Effect Of Conditioned Medium Of Umbilical Cord Mesenchymal Stem Cells And Dental Pulp Stem Cells On The Function Of Umbilical Vein Endothelial Cells

Objective:To explore the proliferation of human umbilical vein endothelial cells(HUVECs)in the conditioned medium of human umbilical cord mesenchymal stem cells(h UCMSCs)and human dental pulp stem cells(h DPSCs)at different cell concentrations.This study compared the effects of h UCMSCs and h DPSCs conditioned medium on the functions of HUVECs during the migration and tube formation assay.The experiment laid the foundation for the subsequent related research.Methods:(1)Isolation,culture and identification of human umbilical vein endothelial cells.The cells were obtained by digesting the umbilical cord vein intima with 0.1% collagenase II at room temperature for 15 minutes.HUVECs were identified by factor Ⅷ immunohistochemistry and tube formation assay.(2)The effect of h UCMSCs and h DPSCs conditioned medium on the function of HUVECs.The proliferation of HUVECs in different cell concentrations of h UCMSCs and h DPSCs conditioned medium was evaluated by CCK-8 assay to screen the conditioned medium concentration for subsequent experiments.HUVECs was cultured in different conditioned medium,and the function of HUVECs was tested by migration assay,scratch assay and tube formation assay.The angiogenesis of HUVECs was measured by the m RNA expression of VEGF,KDR,Ang-2,HGF,HIF-1α.To explore the related mechanisms of h UCMSCs/h DPSCs promoting angiogenesis,the concentrations of VEGF,HGF and HIF-1α in h UCMSCs/h DPSCs conditioned medium were tested by ELISA.Results:(1)HUVECs were round,short fusiform or polygonal,and grew in an adherent monolayer.At the initial stage,cells grew in clusters which form multicellular circular colonies,and then gradually arranged into paving stone-like.The factor VIII immunohistochemistry showed that the cell cytoplasm was brown and the nucleus was obvious.HUVECs could form vascular-like structures on Matrigel,suggested that HUVECs were in good cell viability and function.(2)The proliferation of HUVECs increased with the increase of cell concentration in the conditioned medium.When the cell ratio of HUVECs to h UCMSCs/h DPSCs was greater than 1:100,the proliferation of HUVECs in the conditioned medium was stronger than that of the ECM medium group.Therefore,the cell ratio of 1:100 conditioned medium was used for subsequent experiments.Compared with the ECM medium and the normal medium group,h UCMSCs and h DPSCs conditioned medium groups could promote the proliferation,migration and tube formation of HUVECs.There was no significant difference in the proliferation,migration and tube formation of HUVECs between the h UCMSCs group and h DPSCs group.The h UCMSCs/h DPSCs conditioned medium groups expressed VEGF,KDR,Ang-2,HGF and HIF-1α m RNA which was higher than that of ECM group and normal medium group(P<0.05).The expression of HGF and Ang-2 in h DPSCs conditioned medium group was higher than that in h UCMSCs conditioned medium group.While the expression of HIF-1α in h DPSCs conditioned medium group was lower than that in h UCMSCs conditioned medium group(P<0.05).The results of ELISA found that the concentration of VEGF,HGF and HIF-1α in h UCMSCs/h DPSCs conditioned medium was higher than that in normal medium.The concentration of HGF and HIF-1α in h UCMSCs conditioned medium was higher than that in h DPSCs conditioned medium(P<0.05).There was no statistically significant difference in the concentration of VEGF between the h UCMSCs and h DPSCs conditioned medium.Conclusion : We had successfully isolated and cultured HUVECs by enzymatic digestion,and the cells were in good viability and function.The proliferation of HUVECs was dependent on the cell concentration of h UCMSCs/h DPSCs conditioned medium.The proliferation of HUVECs increased with the increase of cell concentration in the conditioned medium.Both h UCMSCs and h DPSCs conditioned medium could promote the proliferation,migration and tube formation of HUVECs,but there was no significant difference between the h UCMSCs group and h DPSCs group.h UCMSCs and h DPSCs promoted the angiogenesis of HUVECs by secreting a variety of angiogenesis-related factors.However,h UCMSCs and h DPSCs might have different mechanisms for promoting angiogenesis.