Overexpressing Of IL-10 In Adipose Mesenchymal Stem Cells Promotes Wound Healing In Diabetic Mice

BackgroundDiabetic ulcer is a serious chronic wound that is difficult to heal,often leading to amputation and even death,and causing heavy medical and economic burden.In recent years,stem cells have been increasingly used in tissue repair.Because of the advantages of wide source and easy access,adipose-derived mesenchymal stem cells(ADSCs)can accelerate wound healing and reduce scar hyperplasia,giving hope to those patients with chronic wounds,which cannot be cured by traditional treatment.Compared with common wounds,the inflammation stage of diabetic ulcer lasts longer,producing abnormal inflammatory cytokines which contribute to impaired healing.In addition,macrophages play a central role in the inflammatory response of chronic wounds.M1 secrete proinflammatory cytokines(e.g.IL-1?,TNF-?,IL-6),nitric oxide,proteases and reactive oxygen species,inhibiting tissue repair.M2 secrete anti-inflammatory factors(e.g.IL-10),ornithine and polyamines,which promote inflammation relief and tissue regeneration.While in chronic wounds,macrophages present M1 phenotype for a long time,which result in severe tissue damage.Therefore,in order to regulate the transformation of macrophages from M1 to M2,IL-10 is of particular concern to researchers and clinicians due to its powerful anti-inflammatory function.Although IL-10 can effectively reduce inflammation and promote wound healing,its half-life in the wound is only 2h.How to prolong its role in chronic wounds is a problem to be solved.At present,most studies focus on the application of ADSCs alone to promote wound healing.Some researchers combine ADSCs with biomaterials or conditioned medium/concentrate(freeze-dried powder).Others combine ADSCs with anti-inflammatory factors to inhibit the inflammatory response of chronic wounds.Considering the role of ADSCs and IL-10 in wound healing,we intend to overexpress IL-10 in ADSCs with genetic engineering and transplant them into the wound surface of diabetic mice,so as to achieve rapid wound healing of diabetic ulcer.Objective(1)To investigate whether ADSC-IL10(ADSC-IL10)could affect the stem cell characteristics of ADSCs;(2)To investigate whether ADSC-IL10 can promote wound healing in diabetic mice;(3)To study the mechanisms of ADSC-IL10 in promoting wound healing in diabetic mice.Methods1.The effects of overexpression of IL-10 on ADSCs(1)The protein-coding gene IL-10 of mice was integrated into human ADSCs by lentivirus;(2)The expression of IL-10 protein in the supernatant of ADSC-IL10 was detected by the ELISA kit;(3)The surface markers of ADSC-IL10(CD44,CD73,CD90,CD105)were analyzed by flow cytometry;(4)Migration and proliferation of ADSC-IL10 were detected by scraping line method and MTT;(5)Adipogenic and osteogenic differentiation of ADSC-IL10 were induced.The adipogenic gene PPAR-? and osteogenic gene RUNX2 were detected by fluorescence real-time quantitative PCR.2.Effects of conditioned medium of ADSC-IL10 on cells related to wound repair(1)The mouse peritoneal macrophages(Raw 264.7)were cultured in the conditioned medium(CM)of ADSC-IL10 and ADSC-PCDH.DMEM low glucose medium was used as control.After 12h of culture,mRNA of Raw 264.7 cells was detected by qPCR.M1 was detected by iNOS and CD86,and M2 by Arg-1 and CD206.And the expressions of inflammatory factors such as IL-1?,IL-6,IL-10 and MCP-1,as well as growth factors such as EGF,VEGF and TGF?-1 were detected.(2)The effects of ADSC-IL10-CM and ADSC-CM on the migration of normal skin fibroblasts were detected by transwell.The effect of ADSC-CM on the migration of immortalized epidermal cells was detected by the streak method.3.Transplantation of ADSC-IL10 to promote wound healing in diabetic mice and study of its mechanisms(1)The model of diabetic mouse was induced by high-fat and high-sugar diet and a single intraperitoneal injection of 150 mg streptozotocin(STZ)per kilogram of body weight.Mice with random blood glucose higher than 16.7 mmol/L were included in the experimental group;45 hyperglycemic mice were randomly divided into control group,ADSC-PCDH group and ADSC-IL10 group,with 15 mice in each group.Cut 1.5cm×1.5cm skin on the back of mice to form wound.Transplant 1×106 ADSC-IL10 to the wound of mice in ADSC-IL10 group,1×106 ADSC-PCDH to ADSC-PCDH group,and the same volume of normal saline to the control group.Observe the wound healing progress of each mice,and photographs were taken respectively.(2)Histological staining and qPCR were performed on tissues taken from the wounds of mice at day 3 and 7.The wound healing status was observed through HE staining,and immunofluorescence(CD206,Arg-1)was used to identify the number of M2 in mouse skin tissue.The expression of CD206 and Arg-1 were detected by qPCR to identify the type of macrophages in the wound tissue.Inflammatory factors such as IL-1?,IL-6,IL-10 and MCP-1 were detected to determine the inflammatory status of the wound tissue.The expression EGF,VEGF and TGFB-1 in wound tissue were measured.Results:1.The effects of overexpression of IL-10 on ADSCs(1)The concentrations of IL-10 in the supernatant of ADSC-IL10,ADSC-PCDH and ADSCs were 8245±294.6 pg/ml,358.7±30.36 pg/ml and 342.8±13.64 pg/ml respectively.The content of IL-10 in the supernatant of ADSC-IL10 was significantly different from that of ADSC-PCDH and ADSCs.(2)The stem cell markers CD44,CD73,CD90 and CD105 were all positively expressed in ADSC-IL10,ADSC-PCDH and ADSCs.There was no significant difference among them.(3)ADSC-IL10,ADSC-PCDH and ADSCs migrated at 24h,and there was no significant difference in cell proliferation and migration.(4)Adipogenic differentiation of ADSC-IL10,ADSC-PCDH and ADSCs were induced for 7 days.No significant difference was observed in the formation of lipid droplets and oil red O staining.There was no significant difference in the expression of lipogenic gene PPAR-?.Osteoblastic differentiation of ADSC-IL10,ADSC-PCDH and ADSCs were induced for 21 days.No difference was observed with alibi red staining.There was no differenee in the expression of the osteoblastic gene RUNX2 among the three groups.2.Effects of conditioned medium of ADSC-IL10 on cells related to wound repair(1)Raw 264.7 cells were cultured in ADSC-IL10-CM,and the results showed that ARG-1 and CD206 were highly expressed in vitro.(2)Low expression of proinflammatory factor IL-lp,IL-6,MCP-1 and high expression of anti-inflammatory factor IL-10 were detected.In addition,high expression of EGF,TGFp-1,VEGF were also detected.The results showed that Raw 264.7 cells cultured in ADSC-IL10-CM differentiated into M2.The expression of inflammatory factors decreased and growth factors increased.(3)Compared with the control group,ADSC-IL10-CM and ADSC-CM could promote the migration of skin fibroblasts.The migrated fibroblasts in ADSC-IL10 group was more than that of ADSC-PCDH group.ADSC-CM promotes the migration of immortalized epidermal cells.3.Transplantation of ADSC-IL10 to promote wound healing in diabetic mice and study of its mechanisms(1)Diabetic mice in the ADSC-IL10 group(healing in 19 days)had significantly higher healing rate than those in the ADSC-PCDH group(healing in 21 days)and the control group(healing in 25 days);the healing area,calculated with ImageJ,in the ADSC-IL10 group was significantly higher than that of the ADSC-PCDH group and the control group.(2)Immunofluorescence staining of CD206 showed no significant difference in M2 in skin tissue on day 3.On day 7,the expressions of Arg-1 and CD206 in the wound tissue of the ADSC-IL-10 group were higher than those of the ADSC-PCDH group,and the expressions of the ADSC-PCDH group were higher than those of the control group.The expression of Arg-1 and CD206 in tissues detected by qPCR also showed the same results.It suggested that the content of M2 in the wound tissue of the ADSC-IL-10 group was increased.(3)Compared with the control group,the expression of pro-inflammatory factor IL-1? was decreased and anti-inflammatory factors IL-4R and IL-10 were increased in the ADSC-IL10 group.Expression of EGF,TGFp-1 and VEGF were increased.It suggested that inflammatory response of the ADSC-IL10 group decreased and growth factor increased.ConclusionIn summary,our study showed that ADSC-IL10 did not affect the stem cell characteristics of ADSCs.Compared with ADSCs transplantation,ADSC-IL10 can promote wound healing in diabetic mice.The mechanism of ADSC-IL10 to accelerate wound healing may include the following aspects:ADSC-IL 10 promotes the expression of M2 in the wound of diabetic mice;inhibiting the secretion of pro-inflammatory factors such as IL-1?,IL-6 and MCP-1;the expression of EGF,TGFp-1 and VEGF was promoted;the migration of skin fibroblasts and epidermal cells were promoted.