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本研究探讨脑源性神经营养因子(BDNF)促进血管新生的作用及其参与的信号通路,为抗肿瘤血管生成的研究提供新的实验依据。以人脐静脉内皮细胞为对象,采用Western blot方法检测细胞内磷酸化Akt、ERK1/2蛋白质的表达;采用Transwell小室迁移实验和管腔形成实验评价体外内皮细胞血管新生的能力,MTT法检测内皮细胞增殖活性,FITC-Annexin-Ⅴ/PI双染流式细胞术分析细胞调亡。结果表明:BDNF以时间依赖性的方式激活PI3K/Akt和MEK1/ERK信号通路。应用PI3K激酶抑制剂Ly294002、MEK1激酶抑制剂PD98059可以明显阻断BDNF对PI3K/Akt、MEK1/ERK信号通路的激活。100ng/ml的BDNF体外促内皮细胞血管新生能力与25ng/ml血管内皮生长因子(VEGF)相当,其中BDNF诱导的细胞迁移分别被Ly294002和PD98059阻断,其抑制率分别约为74%和36%;同样,Ly294002、PD98059可部分阻断BDNF诱导的小管形成效应,其阻断率分别约57%和37%;而BDNF的促增殖效应仅被PD98059拮抗,抑制凋亡效应仅受Ly294002影响。结论:BDNF在体外有促血管新生的作用。PI3K/Akt和MEK1/ERK信号通路以不同机制共同调节这一过程,其中PI3K/Akt信号通路起着更为重要的调节作用。
This study explored the role of brain derived neurotrophic factor (BDNF) in promoting angiogenesis and its signaling pathways, providing a new experimental basis for the study of anti-angiogenesis. The expression of phosphorylated Akt and ERK1 / 2 was detected by Western blot in human umbilical vein endothelial cells (HUVECs). The ability of endothelial cell angiogenesis in vitro was evaluated by Transwell migration assay and luminal formation assay. Endothelial Cell proliferation activity and apoptosis were analyzed by FITC-Annexin-Ⅴ / PI double staining flow cytometry. The results showed that BDNF activated PI3K / Akt and MEK1 / ERK signaling in a time-dependent manner. Application of PI3K kinase inhibitor Ly294002 and MEK1 kinase inhibitor PD98059 can obviously block the activation of PI3K / Akt and MEK1 / ERK signaling pathway by BDNF. BDNF at 100ng / ml induced endothelial cell angiogenesis in vitro was comparable to that of 25ng / ml vascular endothelial growth factor (VEGF), BDNF-induced cell migration was blocked by Ly294002 and PD98059, respectively, and the inhibitory rates were about 74% and 36% Similarly, Ly294002 and PD98059 partially blocked the BDNF-induced tubule-forming effect with a block rate of 57% and 37%, respectively. However, the proliferative effect of BDNF was only inhibited by PD98059 and the effect of Ly294002 alone was inhibited by Ly294002 and PD98059. Conclusion: BDNF can promote angiogenesis in vitro. The PI3K / Akt and MEK1 / ERK signaling pathways co-regulate this process by different mechanisms, of which the PI3K / Akt signaling pathway plays a more important regulatory role.