An Experimental Study Of Circulating Fibrocytes In Promoting Diabetic Wound Healing

Objective:To explore the promoting effects and biological mechanisms of transplanting circulating fibrocytes in diabetic wound healing,and provide new guidance and theoretical basis for the clinical application of circulating fibrocytes in promoting diabetic wound healing.Methods:Regarding the spontaneous genetic mutational diabetic mice(DB/DB)as diabetic individuals,normal mice(DB/+)as the normal individuals,collecting peripheral blood after anesthesia by 2%chloral hydrate,diabetes mellitus circulating fibrocytes(DMcFb)and normal circulating fibrocytes(NcFb)were obtained by density gradient centrifugation respectively.Twenty-seven DB/DB mices as diabetic models were randomly divided into 3 groups,including DMcFb treatment group(wounds injected DMcFb),NcFb treatment group(wounds injected NcFb),and PBS control group(wounds injected PBS)after being made 1 xlcm full-thickness skin defect models in the back by 2%chloral hydrate.The wounds of experimental mices were measured,dressed changes,took photo and calculated the rate of wound healing at day 3,7,10,14,17,21 post operation.Moreover,wound tissues which were obtained at day 7,14 were mearured new blood vessel formation rate(new blood vessel formation rate(the expression of MVD)by immunohistochemistry,cell proliferation rate of wound tissue(the expression of Ki67)by immunofluorescence,collagen deposition of wound tissue by mason dyeing,and the expression level of relevant genes of wound repairing such as IL-6,VEGF-A,CXCL12,CXCR4,COL-I and ?-SMA by Real time PCR.Results:1)At day 3,7,10 after injury the wound healing of DMcFb treatment group was faster than PBS control group(P<0.05),as well as the wound healing of NcFb treatment group was faster than PBS control group(P<0.05).However,at day 14,17 after injury,compared with the PBS control group,DMcFb and NcFb treatment group had no significant difference(P>0.05).At day10,14,17 after injury,compared with NcFb treatment group,the wound healing of DMcFb treatment group had no significant difference(P>0.05).But at day 3,7 after injury,the wound healing of NcFb treatment group was slightly faster than DMcFb treatment group(P<0.05).The wounds of three groups were close to heal at day 17 after injury and healed completely at day 21 after injury.2)At day 7,14 after injury,the proliferation index of Ki67 of DMcFb treatment group and NcFb treatment group were higher than PBS control group(P<0.05,P<0.05).At day 7 after injury,the proliferation index of Ki67 of NcFb treatment group was higher than DMcFb treatment group(P<0.05).The proliferation index of Ki67 of DMcFb treatment group and NcFb treatment group at day 14 after injury were higher than proliferation index of Ki67 at day 7 after injury(P<0.05).3)At day 14 after injury,the expression of MVD protein(new blood vessel formation rate)of DMcFb treatment group was higher than PBS control group(P<0.05).At day 7,14 after injury,the expression of MVD protein of NcFb treatment group was higher than PBS control group(P<0.05).At same point after injury,which the expression of MVD protein of NcFb treatment group compared with DMcFb treatment group had no statistical significance(P>0.05).The expression of MVD protein of three groups at day 14 after injury were higher than the expression of MVD protein at day 7 after injury(P<0.05).4)At day 7,14 after injury,the relative expression level of IL-6 mRNA of DMcFb treatment group and NcFb treatment group were higher than PBS control group(P<0.05,P<0.05).At same point after injury,which the relative expression level of IL-6 mRNA of NcFb treatment group compared with DMcFb treatment group had no statistical significance(P>0.05).The relative expression level of IL-6 mRNA of three groups at day 14 after injury were higher than the relative expression level of IL-6 mRNA at day 7 after injury(P<0.05).5)At day 14 after injury,the relative expression level of VEGF-A mRNA of DMcFb treatment group was significantly higher than PBS control group(P<0.05).At day 7,14 after injury,the relative expression level of VEGF-A mRNA of NcFb treatment group was higher than PBS control group(P<0.05).At day 14 after injury,the relative expression level of VEGF-A mRNA of NcFb treatment group was higher than DMcFb treatment group(P<0.05).But at day 7 after injury,which the relative expression level of VEGF-A mRNA of NcFb treatment group compared with DMcFb treatment group had no statistical significance(P>0.05).The relative expression level of VEGF-A mRNA of three groups at day 14 after injury were higher than the relative expression level of VEGF-A mRNA at day 7 after injury(P<0.05).6)At day 7 after injury,the relative expression level of CXCL12 mRNA of DMcFb treatment group and NcFb treatment group were significantly higher than PBS control group(P<0.05,P<0.05).At day 7 after injury,the relative expression level of CXCL12 mRNA of NcFb treatment group was slightly higher than DMcFb treatment group(P<0.05).The relative expression level of CXCL12 mRNA of NcFb and DMcFb treatment group at day 7 after injury were higher than the relative expression level of CXCL12 mRNA at day 14 after injury(P<0.05).7)The relative expression level of a-SMA mRNA of DMcFb treatment group was lower than PBS control group at day 7 after injury,but significantly higher than PBS control group at day14 after injury(P<0.05),as NcFb treatment group was(P<0.05).At day 14 after injury,the relative expression level of a-SMA mRNA of NcFb treatment group was higher than DMcFb treatment group(P<0.05).The relative expression level of a-SMA mRNA of NcFb and DMcFb treatment group at day 14 after injury were higher than the relative expression level of a-SMA mRNA at day 7 after injury(P<0.05).Conclusions:The wound tissues at day 7,14 after the injury which were from diabetic mice injected NcFb/DMcFb in wounds had many better advantages in cell proliferation rate,new blood vessel formation rate(MVD),collagen deposition,chemotactic factor CXCL12,promote angiogenesis factor(VEGF-A),inflammation medium(IL-6),extracellular matrix(?-SMA)than PBS control group.It presumably was that DMcFb may regain normal cellular functional activity to some extent because of being cultured in vitro and away from diabetic microenvironment.Furthermore,at day 7/14 after injury,local transplantation of DMcFb/NcFb not only can promote the progress of chronic wound healing in cell proliferation,angiogenesis,mediated cytokine and extracellular matrix deposition and so on,but also improve the rate of wound healing at different time points.Combined with the progress of wound healing at each time point about this experiment,it may facilitate the early progress of wound healing by transplantating cFb in diabetic wound,which can provide new guidance and theoretical basis for the clinical application of cFb treatment of chronic wounds.