Notch1信号通路激活在低氧诱导人脐静脉内皮细胞血管形成中的作用

目的观察低氧条件下HIF-1α/VEGF/Notch信号通路在人脐静脉内皮细胞（HUVEC）血管生成中的作用。 方法将HUVEC进行常氧和低氧二氯化钴（CoCl2），200 μmol/L]诱导，再将常氧和低氧处理的HUVEC应用Notch1信号通路的抑制剂DAPT （30 μmol/L，24 h）和激活剂JAG-1 （30 μmol/L，24 h）干预。通过体外小管形成实验观察低氧对HUVEC血管生成能力的影响。应用RT-PCR和Western blot检测HUVEC中低氧诱导因子-1α （HIF-1α）、血管内皮生长因子（VEGF）、基质金属蛋白酶-9 （MMP-9）和Notch1信号分子（Notch1、Dell4和JAG-1）的mRNA和蛋白表达。通过Transwell迁移实验和伤口愈合实验观察低氧、DAPT、JAG-1对HUVEC迁移能力的影响。应用MTT法检测低氧及Notch1对HUVEC增殖的影响。两组间比较采用t检验，采用析因设计方差分析低氧和DAPT以及低氧和JAG-1对HUVEC迁移能力、距离、小管形成能力和细胞增殖的交互作用。 结果与常氧组比较，低氧组小管总长（8.18±0.62）mm比（15.43±1.32）mm]增高，差异具有统计学意义（P < 0.05）。与常氧组比较，低氧组的HIF-1α、VEGF、MMP-9、Notch1、Dell4和JAG-1的mRNA相对表达量和蛋白相对表达量（1.01±0.03比4.43±0.35，1.02±0.03比3.55±0.28，0.98±0.04比3.24±0.25，1.01±0.03比3.22±0.25，0.99±0.02比2.89±0.22，1.02±0.04比2.43±0.19，0.98±0.01比3.13±0.24，0.98±0.02比2.67±0.21，0.97±0.03比2.45±0.19，1.01±0.03比2.44±0.19，1.00±0.04比2.30±0.18，1.03±0.05比2.27±0.18）均升高，差异有统计学意义（P均< 0.05）。Transwell迁移实验和伤口愈合实验显示，低氧条件下，DAPT干预使HUVEC的迁移能力降低，JAG-1干预使HUVEC的迁移能力升高（P均< 0.05）。小管形成和MTT法测定显示，低氧条件下，DAPT干预使HUVEC的小管形成能力和细胞增殖能力降低，JAG-1干预使HUVEC的小管形成能力和细胞增殖能力升高（P均< 0.05）。析因设计的方差分析结果显示，低氧和JAG-1对迁移细胞数、小管形成和细胞增殖能力交互作用具有协同作用（P < 0.05）。 结论低氧可通过激活HIF-1α/VEGF/Notch1信号通路提高HUVEC的血管生成能力、迁移能力和细胞增殖能力。

Effects of the activation of Notch1 signaling pathway in hypoxia-induced angiogenesis of human umbilical vein endothelial cells

ObjectiveTo investigate the role of HIF-1α / VEGF / Notch signaling pathways in hypoxia-induced angiogenesis of human umbilical vein endothelial cells (HUVEC) . MethodsHUVEC was induced by normoxic and hypoxia mimetic agent-Cobalt dichloride (CoCl₂, 200 μmol/ L) , followed by inhibitor of Notch signaling pathway DAPT (30 μmol/L, 24 h) , and activator of Notch signaling pathway JAG-1 (30 μmol/L, 24 h) . The effect of hypoxia on the angiogenesis of HUVEC cells was investigated by in vitro tubule formation assay. The effect of hypoxia on the angiogenesis ability of HUVEC was observed through in vitro tubule formation experiments.The mRNA and protein expression level of hypoxia inducible factor-1α (HIF-1α) , vascular endothelial growth factor (VEGF) , matrix metalloprotein-9 (MMP-9) and molecules of Notch1 signaling pathway (Notch1, Dell4 and Jagged1) were detected by RT-PCR and Western blot respectively. The effects of hypoxia, DAPT, and JAG-1 on HUVEC migration were investigated by Transwell migration assay and wound healing assay. The effects of hypoxia and Notch1 on HUVEC proliferation were measured by MTT assay. Factorial design variance was used to analyze the interaction of DAPT and JAG-1 on HUVEC migration ability, migration distance, tubule formation ability, and cell proliferation under hypoxic conditions. The t test was used to compare the two groups. ResultsCompared with the normoxia treated cells, the total length of tubules in CoCl₂-treated cells was significantly higher (8.18±0.62) mm vs (15.43±1.32) mm, P < 0.05]. Compared with the normoxia treated cells, the mRNA of HIF-1α, VEGF, MMP-9, Notch1, Dell4 and Jagged1 in CoCl₂-treated cell were significantly up-regulated, (1.01±0.03 vs 4.43±0.35, 1.02±0.03 vs 3.55±0.28, 0.98±0.04 vs 3.24±0.25, 1.01±0.03 vs 3.22±0.25, 0.99±0.02 vs 2.89±0.22, 1.02±0.04 vs 2.43±0.19) , as well as the protein expressions increased (0.98±0.01 vs 3.13±0.24, 0.98±0.02 vs 2.67±0.21, 0.97±0.03 vs 2.45±0.19, 1.01±0.03 vs 2.44±0.19, 1.00±0.04 vs 2.30±0.18, 1.03±0.05 vs 2.27±0.18) (all P < 0.05) Transwell migration experiments showed that compared with the normoxic treated cells, the number of HUVEC migrating cells treated by CoCl₂ was significantly increased (P < 0.05) . Wound healing experiments showed that compared with the normoxic-treated cells, the migration distance of HUVEC treated by CoCl₂ was significantly increased (P < 0.05) . Compared with the hypoxia group, the migration ability of HUVEC treated by CoCl₂ + DAPT was significantly reduced, while the migration of HUVEC treated by CoCl₂+ JAG-1 was significantly increased (P < 0.05) . Tubular formation and MTT assay showed that compared with the hypoxic group, the tubular formation ability and cell proliferation of HUVEC treated by CoCl₂ + DAPT were significantly reduced, while the tubular formation and cell proliferation of HUVEC treated by CoCl₂ + JAG-1 were significantly increased (P < 0.05) . Factorial design analysis of variance showed that hypoxia and JAG-1 had a synergistic effect on the interaction of migrating cell number, tubule formation, and cell proliferation ability (P < 0.05) . ConclusionHypoxia could significantly induced the angiogenesis, migration and proliferation of HUVEC cells by activating HIF-1α/VEGF/Notch1 signaling pathway.