNF activated the phos- phatidylinositol-3-kinase(PI3K)/AKT/ eNOS pathway in HUVEC in a lime-and dose-dependent manner. BDNF-stimulated NO production was blocked by LY294002,a PI3K inhibitor,In vitro,BDNF induced HUVEC migration and tube formation on Matrigel,which could be significantly blocked by LY294002 and N~G- nilro-L-arginine methyl ester(L-NAME)respectively;but BDNF induced HUVEC apoptosis could be blocked only by LY294002.In vivo,BDNF increased capillary ingrowth into subcutaneously implanted Matri- gel plugs in mice,which could be significantly reduced in L-NAME treated mice.Conclusion BDNE in- duces angiogenesis through the AKT/eNOS signaling kiuase pathway.It may be a novel target for the anti-an- giogenesis therapy for multiple myeloma.