The Experiment Of Transplanted Skin Fibroblasts In Wound Healing Of Diabetic Rats

Objective:To investigate the effect and possible mechanism of transplantation of skin fibroblasts in the diabetic mice wounds and to investigate the changes of the activity of diabetic skin fibroblasts after in vitro culture,so as to lay a foundation and provide a theoretical basis for clinical application of skin fibroblasts for the treatment of diabetic wounds.Methods:Diabetic individual models were selected from 10 to 12 weeks old spontaneously genetically modified diabetic mice(DB/DB),normal individual models were selected from normal mice(DB/+),and through tissue block culture method get both diabete skin fibrocytes(DMsFb)and normal skin fibrocytes(NsFb)at the same time.According to the random number table method,twenty-seven DB/DB mices between six and eight weeks old were divided into 3 groups as diabetic models after being made 1 xlcm full-thickness skin defect models in the back,including A.PBS control group(injected PBS in four wound edges of DB/DB mice);B.DMsFb treatment group(injected DMsFb in four wound edges of DB/DB mice);C.NsFb treatment group(injected NsFb in four wound edges of DB/DB mice).In each group nine mices were subjected to wound open size measurement,wound dressing change,image collection,and wound healing rate calculation.The 3rd,7th,10th,14th,17th,and 21st days after injury were taken as time points.Wound dressing at day7,14 and measured wound tissue’s cell proliferation rate(Ki67’s expression)by immunofluorescence,wound tissue’s new blood vessel formation rate(MVD’s expression)by immunohistochemistry,wound tissue’s collagen deposition by mason dyeing.Annexin-FITC/PI flow cytometry was used to detect the apoptotic rate of DMsFb and NsFb after in vitro culture,RT-PCR was used to detect the cell’s TGF-β1,AGEs,MMP-9 and NK-1 mRNA expression levels after cultured in vitro.The protein expression level of TGF-β1 AGEs,MMP-9 and NK-1 was detected by Western blotting.Data were processed with two independent-sample t test and one-way analysis of variance.Results:1)At each time point after injury,both NsFb group and DMsFb group healed faster than PBS control group.Except for the 3rd and 10th days after injury,the wound healing rates of rats in DMsFb group was slower than that in NsFb group(P<0.05).2)At day 7 and 14 after injury,compared with PBS control group,Ki67 proliferation index in both NsFb group and DMsFb group were significantly higher(P<0.05),and the proliferation index of Ki67 in the NsFb treatment group was higher than that in the DMsFb treatment group(P<0.05).The Ki67 proliferation index of the wounds at day14 was higher than that of day7 in these groups.3)The expression of wound MVD protein in NsFb group and DMsFb group was higher than that of PBS control group at the 7th and 14th day after injury(P<0.05).There was also a significant difference in MVD protein expression between NsFb group and DM skin Fb group(P<0.05).4)Masson’s staining showed that the amount of deposited collagen in NsFb group was higher than that in DMsFb group and PBS control group(P<0.05),while the amount of DMsFb group was slightly higher than that of PBS control group(P>0.05).5)The cell viability testing revealed that the apoptotic rate of DMsFb was significantly higher than that of NsFb.When compared to DMsFb P0,DMsFb P4 was much more purified after in vitro culture,but the rate of apoptosis was not improved.The TGF-β1 and NK-1 mRNAin NsFb P0 were significantly higher than those in DMsFb,while NK-1 mRNA in DMsFb P4 showed a downward trend when compared with DMsFb P0(P<0.05).The AGEs and MMP-9 mRNA of DMsFb P0 were much higher than those of NsFb P0,but the DMsFb P4 AGEs mRNA was significantly reduced(P<0.05).Western blotting showed that the expression of the corresponding protein in each group was basically consistent with the mRNA expression trend.Conclusions:After NsFb/DMsFb’s injection in the four wound edges of diabetic mice,these two treatment groups’ indicators during the healing process such as cell proliferation index(Ki67),new blood vessel formation rate(MVD protein),collagen deposition were have improved significantly when compared with the PBS control group.Both the activity of NsFb/DMsFb cells were detected and the DMsFb P4 AGEs mRNA was significantly lower than DMsFb P0.It presumably was that DMsFb can be detached from diabetes(a high-sugar microenvironment)to cultured in vitro and purified,and its cell functional activity can be restored to a certain extent,so that to improve chronic wounds in terms of cell proliferation,neovascularization,collagen deposition,cytokine-mediated,and so on.And to increase the rate of wound healing in diabetic mice during the healing progresses at different time points.Therefore,local transplantation of sFb on diabetic wounds may play an important role in promoting the early wound repair process,which will lay the foundation for the clinical application of sFb cell therapy for chronic wounds,and provide theoretical basis.