| Objective:By adopting joint AMD3100+ G-CSF mobilization of autologous bone marrow Endothelial progenitor cells(EPCs)in peripheral blood,collect the cells by in vitro amplification,and partial transplantation in diabetic mice(db/db)in animal models of wound,and compared with normal mice(db/+)source of EPCs transplantation effect comparison,EPCs promote healing of diabetic wound vascularization and evaluate the method of mechanism and provide reference for clinical treatment.Methods:Male mice db/+(100)and db/db(70)aged 5-6 weeks were used as experimental subjects.Bone marrow EPCs were mobilized by AMD3100+G-CSF,and then amplified and cultured to the second generation EPCs in vitro.Thirty-six diabetic mice were selected and divided into three groups according to random number table:injection of normal saline(NS)group,transplantation of normal EPCs group(db/+-EPCs group)and transplantation of diabetic EPCs group(db/db-EPCs group),with 12 mice in each group.Full-thickness skin defect wounds with an area of 1cmx1cm were made on the back.Immediately after injury,50ul of PKH26-labeled normal EPCs and diabetic EPCs suspension were injected at 4 sites on the wound surface of transplanted mice in db/+-EPCs group and db/db-EPCs group,respectively.Mice in NS group were injected with the same amount of NS at the same location.At 3,5,7,10,14 and 17 days after injury,the surviving mice in the three groups were taken for gross observation of the wound surface and the wound healing rate was calculated.The growth and migration of PKH26-labeled EPCs were observed under fluorescence microscope after frozen sections of the wound tissues of 3 mice in each group 3 days after injury.At 7,14 and 17 days after injury,3 mice in each group were sacrificed after gross observation of the wound surface,and the wound tissue was collected.Granulation formation was observed by HE staining on the wound tissues of each group.Immunohistochemistry was used to detect the density of new blood vessels(CD31)in the wound tissues.Masson staining was used to detect the amount of collagen fiber deposition in the wound tissues.Real-time fluorescence quantitative reverse transcription PCR and Western blot were used to detect the mRNA and protein expressions of VEGF,SDF-1 and CXCR4 in the wound tissues,respectively.SPSS 17.0 statistical software was used to process all the data.The measurement data were expressed as mean ± standard deviation.One-way ANOVA and factorial analysis were used for multi-group treatment,and Bonferroni correction was performed for p values.Results:1)On day 3 and 5 after injury,the wound healing rate of the three groups was similar(p>0.05);On day 10 after injury,the wound healing rate of transplanted db/db-EPCs group was similar to that of injected NS group(p>0.05),and the wound healing rate of transplanted db/+-EPCs group and transplanted db/db-EPCs group was significantly higher than that of injected NS group at other time points(p<0.05 or p<0.01).The wound healing rate of db/+-EPCs group was similar to that of db/db-EPCs group at all time points after injury(p>0.05).2)On day 7 after injury,there was no significant difference in the number of CD31 positive cells in the db/+-EPCs group,db/db-EPCs group and NS group(p>0.05).On day 14 and 17 after injury,the number of CD31 positive cells in the db/+-EPCs and db/db-EPCs groups was higher than that in the NS group(p<0.05).On day 14 after injury,the number of CD31 positive cells in db/+-EPCs group was higher than that in db/db-EPCs group(p<0.05).On day 17 after injury,the CD31 positive cell number in db/+-EPCs group was similar to that in db/db-EPCs group(p>0.05).3)On day 7 after injury,the amount of collagen fiber deposition in the wound tissue of db/+-EPCs group,db/db-EPCs group and NS group was similar(p>0.05);On day 14 and 17 after injury,the amount of collagen fiber deposition in the db/+-EPCs and db/db-EPCs groups was higher than that in the NS group(p<0.05).On day 14 after injury,the amount of collagen fiber deposition in the db/+-EPCs group was slightly more than that in the db/db-EPCs group,but the difference was not statistically significant(p>0.05).On day 17 after injury,the amount of collagen fiber deposition in db/+-EPCs group was higher than that in db/db-EPCs group(o<0.05).4)On day 7 after injury,the mRNA expression levels of VEGF,SDF-1 and CXCR4 in db/+-EPCs group and db/db-EPCs group were significantly higher than those in NS group(p<0.05,p<0.05,p<0.05),the mRNA expression levels of CXCR4 and SDF-1 in db/+-EPCs group were higher than those in db/db-EPCs group(p<0.01,p<0.05),and the mRNA expression level of VEGF in db/+-EPCs group was slightly higher than that in db/db-EPCs group.There was no statistically significant difference(p>0.05).On day 14 after injury,the mRNA expression levels of VEGF,SDF-1 and CXCR4 in db/+-EPCs group and db/db-EPCs group were significantly higher than those in NS group(p<0.005,p<0.005,p<0.01),and the mRNA expression levels of CXCR4 and SDF-1 in db/+-EPCs group were higher than those in db/db-EPCs group(p<0.05,p<0.05).On day 17 after injury,the mRNA expression levels of VEGF,SDF-1 and CXCR4 in db/+-EPCs group and db/db-EPCs group were significantly higher than those in NS group(p<0.05,p<0.01,p<0.01),and the mRNA expression levels of VEGF,SDF-1 and CXCR4 in db/+-EPCs group and db/db-EPCs group were similar(p>0.05).5)At 7 days after injury,the protein expression levels of VEGF,SDF-1 and CXCR4 in db/+-EPCs group and db/db-EPCs group were significantly higher than those in NS group(p<0.05,p<0.01,p<0.05),the protein expression levels of CXCR4 and SDF-1 in db/+-EPCs group were higher than those in db/db-EPCs group(p<0.01,p<0.05),and the protein expression level of VEGF in db/+-EPCs group was slightly higher than that in db/db-EPCs group,but the difference was not statistically significant(p>0.05).On day 14 after injury,the protein expression levels of VEGF,SDF-1 and CXCR4 in db/+-EPCs group and db/db-EPCs group were significantly higher than those in NS group(p<0.05,p<0.01,p<0.05),and the protein expression levels of CXCR4 and SDF-1 in db/+-EPCs group were higher than those in db/db-EPCs group(p<0.05,p<0.05).On day 17 after injury,the protein expression levels of VEGF,SDF-1 and CXCR4 in db/+-EPCs group and db/db-EPCs group were significantly higher than those in NS group(p<0.05,p<0.01,p<0.05),and the protein expression levels of VEGF,SDF-1 and CXCR4 in db/+-EPCs group and db/db-EPCs group were similar(p>0.05).Conclusions:Both amplified diabetic EPCs cultured in vitro and allogeneic normal EPCs transplanted can promote wound healing in diabetic mice by promoting angiogenesis,wound granulation tissue formation,collagen fiber deposition and other mechanisms.Dynamic test results showed that diabetic EPCs could only partially restore the biological activity of cells in vitro culture,while allogeneic normal EPCs transplantation had a short effect,which may be the reason for the comparison of the wound healing rate between the two groups.Further research and breakthrough are expected to achieve better results. |
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