Study On The Treatment Of Skin Scald In Mice With Fetal Bovine Epidermal Stem Cells

This study was aimed to study the isolation,culture and identification of simmental bovine epidermal stem cells（Ep SCs）,explore their in vitro induction and differentiation potential,and investigate their therapeutic effect on scald model in mice.Simmental cattle Ep SCs were isolated from 3 to 4 months old simmental cattle epidermis by enzyme digestion method and tissue adhesion method,and were continuously cultured and cryopreserved in vitro.The proliferation ability of Simmental cattle Ep SCs was investigated by cloning formation ability detection and growth curve drawing.The migration ability of Ep SCs was detected by scratching test.Ep SCs surface markers（ITGβ1,P63 and CD71）were identified by immunofluorescence,and the expression analysis of specific genes（ITGβ1,KRT19 and ITGα6）were performed by RT-PCR.Simmental Ep SCs were induced into adipogenesis,osteogenesis and cartilage differentiation in vitro,and their multidirectional differentiation potential was detected.The second degree scald model of ICR mice was made by scalding the epidermis with100℃ boiling water.After 1 day of scald on the epidermis,Ep SCs were injected subcutaneously into the damaged epidermis through cell transplantation.The mouse epidermis was taken,and the repair of epidermis was observed by observing HE pathological sections,and the transplanted stem cells in the epidermis were located.Il-6 and TNF-αinflammatory cytokines were detected by ELISA at molecular level.The results showed that the purity of Ep SCs obtained by tissue block adhesion method was lower than that obtained by enzyme digestion method.The growth curve of Ep SCs was "S" type,and the average population doubling time was 32.64 h.Immunofluorescence showed positive expression in surface specific markers of Ep SCs such as ITGβ1,P63 and CD71,RT-PCR showed that Ep SCs highly expressed gene ITGβ1,KRT19 and ITGα6 but CD31.The induced differentiation of adipocytes produced a large number of lipid droplets with strong refraction,which were dark red by oil red O staining and expressed PPAR-γ and LPL.The induced differentiated osteoblasts could form a large number of calcium nodules.Calcium deposits stained by alizarin red were orange-red with a certain thickness,and Collagen Ι and RUNX2 genes were positively expressed in the cells.The chondrocytes induced differentiation could form cartilage matrix stained with Alcian blue and positively expressed Collagen II and BGN.The results of Ep SCs cell transplantation in the treatment of scald in mice showed that Ep SCs can promote the repair of epidermis and hair follicles,which may stimulate the proliferation of surrounding cells to promote skin repair through Ep SCs proliferation and exosomes.Meanwhile,Ep SCs inhibited IL-6 and TNF-αinflammatory cytokines.Conclusion:In this study,SIMmental bovine Ep SCs were successfully isolated from simmental fetal bovine epidermis in good growth state,and had the potential to differentiate into osteogenesis,cartilage and adipose cells.Simmental Ep SCs cell line and its isolation and culture system in vitro were established.Ep SCs can promote the repair of skin injury and inhibit inflammation.