Study On The Mechanism Of Canine MSC-Exo And MiR-143 In The Repair Of Chondrocyte Damage

Osteoarthritis(OA)is a degenerative joint disease and there is an obvious increase in incidence with age.Decreased number of chondrocytes in joint tissues,metabolic disorder of extracellular matrix,inflammatory reaction of synovial joint and remodeling of subchondral bone can lead to joint dysfunction in OA patients.The articular cartilage has poor self-repairing ability and the existing treatment methods mainly focus on antiinflammatory,analgesic and other symptoms to relieve symptoms,there is no exact and effective treatment method since there are no blood vessels and nerves in the articular cartilage.It has been reported that Mesenchymal stem cells(MSC)and their derived exosome(MSC-Exo)have a significant effect on the repair of cartilage damage,but how it works is still unclear.In order to clarify the mechanism of MSC and MSC-Exo promoting cartilage damage and repair,we did the following research:1.Isolation and identification of MSCs and their exosomes from three tissues and organs of fat,bone marrow and umbilical cord:Flow cytometry was used to identify the surface markers of MSCs isolated from canine fat,bone marrow and umbilical cord.The results showed that MSCs isolated from this three tissues and organs all expressed MSC surface markers CD90,CD44 and CD29,and low expression of white blood cell surface marker CD45,and the results were consistent with the characteristics of MSCs.The MSC-Exo was separated from the MSC culture supernatant by ultracentrifugation,and the morphological structure,particle size and the specific protein expression level of MSC-Exo were detected by transmission electron microscopy,Nanoparticle Tracking Analysis(NTA)and Western blot.The results showed that the MSC-Exos were cup-shaped in shape,with a particle size distribution between 40 nm and 120 nm,and all expressed specific surface marker proteins CD81,CD9 and TGS101.The above results suggest that the cell vesicles isolated in this experiment meet the characteristics of exosomes.2.The effects of three different sources of MSC-Exos on the proliferation,migration,apoptosis and extracellular matrix metabolism of canine chondrocytesIn order to study the mechanism of MSC-Exo’s repair effect on OA after injury,we first established the chondrocyte injury model induced by the inflammatory factor Interleukin-1β(IL-1β)combined with Tumor Necrosis Factor-α(TNF-α),and then Cell Counting Kit-8(CCK-8),scratch test,flow cytometry,q PCR and Western blot were used to detect the effects of MSC-Exos on canine chondrocyte proliferation,migration,apoptosis and the level of extracellular matrix metabolism.The results show that MSCExos from the above three sources at a concentration of 50 and 100 μg/m L can significantly promote the proliferation and migration of chondrocytes in the 48 and 72 hours.Inhibit the chondrocytes apoptosis induced by IL-1β combined with TNF-α.In addition,it can up-regulate the expression of type II collagen and proteoglycan in the extracellular matrix of chondrocytes in damaged chondrocytes,and down-regulate Matrix Metalloproteinase 13(MMP13)and A Disintegrin and Metalloproteinase with Thrombospondin motifs-5(ADAMTS5)expression levels.These results suggest that MSC-Exos have the ability to promote the proliferation and migration of chondrocytes and the synthesis of extracellular matrix,and it can also inhibit chondrocyte apoptosis and extracellular matrix decomposition.3.The expression analysis of miRNA in umbilical cord MSC-Exo and the mechanism of miR-143 in promoting chondrocyte damage and repairRefer to the laboratory’s high-throughput sequencing results of MSC-Exo from umbilical cord tissue,select miR-143 with high expression level to explore its role in OA and its possible mechanism.CCK-8,q PCR and Western blot were used to study its effects on the proliferation of chondrocytes and the level of extracellular matrix metabolism..And then the bioinformatics target gene prediction software Targetscan was used to analyze the possible target genes of miR-143 and their possible binding sites.Finally,the target genes were verified by dual luciferase reporter gene,q PCR.The results show that transfection of miR-143 mimic can reverse the expression levels of extracellular matrix type II collagen and proteoglycan down-regulated by IL-1βcombined with TNF-α,while reducing the levels of extracellular matrix degrading enzymes MMP-13 and ADAMTS-5.Transfection of miR-143 mimic can also promote cell proliferation.Finally,the dual luciferase reporter gene and q PCR method verified that IL1RL1 is the direct target of miR-143.Conclusion: This study shows that MSC-Exo is similar to MSC in promoting articular chondrocyte injury and repair.In addition,it further reveals the mechanism of non-coding RNA miR-143 promoting chondrocyte injury and repair at cellular and molecular levels.It is suggested that miR-143/IL1RL1 can participate in the regulation of chondrocyte proliferation and extracellular matrix metabolism.