| Research background and purpose:Type 2 diabetes(T2D)is one of the most common metabolic diseases,characterized by chronic hyperglycemia.This occurs when insulin secretion is impaired or insufficient to meet the body's physiological needs for maintenance.Currently,many traditional risk factors for diabetes have been identified,such as fasting glucose,lifestyle,and obesity.Diabetes mellitus(DM)is a metabolic disease characterized by chronic hyperglycemia due to inadequate insulin secretion or insulin resistance.Chronic wounds have characteristic pathological associations and cannot be repaired in an orderly and timely manner to restore structural and functional integrity.Foot ulcers occur in 2 to 3 percent of diabetics each year,a major incidence that often leads to pain,distress and poor quality of life.Endothelial colony-forming cells(ECFCs)are considered to be lated Endothelial progenitor cells(EPCs),and their role in wound repair is widely concerned.ECFCs are primitive cells derived from bone marrow,which can be induced to differentiate into mature endothelial cells(ECs)under certain conditions.They are not only involved in angiogenesis in embryo,but also play an important role in angiogenesis,endothelial repair and functional maintenance after birth.To this end,we speculated that endothelial colony-forming cells can promote the healing of diabetic patients with chronic wounds and lesions angiogenesis through paracrine mechanism.This experiment discussed the function of ECFCs conditioned medium(CM)on biological effects of vascular endothelial cells and skin dermal fibroblasts.It provides new ideas and theoretical support for the effect of ECFCs on wound repair in diabetic patients.Methods:1.Extraction,isolation,culture and identification of ECFCs1.1 extraction,isolation and culture of ECFCs cells:umbilical cord blood of healthy volunteers was collected,and mononuclear cells were obtained by density gradient centrifugation.Human fibrin was added to a six-well cell culture plate,incubated in a 37? incubator for 3h,and then inoculated into a cell culture plate containing human fibrin for culture.1.2 ECFCs cell identification:flow cytometry and immunofluorescence were used to detect surface antigen of ECFCs cells,Matrigel tube experiment was used to detect ECFCs cells in vitro,and confocal method was used to detect the binding ability of ECFCs cells to Dil-acLD and FITC-UEA-I.2.1 Preparation of cell conditioned medium ECFCs-CM:ECFCs cells in logarithmic growth phase were continuously cultured in serum-free M199 basic medium with normoxia and hypoxia condition for 24h and 48h,and the supernatant was collected,that is,the conditioned medium(ECFCs-CM).2.2.Cytokine detection in ECFCs-CM:antibody chip and ELISA were used to detect cytokines in ECFCs-CM.3.Effects of ECFCs-CM on HUVECs and HDFs cells:3.1 The effect of ECFCs-CM on the proliferation of HUVECs and HDFs cells was detected by cck-8 method.3.2 Cell scratch method was used to detect the effect of ECFCs-CM on the migration ability of HUVECs and HDFs.3.3 Influence of ECFCs on migration ability of HUVECs and HDFs was detected by Transwell indirect co-culture method.3.4 The effect of ECFCs-CM on cell cycle of HUVECs and HDFs cells was detected by flow cytometry;3.5 The effect of ECFCs-CM on apoptosis of HUVECs and HDFs cells was detected by flow cytometry.4.Preparation of silk fibroin protein/sodium alginate hydrogel;Results:1.Extraction and culture of ECFCs cell:on the 4th day of EPC primary culture,a small number of spindle-shaped adherent cells were found,and a colony-like growth colony was found with round cells in the middle and spindle cells in the periphery,whose structure was very similar to the shape and blood island.On the 8th day of ECFCs primary culture,the cell colony began to contact and fuse.On the 14th day of ECFCs primary culture,the cells showed typical "paving stone" growth.2.Identification of ECFCs cells:flow cytometry showed high expression of KDR and CD144(vWF),partial expression of CD34,and low expression of CD133 and CD45.Matrigel matrix on the formation of lumen like structure,showing typical characteristics of endothelial cells;3.Antibody microarray detection showed that the expression of active factors in supernatant of cell culture medium was more favorable under hypoxia condition and continuously cultured for 48h.ELISA showed that PDGF-BB,IL-8 and EGF were not expressed in the cell superplasmid of EBM-2 group,while the expression levels of PDGF-BB,IL-8 and EGF were higher in the cell superplasmid of ECFCs-CM group.4.Effect of ECFCs-CM on HUVECs and HDFs cells:CCK-8 method showed that the proliferation of HUVECs and HDFs cells in ECFCs-CM group was significantly higher than that in EBM-2 group(P<0.05).Cell scratch assay showed that HUVECs and HDFs cells cultured in ECFCs-CM group at 6h,12h and 24h were significantly larger than those in EBM-2 group(P<0.05).The number of HUVECs and HDFs cells in the ECFCs-CM co-culture group was significantly higher than that in the EBM-2 group(P<0.05).Flow cytometry showed that the proportion of HUVECs and HDFs cells in the EBM-2 group in G0/G1 phase was significantly lower than that in the EBM-2 group(P<0.05).5.Preparation of silk fibroin protein/sodium alginate hydrogels:the pore diameter of SF/ALG composite hydrogels was 50-120 ?m,and the FTIR spectra showed that there was no significant difference in the vibration mode of important functional groups compared with pure ALG and SF.SF/ALG analysis of hydrogel porosity showed high porosity and good uniformity;SF/ALG hydrogel swelling increased with time.Conclusion:ECFCs-CM rich in multiple cytokines can promote cell proliferation,migration and tubation of HUVECs and HDFs cells,and reduce apoptosis of HUVECs and HDFs cells.ECFCs promotes neovascularization through paracrine,providing a new idea for "cell-free" treatment strategies based on paracrine mechanisms of stem cells/progenitor cells to promote diabetes and refractory wound repair. |
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