| Objectives:To observe the effect of Human Umbilical Cord Mesenchymal Stem Cell Exosomes(hUCMSCs-ex)combined with escharectomy and skin grafting on the repair of scalded wounds in rats,and to explore its dosage and mechanism.This study provides theoretical basis and new ideas for the clinical application of hUCMSCs-ex in the future.Methods:A rat model of escharectomy and skin grafting after scald was established,and hUCMSCs-ex was used as an intervention measure.Label-free quantitative proteomics technology was used to find the changes of differential proteins in the repair effect of hUCMSCs-ex on escharectomy and skin grafting in rats.The molecular signaling pathways involved in wound repair were preliminarily discussed.1.hUCMSCs were isolated from neonatal umbilical cord tissue by tissue block adherence method.By observing the morphological structure of the cells;Oil red O staining and alizarin red staining were used to identify adipogenic and osteogenic differentiation of stem cells.The conditioned medium of passages P3-P6 of hUCMSCs was collected,and huc MSCs-Ex was extracted by ultracentrifugation.The morphology,size and structure of exosomes were observed by Transmission Electron Microscope(TEM).Nanoparticle Tracking Analysis(NTA)was used to detect the particle size and concentration of exosomes.Western blot was used to detect the expression of exosome surface markers CD63,CD81 and TSG101.2.To establish a rat scald model and observe the effect of metal coils on preventing wound contraction and healing by comparing the wound healing rate.The rats were divided into 5 groups:blank control group,H-EXO group,L-EXO group,PBS group,and Exosomes-free supernatant group.The wound healing rate of each group was compared 14,21 and 28 days after operation.HE staining and Masson staining were used to observe the pathological changes of wound skin tissue in each group after wound healing.3.The wound skin tissues of H-EXO group,PBS group,and blank control group were collected,and the raw data of mass spectrometry were obtained and qualified by Label-free quantitative proteomics technology,and then the repeatability of sample quantitative results was tested,significant difference analysis,and bioinformatics analysis were performed.To explore the differentially expressed proteins of hUCMSCs-ex in the repair of scald wound after escharectomy and skin grafting in rats.Western Blot was used to detect Cystatin A/Stefin A(CSTA/STFA)and Ig gamma 1 chain C region protein(Ig gamma 1 chain C region)in H-EXO group,PBS group and blank control group.IGHG1)expression.Results:1.Primary hUCMSCs were obtained by tissue block adherence method.Under inverted phase contrast microscope,the primary hUCMSCs adhered to the bottom of the tissue block and grew and fused.At P1 generation,the cells were polygonal.After P2 generation,the morphology of the cells gradually elongated and showed a long spindle shape.The morphology of P3 generation cells tended to be stable and long fusiform,and 90%of the cells were tightly arranged,showing a vortex shape,and the growth condition was good.Alizarin red staining showed that the calcium salt particles of osteogenic induced hUCMSCs appeared as dense red nodules.Oil red 0staining was used to form adipogenic hUCMSCs,and the fat particles were orange-red.Transmission electron microscopy showed that the extracted exosomes were well dispersed and uniform in size,with a saucer-like double capsule structure,a low electron density component in the center,a relatively clear boundary of the membrane,and a diameter of about 100nm,which was consistent with the morphological characteristics of mesenchymal stem cell-derived exosomes.NTA Analysis results showed that the Original Concentration of Particles in the original solution was 5.0×10~8Particles/ml,and the Peak Analysis was 127.4nm,which was in line with the diameter range of hUCMSCs-ex.Western Blot results showed that hUCMSCs-ex surface specific markers CD63,CD81,and TSG101 were highly expressed.2.In the experiment of preventing wound margin contraction with metal coils,the wound healing rate of group 1(escharectomy+metal coils fixing wound margin+autologous skin grafting+allogeneic skin covering)was higher than that of group 2(escharectomy+metal coils fixing wound margin+allogeneic skin covering).The wound healing rates of groups 3 and 4 without metal ring fixation were significantly higher than those of groups 1 and 2 with metal ring fixation.In the experiment on the repair effect of hUCMSCs-ex on escharotomy and skin grafting of scalded wounds,the survival and fusion of skin grafts,wound healing speed and quality in EXO group were better than those in EXD group and PBS group,and H-EXO group was better than L-EXO group.The EXD group also showed a tendency to promote wound healing compared with the PBS group.HE staining and Masson staining showed that regenerated epidermis was observed in both H-EXO and L-EXO groups,accompanied by skin appendages such as hair follicles and sebaceous glands,with distinct layers.The H-EXO group had more collagen production,more orderly arrangement of collagen fibers,and more new blood vessels.In the EXD group,there was a small amount of skin appendage formation,but more angiogenesis,no keratinized epithelium,and inflammatory cell infiltration were observed than in the L-EXO group.In the PBS group,a large number of inflammatory cells were infiltrated,the skin layers were not clear,and no skin appendages were observed.3.The log FC of PBS group and EXO group was 1.2 times and the P-value was less than 0.05.A total of 332 differentially expressed genes were screened out,including 162 up-regulated genes and 170 down-regulated genes.Two differentially expressed proteins,IGHG and CSTA,were identified,among which IGHG showed the most significant differential expression.The results of Western blot showed that the protein synthesis of IGHG1 and CSTA in EXO group was significantly higher than that in PBS group(p<0.05).The protein synthesis of IGHG1 and CSTA in EXO group was significantly higher than that in NORMAL group(p<0.05).There was no significant difference in the protein content of IGHG1 and CSTA between the PBS group and the NORMAL group.Conclusions:1.hUCMSCs can be obtained by tissue block adherence method;hUCMSCs-Ex can be isolated from conditioned medium by ultracentrifugation.2.hUCMSCs-Ex can accelerate the repair process of escharectomy and skin grafting of scald wounds in rats and improve the quality of wound healing.3.hUCMSCs-Ex may participate in the process of wound healing by highly expressing IGHG1 and CSTA. |
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