| Objective: To investigate whether the up-regulation of TGF-β1 / CXCR7 signal axis can improve the angiogenesis of endothelial progenitor cells in the diabetic environment,and provide new theoretical and experimental basis for the study of chronic diabetes wound angiogenesis disease.Methods:(1)Establish a C57 BL / 6 mouse diabetic wound model and observe the effect of TGF-β1 on wound healing,and verify that the expression of TGF-β1 in diabetic wounds is reduced.(2)Obtain and fix wound tissue samples,and observe the effect of TGF-β1 on wound angiogenesis by HE staining sections.(3)Observe the effect of TGF-β1 on the number of wound endothelial progenitor cells by immunofluorescence staining.(4)Extract bone marrow EPCs from C57 BL / 6 mouse by density gradient centrifugation,identify by flow cytometry and differentiation experiments.(5)EPCs with down-regulation of CXCR7 were obtained by manufacturing a high glucose environment and lentiviral transfection methods,and the effect of CXCR7 down-regulation was verified using the Western Blot method.(6)The effect of TGF-β1 on the proliferation of CXCR7 down-regulated EPCs was tested by Ed U cell proliferation experiments.(7)The effect of TGF-β1 on the migration of CXCR7 down-regulated EPCs was tested by Scratch Test and Transwell method.(8)The effect of TGF-β1 on the ability of CXCR7 to down-regulate EPCs by the tubule formation experiment.(9)The effect of TGF-β1 on the expression of CXCR7 m RNA in EPCs was tested by RT-PCR experiments.(10)The effect of TGF-β1 on the protein expression of CXCR7 in EPCs was detected by Western blot.Results:(1)The C57 BL / 6 mouse model of diabetes wounds was successfully made,and it was observed that diabetic wounds with TGF-β1 heal faster,and verified that the expression of TGF-β1 in diabetic wounds was reduced.(2)HE staining revealed that the blood vessel density of diabetic wounds with TGF-β1 was higher.(3)Immunofluorescence staining of wound tissue sections showed that the number of EPCs in diabetic wounds with TGF-β1 was increased.(4)EPCs were successfully isolated and purified from the bone marrow of C57 BL / 6 mice by density gradient centrifugation and identified by flow cytometry,it has the phenotype of mesenchymal stem cells and can differentiate into endothelial cells.(5)The addition of glucose and lentiviral transfection to the cell culture medium successfully obtained EPCs down-regulated by CXCR7.(6)High glucose environment reduces the cell proliferation,migration and tube formation ability of EPCs,and the addition of TGF-β1 can partially restore the proliferation and tube formation ability of EPCs,but has no significant effect on migration ability.(7)sh CXCR7 lentivirus transfection is reduced The cell proliferation and tube-forming ability of EPCs were not significantly affected.(8)The high glucose environment reduced the expression of CXCR7 m RNA and protein in EPCs.The addition of TGF-β1 partially reversed the inhibitory effect.Conclusions:(1)TGF-β 1 can increase the number of local EPCs on diabetic wounds,promote angiogenesis,and ultimately accelerate healing of diabetic wounds.(2)Density gradient centrifugation method can successfully isolate EPCs from bone marrow,which can be cultured and proliferated stably in vitro.EPCs have the phenotype of stem cells and endothelial cells,and can be differentiated into endothelial cells and have the ability to form tubules.(3)TGF-β1 partially restores the inhibited proliferation and tubular capacity of EPCs in high-glycemic environments.(4)TGF-β 1partially restored the down-regulated expression of CXCR7 in diabetic EPCs. |
