轴突导向因子Netrin-1在血管生成中调节作用的实验研究

Regulation of axon guidance cue netrin-1 on angiogenesis

目的研究神经轴突导向因子 Netrin-1在血管生成中的作用及其机制。方法原代培养人脐静脉内皮细胞(HUVEC),用逆转录多聚酶链反应检测其上 Netrin-1的各种受体表达,并用体外实验分别检测 Netrin-1对 HUVEC 增殖,迁移和管状结构形成的作用,并用兔角膜新生血管实验研究在不同浓度 Netrin-1作用下体内血管新生的情况。结果在 HUVEC 上仅检测到一种受体 UNC5B的表达。在体内和体外实验中均发现 Netrin-1不仅是个促血管生成因子,也是一个抑制血管生成因子,在浓度低于500 ng/ml 时均表现为促进作用,而在1000～5000 ng/ml 时表现为抑制作用。其中,当 Netrin-1为50 ng/ml 时,HUVEC 的增殖能力最强,当 Netrin-1浓度为100 ng/ml 时,HUVEC 的迁移能力最强。结论 Netrin-1在血管生成中起着促进和抑制的双重调控作用,这种双重调控作用取决于Netrin-1浓度,其中抑制作用和受体 UNC5B 相关。

Objective To study the effect of netrin-1, an axon guidance cue, on angiogenesis. Methods Human umbilical cord vein endothelial coils （HUVECs） were isolated and cultured. Reverse transcription and polymerase chain reaction （RT-PCR） was used to detect all the known receptors of netrin- 1 in the HUVECs. Netrin-1 and vascular endothelial growth factor （VEGF） of the concentration of 10 ng/ml were added into the culture fluid respectively for 72 h, cholecystokin-8 （CCK-8） was added, and then enzyme mark instrument was used to calculate the relative absorbance （A value）. Other HUVECs were added into the upper chamber of the Transwell co-culture system and netrin-1 of different concentrations and VEGF of the concentration of 10 ng/ml were added into the lower chamber respectively for 6 h, and then invert microscopy were used to observe the migration of HUVECs. Matrigel was added into the 96-well plate, and HUVECs and netrin-1 of different concentrations were added into the wells, then contrast microscopy was used to calculate the tube formation. Corneal micropecket assay was performed on 96 rabbits to determine the corneal neovascularization （CNV） with treatment with different concentrations of netrin-1. Results Of the 6 ligands only the receptor UNC.SB was expressed in the HUVECs. The cell counting kit-8 assay showed that the proliferation of HUVECs was promoted when the concentration of netrin-1 was under 500 ng/ml, especially when the concentration was 50 ng/ml, however, when the netrin-1 concentrations were 1000 to 5000 ng/ml the proliferation of the HUVECs was inhibited （ P 〈 0.05 ）. The migration of HUVECs was promoted when the concentration of netrin-1 was under 500 ng/ml, especially when the concentration was 100 ng/ml, however, when the netrin-1 concentrations were 1000 to 5000 ng/ml the migration of the HUVECs was inhibited （ P 〈 0.05 ）. Normally HUVECs formed branch-like and tube-like structure in the culture plate, netrin-1 did not influence the tube formation when the concentration was under 500 ng/ml, and inhibited the tube formation when the concentration was 1000 -5000 ng/ml （P 〈0.05 ）. Rabbit corneal micropecket assay showed that netrin-1 of the concentration of 100 ng/ml and VEGF of the concentration of 10 ng/ml promoted the angiogenesis, however, the netrin-1 of the concentration of 5000ng/ml inhibited the angiogenesis. Conclusion Netrin-1 shows has a dual function, both promotive or inhibitory effects, on angiogenesis, depending on the concentration and its inhibitive effect is mediated by UNC.SB.