Abstract:

Objective To investigate the effects of neurotrophic factor Neuritin overexpression on the angiogenic effects of chicken embryonic allantoic membrane （CAM） and human umbilical vein endothelial cells （HUVECs）， and to provide a new direction for the treatment of angiogenic diseases. Methods Thirty fresh yellow-skinned breeding eggs were selected to establish a CAM model， which were divided into three groups by randomized numerical table method： positive control group （bFGF）， negative control group （NS） and experimental group （Neuritin）， with 10 eggs in each group. The positive control group was loaded with 2 500 U/mL of bFGF， the experimental group was loaded with 10 μg/mL of Neuritin protein， and the negative control group was loaded with NS. 10 μL loading volume was loaded into each group， and all CAMs were incubated at the same temperature， relative humidity， and time， and the vascular branching， number， and size of the CAMs in each group were recorded after 72 h of incubation. Fresh umbilical cords from healthy pregnant women were selected to produce primary HUVECs， which were divided into three groups： transfected with recombinant plasmid （HUVEC-neu group）， transfected with empty vector （HUVEC-3.1 group）， and untransfected （HUVEC group）. Primary HUVECs in the HUVEC-neu group were transfected with the recombinant plasmid Neuritin， and those in the HUVEC-3.1 group were transfected with the empty vector. HUVEC-3.1 group was transfected with the empty vector plasmid， and HUVEC group was not given any special treatment， and all three groups received the same culture regimen. Western blot was used to detect the protein expression level of Neuritin in HUVEC-3.1 and HUVEC-neu groups. CCK-8 assay， cell scratch assay， Transwell assay， and tube formation assay were used to detect protein expression level of Neuritin in HUVEC-3.1 group and HUVEC-neu group， and HUVEC-neu groups for cell proliferation， migration and tube formation. Results （1） The number of CAM vessel branch points and microvessels in the experimental group was significantly increased compared with that in the negative control group （P < 0.01）， but there was no statistically significant difference in the number of large and medium-sized vessels between the two groups （P > 0.05）； （2） Neuritin was successfully overexpressed in HUVECs in the HUVEC-neu group. （3） Compared with the HUVEC-3.1 group， the proliferation vigor of cells in the HUVEC-neu group was decreased （P < 0.05）， but their migration and tube formation abilities were significantly enhanced （P < 0.01）. Conclusion Neuritin overexpression promotes angiogenesis and participates in the regulation of neovascularization by affecting cell proliferation， migration， and tube formation ability.

Key words: angiogenesis, Neuritin, CAM, HUVECs