Co-Culture Of Deer Antler MSCs And EpSCs And The Therapeutic Effect On Skin Trauma In Mice

Antler is a regenerative appendage that grows on the forehead of male deer and its rapid growth is based on the proliferation and differentiation of stem cells.The antler skin is attached to the antler bone,and the wound after dislodgement during annual antler regeneration starts from the rapid healing of the antler skin tissue and stimulates rapid antler growth by pulling on it during the antler growth process.Antler skin is the microenvironment for the growth of antler MSCs,and the growth and development of antler as well as the regenerative wound healing after antler bone shedding are closely related to antler skin.In order to investigate the basic characteristics of antler MSCs and velvet skin EpSCs and their effects on the treatment of skin trauma,this experiment used antler MSCs and velvet skin EpSCs as experimental materials to study their growth characteristics,basic morphological indices and other biological characteristics through in vitro co-culture;established a co-culture system to analyze the effect of antler MSCs on the proliferation and migration of velvet skin EpSCs;used intravenous injection of co-culture cells The effect of intravenous injection of co-cultured cells on the treatment of murine skin trauma was investigated.The results of the study laid the foundation for further development and utilization of antler MSCs,and also provided new theoretical basis and research ideas for clinical repair of skin trauma by antler skin EpSCs.In Experiment 1,To study the biological characteristics of Tarim antler mesenchymal stem cells(MSCs).Two sources of MSCs,mouse bone marrow and deer antler,were cultured in vitro to compare their growth characteristics,and the morphological features,structure,organelle composition and number of cells were compared using light microscopy and transmission electron microscopy.The antler MSCs were shuttle-shaped with regular arrangement and homogeneous morphology;the morphology of murine BMSCs was irregular,triangular,polygonal,etc.The cytosolic and nucleolar areas of antler MSCs were963μm~2and 134μm~2,respectively,which were significantly higher than those of murine BMSCs at 551μm~2and 79μm~2(P<0.01);the nucleoplasmic ratio of antler MSCs was 0.167,which was significantly lower than that of murine BMSCs at 0.206(P<0.01).The nucleoplasmic ratio of antler MSCs was 0.167,which was significantly lower than that of murine BMSCs at 0.206(P<0.05);the in vitro culture of antler MSCs showed an"S"-shaped growth trend,and the proliferation rate was significantly higher than that of murine BMSCs(P<0.01).The ultramicroscopic results showed that antler MSCs had a large cytosolic area,simple organelle types,and organelle morphology such as mitochondria and endoplasmic reticulum with characteristics of early na(?)ve cell development.The proliferative capacity of Tarim antler MSCs was higher than that of mice,which has wider application advantages in clinical treatment.In experiment 2,EpSCs were obtained by type IV collagen sorting,and the in vitro culture system of EpSCs was successfully established.P3 and P5 cells started to grow slowly from the first day and entered the exponential growth phase by the fifth day.The growth trend of P1,P3 and P5 cells was the same with the same inoculation density,but the growth trend of P1 cells was faster than that of P3 and P5.In vitro culture showed that there was no difference in the speed of wall attachment between antler skin EpSCs and antler MSCs,but the mitotic rate of antler skin EpSCs was slower and the cell fusion time was longer.cell fusion time was longer.In experiment 3,to compensate for the slow growth of antler skin EpSCs and improve the efficiency of cell culture in vitro,antler MSCs were co-cultured with EpSCs in different cell ratios(i.e.1:1,1:3 and 3:1).It was found that the cell compatibility and number of cells in the co-culture group were better and more in the ratio of 3:1,and the cell proliferation rate was significantly higher than that of EpSCs at 24h-72h(P<0.01),and its significant effect on the proliferation of EpSCs was confirmed by comparing the fluorescence intensity ofβ1 integrin,and CK19 and the number of CCK8 and positive cells.The scratch test showed that antler MSCs significantly promoted the migration of EpSCs in the antler skin during 24h-72h of scratching(P<0.01).Antler MSCs had a significant pro-proliferation and migration effect on antler skin EpSCs.In experiment 4,we found that co-culture of deer antler MSCs and antler skin EpSCs in vitro had a significant promotion effect on the proliferation and migration of EpSCs,so we chose the optimal group of co-culture(3:1)to treat the mouse back full skin defect model,compared with the saline group and natural healing group,the wound area of the 3d co-culture cell treatment group(20.1±0.75 mm~2)was significantly smaller than that of the The wound area of the co-culture cell treatment group(20.1±0.75 mm~2)was significantly reduced compared with the natural healing group(27.5±2.7 mm~2)and the saline group(26.3±1.7 mm~2)in the 3rd day(P<0.01);the wound area of the co-culture cell treatment group(10.8±3.1 mm~2)continued to be significantly reduced in the 7th day(P<0.01),and the wound area of the treatment group healed to 5.4±2.7 mm~2and 2.5±0.97 mm~2in the 9th and 12th day,respectively.0.97 mm~2,both significantly lower than the control two groups(P<0.01).This indicates that the in vitro co-culture group of antler MSCs and antler skin EpSCs can significantly promote the healing of mouse skin wounds.In summary,deer antler MSCs have early naive cell development characteristics and strong proliferation ability,and co-culture with antler skin EpSCs can significantly promote the proliferation and migration of EpSCs;in the treatment of skin trauma with reference to the therapeutic effect of deer antler MSCs,co-culture of deer antler MSCs with antler skin EpSCs can significantly promote the healing of skin trauma.