| Corneal epithelium acts as a mechanical barrier to maintain corneal homeostasis.Once the corneal epithelium and basement membrane are destroyed,the surrounding corneal epithelial cells will secrete various cytokines,activate fibroblasts to differentiate into myofibroblasts,and cause irreversible corneal opacity and even blindness.The migration rate of corneal epithelial cells directly affects corneal epithelial closure rate and corneal repair.Accelerating corneal epithelial healing is essential for corneal repair.Common clinical methods to treat corneal epithelial wound include artificial tears,autologous serum,anti-inflammatory drugs,amniotic membrane covering,etc.Clinical treatment has shown certain efficacy,but it is also easy to cause different complications.New methods such as gene therapy,especially noncoding micro RNA(miRNA),have attracted much attention in the treatment of clinical diseases.It is of great significance to use miRNAs to repair corneal epithelial wound.miRNA is a natural and powerful regulator of gene expression and a key medium of basic biological processes of organisms.Studies have shown that miRNA 24-3p can enhance epidermal growth factor signal transduction and CTGF transcription by activating EGFR and/or CTGF phosphorylation to achieve cell migration.Accordingly,we hypothesized that controlling miRNA 24-3p level in corneal epithelial cells to activate phosphorylation of EGFR and/or CTGF could provide a new clinical therapeutic method for regulating corneal epithelial wound healing and epithelial homeostasis.At present,there are few reports on the role of miRNA 24-3p in the corneal epithelial wound repair.We plan to verify the therapeutic effect of miRNA 24-3p on corneal epithelial wound diseases through in vitro cell biology and in vivo experimental animal models,and develop miRNA 24-3p multistage delivery system to promote the healing of alkali burned corneal epithelial wound,providing a new therapeutic idea for the corneal epithelial wound repair.Firstly,we studied the biological function of miRNA 24-3p by transfecting it into corneal epithelial cells.The results of cell proliferation,cell chemotaxis,cell migration and RT-PCR experiments showed that miRNA 24-3p could not promote the proliferation of corneal epithelial cells,but could improve the chemotaxis of corneal epithelial cells and promote the migration of corneal epithelial cells.In the in vitro cell wound closure experiment,the miRNA 24-3p group completed more than 50% of the closure,while the negative control miRNA NC group did not.miRNA 24-3p also upregulated the expression of migration related genes FRS2,CDC42,CTGF,EGFR and MMP9,which were significantly different from the blank control group and the negative control group.Secondly,to solve the problem that miRNA 24-3p itself cannot directly enter cells,we propose to use the adipose-derived mesenchymal stem cell derived exosomes as the outercarriers of miRNA 24-3p to carry miRNA 24-3p into the corneal epithelial cells for action.Exosomes were extracted from adipose-derived mesenchymal stem cells using gradient ultracentrifugation,and adipose-derived mesenchymal stem cells and exosomes were identified.The exosomes met the ‘gold standard’ of exosomes evaluation.Corneal epithelial cells were treated with different concentrations of exosomes.The experimental results of cell proliferation,cell chemotaxis,cell migration,and vinculin protein expression showed that the cells were concentration dependent on the exosomes.With the increase of exosomes concentration,the proliferation,cell chemotaxis,and cell migration of corneal epithelial cells were enhanced,the expression of vinculin protein was increased,and the expression of migration related genes was increased.Therefore,the exosomes themselves also have the ability to promote cell proliferation,cell adhesion and cell migration in the process of corneal epithelial wound healing.Then we prepared miRNA 24-3p-rich exosomes by electroporation.In the treatment of rabbit corneal epithelial wound disease animal experiment in vivo,the miRNA 24-3p-rich exosomes was injected under the conjunctiva.After 12 days of treatment,compared with the pure exosomes group(85%),the miRNA 24-3p-rich exosomes group can accelerate the rapid closure of rabbit corneal epithelial wound on day 2 after surgery,with a healing rate of more than 90%.Meanwhile,it can regulate the thickness(25–30 μm)and layer number(3–4 layers)of corneal epithelium close to the natural cornea on day 7 after surgery,upregulate the expression of CK3,ZO-1,EGFR and MMP9,regulate the maturation of corneal epithelium and promote corneal tissue repair on day 12 after surgery.Because subconjunctival administration will destroy conjunctival tissue and bring new trauma,we further improved the delivery vector of miRNA 24-3p and proposed to improve the bioavailability of miRNA 24-3p-rich exosomes on the ocular surface by constructing a multistage delivery system.Based on the lubrication and moisturizing advantages of hyaluronic acid,we modified hyaluronic acid with thermosensitive monomer material DEGMA.The successfully prepared thermosensitive hyaluronic acid hydrogel can form a weak hydrogel after eye surface dripping.The pore size of hyaluronic acid hydrogel decreased with the increase of its concentration,showing a tubular network structure.Among them,the critical temperature of hyaluronic acid hydrogel with a concentration of 3 wt% is 37℃,which has shear thinning,injectability and stability.Hyaluronic acid hydrogel with a concentration of 3 wt% can be formed into a highly uniform and transparent thin layer on the ocular surface of rabbits,which can be used to prolong the retention time of miRNA 24-3p-rich exosomes on the ocular surface.In the ophthalmic safety evaluation of the hydrogel,compared with the natural conjunctiva group,the conjunctiva of the hyaluronic acid hydrogel with a concentration of 3 wt% group demonstrated good ophthalmic safety,no multinucleated cell formation,inflammatory cell infiltration and many conjunctival specific goblet cells in its conjunctiva.Based on the biological function of miRNA 24-3p-rich exosomes and the thermosensitive of hyaluronic acid hydrogels,miRNA 24-3p-rich exosomes were loaded into hyaluronic acid hydrogels with a concentration of 3 wt%,and a miRNA 24-3p multistage delivery system was prepared.Exosomes were release by hyaluronic acid hydrogel with a concentration of 3 wt% for more than 21 days,and the miRNA 24-3p-rich exosomes released by hyaluronic acid hydrogel with a concentration of 3 wt% can successfully enter corneal epithelial cells.In the treatment of rabbit corneal alkali burn disease animal model,compared with other groups,the miRNA 24-3p multistage delivery system group accelerated the healing rate of alkali burned corneal epithelium fastest,and the epithelial closure area exceeded 87% after 1 day of treatment.Moreover,after 28 days of treatment of rabbit corneal alkali burn disease animal model,miRNA24-3p multistage delivery system reduced corneal edema,regulated corneal epithelial maturation,inhibited corneal stromal fibrosis and macrophage activation,regulated the microenvironment of alkali burned corneal tissue,and promoted corneal tissue repair.Therefore,the miRNA 24-3p multistage delivery system constructed based on exosomes and thermosensitive hyaluronic acid hydrogels is expected to be used to solve the problem of corneal epithelial wound healing,which provides a new method and idea for the combined treatment of gene therapy and cell-free therapy for the treatment of corneal epithelial wound. |
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