Mechanism Research Of Exosomes Derived From Canine Umbilical Cord Mesenchymal Stem Cell Repair Of Skin And Vascular Injury

Mesenchymal stem cells(MSCs)are adult stem cells which present in the interstitial tissues of the body.MSCs have become ideal seed cells for clinical treatment of stem cells due to its advantages of wide sources,strong proliferation ability,multidifferentiation potential and low immunogenicity.Exosomes(Exo)are nano-sized vesicles rich in microRNAs and other biologically active molecules produced by cells paracrine,which can be fused by target cells and release inclusion such as microRNAs to regulate physiological activities of target cells such as proliferation,differentiation,migration and apoptosis.MSC-Exo is of great significance in the diagnosis and treatment of clinical diseases.Compared with directly transplantation of MSCs,there is no sensitization and carcinogenic risk,and the dosage is easy to control.At present,MSCs research are more focus on human and mouse sources,while less canine MSCs research.But dogs as the preferred domestic pets,their health problems have also received attention.Skin injury is the most common trauma in dogs,which its conventional treatments can easily lead to secondary infections,long wound healing time and scars formation and so on.However,MSC-Exo has the advantages of inhibiting inflammation and speeding up healing,providing a new way to treat skin injury.In order to select the clinically treated seed cells of dogs and study the repair effect of umbilical cord mesenchymal stem cell exosomes(UC-MSC-Exo)on skin and vascular injury and to clarify its mechanism,the following studies were carried out in this experiment:1.Comparative study on the biological and transcriptome characteristics of MSCs from five different sources of umbilical cord,amniotic membrane,placenta,bone marrow and adipose tissue.Five different tissue-derived MSCs were collected and cultured from Unbilical Cord(UC),Amniotic(AM),Placenta(P),Bone Marrow(BM)and Adipose(AD),And its growth ability,adipogenic and osteogenic differentiation ability,cell surface specific markers and transcriptome characteristics were compared.The results showed that MSCs from five different tissue sources were in a fibrous adherent state,and all the cells expressed CD44 positively and expressed CD34 negatively.All MSCs had the highest proliferative capacity in the third generation.The average population doubling time of AD-MSC,P-MSC,BM-MSC,UC-MSC and AM-MSC were(15.8 ± 0.318)h,(21.2 ± 0.629)h,(26.2 ± 0.242)h,(35 ± 0.588)h and(41.9 ± 0.954)h respectively,which AD-MSC had the strongest proliferative activity.MSCs from five different sources can be induced to differentiate into adipocytes and osteoblasts in vitro,and AD-MSC has the highest adipogenic and osteogenic efficiency.However,UC-MSC has superior cytological properties in terms of postpartum waste tissue-derived MSCs.The MSCs transcriptome profile showed that AD-MSC and BM-MSC had the highest homology,while P-MSC was significantly different from the other four types of MSCs.UC-MSC-Exo was chosen as a drug for the repair of skin and vascular injury due to its unique source advantage which is derived from postpartum discarded umbilical cord.2.Canine UC-MSC-Exo promotes the repair of skin woundsIn this experiment,exosomes were isolated from conditional medium of canine UC-MSC by low temperature ultracentrifugation(100,000 g centrifugal force).Transmission electron microscopy showed that the exosomes were concentrated at about 84 nm in an oval cup holder.Western blot analysis showed that the exosomes expressed surface markers CD9,CD63 and CD81.The results suggested that our experiment successfully separated the canine UC-MSC-Exo.In order to study the mechanism of mesenchymal stem cells promoting skin wound repair,this experiment investigated the repair effect of UC-MSC-Exo on canine skin wounds.Twelve healthy Chinese rural dogs in similar age were selected and a 4 cm × 4 cm square incision was made in the skin below the medial scapula of the back,and then randomly divided into a treatment group and a model group,with 6 dogs in each group.The treatment group was injected with a 1 m L exosomes solution at a concentration of 300 μg/m Laround the wound,and the model group was injected with an equal amount of physiological saline.The mental state and wound healing of the dogs were observed daily before and after modeling.The routine blood test and the wound area were measured regularly,and the pathological sections of the callus were observed at the first,second and fourth weeks.It was found that in the first week after modeling,there was a significant inflammatory reaction in the model group,and the number of white blood cells was increased by 30% compared with that before modeling.However,the inflammatory reaction was not obvious in the treatment group,and the total number of white blood cells and neutrophil increased more slowly than that in model group.There was no significant difference in the number of lymphocytes and red blood cells between the two groups.The growth rate of granulation tissue and wound healing in the treatment group was faster than that in the model group,but there was no significant difference.The results suggest that canine umbilical cord mesenchymal stem cell derived exosomes have a certain role in inhibiting inflammation and promoting skin wound healing.3.Regulation of canine UC-MSC-Exo on proliferation,migration and apoptosis of vascular endothelial cell(VEC).Angiogenesis is an important process of wound healing,but the mechanism of canine UC-MSC-Exo on vascular injury repair is still unclear.In order to investigate whether UC-MSC-Exo promotes skin wound healing by altering the biological behavior of VEC,this study used mechanical exfoliation and type I collagenase digestion to separate canine VEC and identify it by surface markers and differentiation characteristics.The effects of canine UC-MSC-Exo on VEC proliferation,migration and apoptosis were determined by CCK-8 method,scratch test and Annexin V flow assay.The results showed that the morphology of canine VEC was paving stone,and the cells positively expressed CD34 and CD31 surface proteins,suggesting that the dog VEC was isolated.The results of CCK-8 test showed that canine UC-MSC-Exo promoted the proliferation of VEC,and this effect was positively correlated with the dose.The results of scratch test showed that canine UC-MSC-Exo can significantly promote VEC migration after 24 hours of co-culture.,which the cell migration rate of UC-MSC-Exo group was increased by 35.56% compared with the control group.The apoptosis rate of UC-MSC-Exo group was 1.5% lower than that of the control group by flow cytometry,and the difference was extremely significant.Therefore,the above results suggest that canine UC-MSC-Exo can significantly promote the proliferation and migration of VEC and inhibit its apoptosis.4.The effect and molecular mechanism of non-coding RNA on the proliferation of VEC in canine UC-MSC-Exo.In this experiment,the microRNAs in canine UC-MSC-Exo were first sequenced,and then four miRNAs related to cell proliferation(cfa-miR-34a/-143/-181/-378)were selected according to the results,and finally their mimics were synthesized and transfected into VEC.It was found that cfa-miR-34 a and cfa-miR-143 significantly promoted VEC proliferation in vitro.Target gene prediction of cfa-miR-34 a and cfa-miR-143 by Target Scan and miRBD software and KEGG signaling pathway analysis of target genes using String software.The results showed that cfa-miR-34 a may promoted VEC proliferation by the Notch signaling pathway,while cfa-miR-143 may by the P13K-Akt,Erb B and MAPK signaling pathways.