| Skin trauma is a clinically frequent surgical disease,which often leads to skin tissue trauma as well as inflammation and oxidative stress at the trauma site,and even systemic infection,and how to promote skin trauma healing has become a focus of attention.Adipose derived mesenchymal stem cells(ADSCs)have the potential for multidirectional differentiation and paracrine secretion,and have the biological properties of anti-inflammation,anti-oxidative stress and promoting the repair of damaged tissues.Stromal cell derived factor-1(SDF-1)was found to have chemotactic effects on stem cells.At present,there are few reports on the studies of SDF-1/ADSCs in the healing of small pig skin wounds in China and abroad.Therefore,this experiment aimed to investigate the effect of SDF-1 pretreatment of ADSCs on the healing of skin wounds in miniature pigs.The main studies were as follows:(1)Effect of SDF-1 pretreatment on ADSCs:The 3rd generation ADSCs were spread in 96-well plates,and 0,50,100,200 and 300 ng/m L SDF-1 were selected to pretreat the ADSCs,and the optimal concentration of SDF-1 pretreatment was determined by detecting cell viability using CCK-8 method.ADSCs were randomly selected to be pretreated with the optimal concentration of SDF-1,and the changes of regeneration-related factors b FGF,TGF-βand EGF in the culture supernatant of ADSCs were detected by ELISA,and the changes of proliferation and paracrine capacity of ADSCs after pretreatment with untreated ADSCs and SDF-1 were compared and analyzed.(2)Effect of ADSCs and SDF-1/ADSCs on skin wound healing in miniature pigs:Nine miniature pigs were randomly divided into three groups,three in each group:the PBS buffer group(PBS group),the adipose mesenchymal stem cell group(ADSCs group),and the stromal cell-derived factor-1 pretreated adipose mesenchymal stem cell group(SDF-1/ADSCs group).In each group,8square wounds with 40 mm sides were created on both sides of the back spine of the pig,40 mm apart unilaterally,and each wound was 25 mm from the dorsal midline.2 m L of PBS buffer,2 m L of ADSCs(about 8×10~5)in PBS mix,and 2 m L of SDF-1/ADSCs in SDF-1/ADSCs mix were injected into each wound at four points immediately after surgery.PBS mixture,and 2 m L PBS mixture containing SDF-1 pretreated ADSCs(approximately 8×10~5)in each wound.Preoperatively,blood samples and skin tissue samples were collected at 7th,14th,21st and 28th days after surgery,and the changes of wound healing were recorded by photographing,pathological histological changes were observed by HE and Masson staining,changes of inflammation-related factors and regeneration-related factors in skin tissues were detected by ELISA,and changes of oxidative stress-related indexes were detected by the kit,and the comparative analysis of ADSCs and SDF-1/ADSCs on the healing of skin wounds in miniature pigs.The test results were as follows:(1)Effect of SDF-1 pretreatment on ADSCs:CCK-8 results showed that pretreatment of ADSCs with 200 ng/m L SDF-1 was the optimal concentration of SDF-1 pretreatment for this experiment.Compared with the NC group,the b FGF and TGF-βcontents in the culture supernatant of ADSCs in the 200 ng/m L SDF-1 group were significantly increased,and there was no significant difference in EGF content.(2)Effects of SDF-1/ADSCs on skin trauma in miniature pigs:1)Clinical examination and pathological histological observation:At 14th and 21st days after surgery,the wound healing rate was significantly increased in the ADSCs group compared with the PBS group,and the wound healing rate was highly significantly increased in the SDF-1/ADSCs group;compared with the ADSCs group,the wound healing rate was significantly increased in the SDF-1/ADSCs group.Compared with the PBS and ADSCs groups,the SDF-1/ADSCs group showed a large amount of granulation tissue growth in the wound,significantly faster epithelialization of the wound,significantly more collagen fiber deposition and neat and orderly arrangement,and the most dense collagen fibers.2)Results of inflammatory indexes:At 7th and 14th days after surgery,compared with the PBS group,the ADSCs group showed significantly lower numbers of WBC,NE and LY,significantly higher levels of ALB and GLB,significantly lower levels of TNF-αand IL-1β,and significantly higher levels of IL-10;compared with the ADSCs group,the SDF-1/ADSCs group showed significantly lower numbers of WBC,NE and LY,significantly higher levels of ALB and GLB The levels of WBC,NE and LY,ALB and GLB were significantly increased,TNF-αand IL-1βwere significantly decreased,and IL-10 was significantly increased in the SDF-1/ADSCs group.At 7th day after surgery,compared with the PBS group,WBC,NE and LY numbers and TNF-αand IL-1βlevels in the SDF-1/ADSCs group were significantly lower,and ALB and GLB levels and IL-10levels were significantly higher.At 14th day after surgery,compared with the PBS group,the SDF-1/ADSCs group showed a significant decrease in the number of WBCs,NE and LY,a highly significant decrease in the levels of TNF-αand IL-1β,and a highly significant increase in the levels of ALB and GLB and IL-10.At 21st day after surgery,TNF-αand IL-1βlevels were significantly lower and IL-10 levels were significantly higher in the SDF-1/ADSCs group compared with the PBS group.3)The results of oxidative stress indexes:At 7th and 14th days after surgery,compared with the PBS group,SOD and GSH activities were significantly increased and MDA content was significantly decreased in the ADSCs group,and SOD and GSH activities were extremely significantly increased and MDA content was extremely significantly decreased in the SDF-1/ADSCs group;compared with the ADSCs group,SOD and GSH activities were significantly The SDF-1/ADSCs group showed significantly higher SOD and GSH activities and significantly lower MDA content compared with the ADSCs group.At 21st day after surgery,GSH activity was significantly higher and MDA content was significantly lower in the SDF-1/ADSCs group compared with the PBS group.4)Regeneration index test results:At 7th and 14th days after surgery,compared with the PBS group,VEGF,b FGF,TGF-βand CD31 contents were significantly increased in the ADSCs group,and VEGF,b FGF,TGF-βand CD31 contents were highly significantly increased in the SDF-1/ADSCs group;compared with the ADSCs group,VEGF,b FGF,TGF-βand CD31 were significantly higher in the SDF-1/ADSCs group compared with the ADSCs group.At 21st day after surgery,the expression of VEGF,b FGF,TGF-β1 was significantly higher in the ADSCs and SDF-1/ADSCs groups compared with the PBS group,and the expression of CD31 was significantly higher in the SDF-1/ADSCs group.In summary,both local injection of ADSCs and SDF-1/ADSCs in skin wounds can improve the local lesions and microstructural damage of wounds,increase the healing rate of skin wounds,reduce the inflammatory response and oxidative stress,accelerate the regeneration of skin tissues,promote the healing of skin wounds,and shorten the healing time of wounds.Compared with untreated ADSCs,the proliferative and paracrine abilities of ADSCs were improved after SDF-1pretreatment,and the treatment of skin wounds was more effective. |
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