Effects And Mechanism Of Exosomes From Umbilical Cord Mesenchymal Stem Cells Of Giant Panda On Skin Wound Healing

The captive giant pandas are facing problems such as poor wound healing ability,susceptibility to infection and resistance to antibiotics post-surgery.Therefore,it is necessary to develop a new method to promote wound healing for giant pandas.Mesenchymal stem cells(MSCs),with self-renewal and differentiation abilities,play an important role in the research and treatment of wound healing in both human and veterinary clinics.Umbilical cord mesenchymal stem cells(UC-MSCs)are currently the only kind of MSCs that can be obtained from living giant pandas,and have the advantages of sufficient sample sources,low cell immunogenicity and strong stemness.Therefore,UC-MSCs have a high application value in giant panda wound treatment.In this study,we isolated giant panda UC-MSCs and their exosomes and found UC-MSC exosomes can promote the proliferation of giant panda dermal fibroblasts(DFs).We identified the types of protein and small RNA in the exosomes,especially the key factors that can promote the proliferation and migration of DFs,and investigated the regulatory mechanisms of exosomes on DFs proliferation,laying a foundation for the further study and application of giant panda UC-MSCs exosomes.The main conclusions are as follows:1.Collagenase type Ⅳ and trypsin were used to digest the umbilical cord of giant pandas.The UC-MSCs from the umbilical vein of giant pandas were successfully isolated,UC-MSCs with the adherence characteristic were displayed as a spindle-or triangular shape,and were arranged in a vortex or cluster orderly structure under high confluence.The UC-MSCs of the giant pandas had a normal karyotype,expressing MSC markers such as CD105,CD73,and CD49 f.They also expressed stem cell markers including SOX2,KLF4,and THY1.Giant panda UC-MSCs could be induced into adipocytes,chondrocytes,and osteoblasts in vitro.2.The exosomes were isolated from the giant panda UC-MSCs culture supernatant by ultracentrifugation,with an average diameter of 79.15 ± 4.81 nm.The exosomes expressed specific surface markers TSG101 and CD81 but did not express endoplasmic reticulum-specific markers CALNEXIN.Exosomes had a significant effect on the proliferation of giant panda DFs in a dose-dependent manner(0-50 μg/m L).Using the mouse skin wound model,we confirmed the effects of improvement on wound healing.The healing rate was 90.42%,which was 1.24 times higher than that of the control group(P<0.05).3.Label-free protein mass spectrometry and RNA sequencing technologies were used to analyze the composition of the exosomes from the giant panda UC-MSCs at P3.In this study,a total of 3466 proteins and 206 miRNAs were identified.The top five proteins which had the highest level were fibronectin(12.217%),annexin(8.253%),histone(4.492%),60 S ribosomal protein RPL(3.338%),and collagen(3.085%);The top five miRNAs were miR-148a-3p(30.28%),miR-21-5p(21.72%),miR-100-5p(10.46%),miR-99a-5p(4.86%),and miR-378a-3p(4.49%).The differences of protein and small RNA were compared between the exosomes derived from low-passage(P3)and higher-passage(P15)UC-MSCs groups.It was found that the types of protein and small RNA in the exosomes derived from P3 UC-MSCs were significantly higher than that of P15 UC-MSCs.Compared with P15 UC-MSCs group,the proliferation-related signaling pathways such as WNT,MAPK,and VEGF were more active in exosomes of UC-MSCs at P3,while the expression of miR-21-5p is higher in exosomes of UCMSCs at P3.4.The function of the identified proteins of exosomes was preliminarily studied.We found bFGF,PDGF-DD,TGF-β1,and Fibronectin could promote the proliferation of DFs.PDGF-DD promoted the proliferation of giant panda DFs by increasing the proportion of cells at S-phase.Fibronectin,which had the highest expression level in exosomes,could also promote the migration of giant panda DFs.5.The mechanism of proliferation and regulation of DFs by miR-21-5p in giant panda UCMSCs exosomes was explored.Firstly,the miR-21-5p overexpression experiments showed that it significantly promoted the proliferation of DFs in giant pandas.Secondly,miRanda and RNAhybrid were used to predict the target genes of miR-21-5p,and the two potential genes including PDCD4 and RECK were identified by combining with the results of DFs m RNA sequencing after miR-21-5p mimic treatment.Double luciferase assay confirmed that PDCD4 and RECK were the target genes of miR-21-5p.Cell RNA interference experiments of the two genes further showed that miR-21-5p promoted the proliferation of DFs by negatively regulating the expression of PDCD4 and RECK,which further clarified the regulatory mechanism of miR-21-5p on DFs proliferation.Finally,miR-21-5p reduced the expression ofα-SMA during the TGF-β1 induction and inhibited the differentiation of DFs,suggesting that it had the function of inhibiting scarring.In conclusion,giant panda UC-MSCs and their exosomes were isolated and identified for the first time.UC-MSCs exosomes were rich in a variety of proteins and miRNAs,which had positive effects on the proliferation of DFs and skin wound healing in mice.Fibronectin,which was the most abundant protein in exosomes,could significantly promote the migration of DFs.In addition,miR-21-5p negatively regulated its target genes PDCD4 and RECK to promote the proliferation of DFs.On one hand,the results of this study laid a theoretical basis for the functional study of giant panda UC-MSCs exosomes.On the other hand,it provided technical support for exploring new methods for giant panda skin wound healing.