Preparation Of Exosomes Derived From Myoblast And Its Effect In The Treatment Of Deep Second Degree Scald

Clinical treatment effects of large areas of burns are far from people's expectations.The scars left after the burns treatment also affect the patient's mental health.Therefore,it is very urgent to develop a more effective treatment for burns.Cells can secrete a30-150 nm vesicle with a double-membrane structure called exosomes,which plays an important role in cells communication and immune response,and its repair function for tissues also received extensive attentions in recent years.Compared with exosomes derived from stem cells,exosomes derived from myoblasts have a higher yield and more stable,and can meet clinical needs better.Therefore,this research selected myoblast derived exosomes to treat burns,and studied its treatment effect and mechanism in vitro and in vivo.The main research contents of this article are as follows:Firstly,We established heat shock mouse fibroblast injury model in vitro and found that exosomes derived from C2C12(C2C12-Ex)had significant proliferative and anti-apoptotic effects on the cells of this model.We extractd of exosomes by ultracentrifugation from the conditioned medium of differentiated myoblast C2C12.And the yield of exosomes was 2 mg exosomes protein per 1000 mL conditioned medium quantified by BCA kits.The particle size of the exosome obtained by TEM and DLS was about 100 nm.Western blot results showed that the extracted product contains a large number of exosomes marker CD63 and TSG101.All these results show that the differentiated myoblast-derived exosomes can be successfully obtained by ultracentrifugation.Secondly,We established the mouse fibroblast injury model in vitro and evaluated the treatment effect of C2C12-Ex.Stimulate mouse fibroblasts 3T3-L1 or L929 at 40?,43?or 46? for 40 min,simultaneous detection the cell numbers at different time,and we finally selected 43 °C 40 min for the next research.Under the selected conditions,cell counting results indicated that C2C12-Ex had a significant proliferative effect on heat-damaged fibroblasts 3T3-L1 and L929.Western blot results showed that C2C12-Ex promoted the expression of proliferating protein PCNA and inhibitory apoptotic proteinBcl-1.Thirdly,We established a deep second degree burns model for SD rats and studied the therapeutic effect of C2C12-Ex on this model and its possible mechanism.Experiments found that C2C12-Ex can significantly promote wound healing in rats and is dose-dependent.At different time,stripped wound tissue and analysis the expression of keratinocyte surface markers CK19,proliferating nuclear antigen PCNA and neovascular marker CD31,found that C2C12-Ex can accelerate the regeneration of the epidermis more effectively,also promoted the regeneration of cells and blood vessels in advance.Real-time quantitative PCR results about collagen ? and collagen ? revealed that C2C12-Ex can optimize the distribution of collagen in the skin and reduce the formation of scars during wound healing.In summary,all our results provided experimental basis for the clinical application of the exosomes derived from myoblasts C2C12-Ex in the treatment of deep second degree scald.