| Objective : The skin has various physiological functions such as barrier,absorption,sensation,body temperature regulation,metabolism,and immunity.The wound of the skin destroys the barrier function of the skin,and also damages the physiological functions such as perspiration,pain,temperature,and touch,causing great pain to the patients and placing a heavy burden on the family and society.However,due to the current technical level and treatment conditions,nearly half of the patients’ wounds cannot heal in time,forming sequelae such as chronic wounds and pathological scars.Although a lot of research has been carried out to promote skin wound healing,there is no rapid treatment method of wound healing.Mesenchymal Stem Cells(MSCs)are a class of primitive undifferentiated cells with multipotential differentiation and self-renewal capacity,with the ability to differentiate into mesoderm,et aloderm and endoderm.MSCs play an important role in skin wound repair and tissue regeneration,and are the most clinically valuable medical tools.Because of its extensive source organization,MSCs are easy to expand in vitro,and low rejet alion after transplantation,they can accelerate wound closure and reduce scar formation by inducing angiogenesis and tissue formation to promote wound repair,they have great clinical application prospet als in skin tissue regeneration.Although stem cell therapy can improve the quality of healing of chronic wounds,some basic questions about optimal cell populations,appropriate cell concentrations,survival of transplanted cells,and the ability of cells to maintain their properties under new conditions remain to be resolved,limitations exist in autologous skin grafts.The mechanisms by which MSCs are used for cell therapy is still unclear.It was previously believed that stem cells work like this: after reaching the damaged site after cell transplantation,they undergo a differentiation process and take over damaged cells.However,the latest research confirms that after transplantation,stem cells migrate to damaged tissues for a long time,and then undergoing differentiation process for treatment is very rare,because immune system will discharge these transplanted stem cells out of the body.Now,the paracrine effet al of stem cells has attracted more and more scientific research.Stem cell paracrine components mainly include growth factors and exosomes.Exosomes can carry specific information to target cells,enabling information exchange between distant cells.The role of stem cells in wound repair and tissue regeneration is primarily related to their paracrine capacity,rather than their ability to differentiate.Paracrine secretion of stem cells is mainly achieved by exosomes.Exosomes are microvesicles secreted by cells,about 30-150 nm in diameter,and the most important role is to transmit communication signals between cells.Exosomes can regulate immune response,regulate the physiological responses such as proliferation,migration,differentiation and apoptosis of target cells,and also promote angiogenesis,maintain the homeostasis of the body’s internal environment,and even serve as a specific diagnostic marker for disease development.It has potential medical application value in the field of regenerative medicine such as wound repair.The exosomes secreted by mesenchymal stem cells have the characteristics of mother cells,which can accelerate self-repair and tissue regeneration of the wound area,restore the homeostasis of the internal environment,and accelerate wound healing.The rapid development of stem cell research has provided hope for improving the scar-free healing of wounds,especially the use of human amniotic mesenchymal stem cells(hAMSCs)in wound regeneration.Therefore,the study of human amniotic mesenchymal stem cell-derived exosomes(hAMSCs-Ex)in the application of ski n wound healing has become one of the hotspots of medical research and application in recent years.Methods: 一、 The role of hAMSCs-derived exosomes in wounds healing 1.Isolation,culture and identification of hAMSCs cells.2.Separation,extraction and identification of exosomes.3.Making a full-thickness skin defet al model in the back of KM mice.4.Preparation of frozen set alion and HE staining.5.Internalization of PKH-26 labeled Ex.6.Effet al of hAMSCs-derived exosomes on proliferation and migration of HaCaT.二、Screening of related miRNAs for promoting wound repair in hAMSCs-Ex 1.Separation and extraction of exosomes.2.Selet aling of miRNAs associated with skin wound healing from hAMSCs and hAMSCs-Ex.3.The selet aled miRNAs were verified by qRT-PCR in exosomes secreted by hAMSCs,hAMSCs-Ex.三、New miRNA N-72 regulates EGF-induced migration of human amniotic mesenchymal stem cells by targeting MMP2 1.Bioinformatics analysis and prediction of new miRNA N-72 target gene.2.Dual luciferase reporter assay to verify target genes.3.Effet al of EGF on the expression of new miRNA N-72.4.Effet al of new miRNA N-72 on EGF-induced cell migration.5.Relationship between MMP2 and EGF-induced cell migration.Results: 一、hAMSCs-derived exosomes promote skin wound healing 1.Successfully separated and identified hAMSCs and hAMSCs-Ex.2.hAMSCs-Ex promotes wound healing in KM mice.3.HE staining showed that hAMSCs-Ex shortened the time of wound repair in mice,promoted re-epithelialization of skin wounds,and formation of skin appendages.4.PKH-26-labeled hAMSCs-Ex can be internalized to HaCaTs,and then transport the contents into the cells.5.hAMSCs-Ex promotes proliferation and migration of HaCaTs.二、 Related miRNAs in hAMSCs-Ex to promote wound repair 1.miRNAs associated with skin wound healing and re-epithelialization were screened from hAMSCs,hAMSCs-Ex,including the novel miRNA N-72 and known miRNAs such as miR-31,miR-210.2.In hAMSCs and hAMSCs-Ex,qRT-PCR not only verified the selet aled miRNAs,but also found that the expression levels of miR-31,miR-210 and miRNA N-72 were higher in hAMSCs-Ex than hAMSCs.三、New miRNA N-72 regulates EGF-induced migration of human amniotic mesenchymal stem cells by targeting MMP2 1.Validated the new discovered new miRNA N-72.2.EGF down-regulates the expression of new miRNA N-72.3.New miRNA N-72 inhibits EGF-induced cell migration.4.MMP2 is the target gene of N-72.5.MMP2 is involved in EGF-induced cell migration.6.Animal experiments showed that exosomes secreted by hAMSCs transfet aled with new miRNA N-72 mimics did not shorten the time of wound healing,but can promoted re-epithelialization,arranged collagen more neatly,and increased newborn hair follicles than exosomes secreted by hAMSCs transfet aled with NC.Conclusion: The isolation and culture method of human amniotic mesenchymal stem cells and the method of exosomes over-extension were established.It was confirmed that hAMSCs-Ex can promote wound healing in KM mice.And hAMSCs-Ex of hAMSCs respet alively carry out the sequencing of miRNA expression profiling,and found miR-210,miR-31,and new miRNA N-72,which are different in hAMSCs and hAMSCs-Ex,which can promote wound repair.We explored their molecular mechanisms that affet al wound healing.. |
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