The Role Of The Induced Pluripotent Stem Cell-derived Keratinocytes Mediated Skin Wound Healing

Skin is the body from dehydration,injury and infection of the first line of defense,is to maintain the stability of the internal environment and resistance micro-organisms,chemical substances such as the invasion of the barrier[1].Skin tissue trauma repair and tissue regeneration is a plastic surgery and trauma surgery in the common problems and challenges,clinical commonly used repair methods there are many shortcomings,such as:Treatment for a long time,resulting in additional damage to the area,a large area of burn patients lack sufficient self-skin and so on.Stem cell research and its basis for regenerative medicine,especially iPS cell technology and its own superiority for the repair of the wound provides a new way and hope.But because of iPS cell technology has only recently eight years,it is applied to the study of skin trauma is still a new field.Current studies have shown that iPS cells can effectively differentiate into keratinocytes under the action of inducers,but their effects on skin wound healing have not been reported.Therefore,this study intends to observe the role of iPS cell-derived keratinocyte transplantation in the treatment of skin trauma in mice,and to reveal the mechanism of its role.Thereby deepening the iPS cell technology used in skin trauma research.Part one Effects of miPSc-derived keratinocytes(miPSc-KCs)transplantation on skin healing in mice(A)PurposeThe effect of miPSc-KC transplantation on skin wound healing in mice was investigated at the level of vector(in vivo).(B)Methods1.miPSc differentiation into keratinocytes(KC)was induced by differentiation medium containing retinoic acid(RA)and bone morphogenetic protein 4(BMP4).The protein expression of K14 and P63 in miPSc-KC was detected by immunofluorescence staining.2.The model of C57BL/6 mice was established by surgical resection,Were divided into two groups,PBS or miPSc-KCs were injected into the mouse wound around the PBS group and miPSc-KCs group.3.at different time points(postoperative 3,5,7 and 14 days)drawn,statistical wound healing situation.(HE)staining,Masson trichrome staining and immunohistochemical staining were performed to observe the wound epithelization,dermal remodeling and collagen accumulation,appendage development and so on.Compared.4.miPSc-KCs labeled with green fluorescent dye CSFE were injected into the mouse skin trauma for four weeks.The transplantation of miPSc-KCs in the wound was observed on the 14th day after operation.(C)Results1.miPSc-KCs express the normal keratinocytes of the marker protein,2.the successful establishment of the mouse back full-thickness skin trauma model,the normal survival of mice,no systemic adverse reactions and serious infection.3.The skin area of mice in miPSc-KCs group was significantly lower than that in PBS group.4.HE staining,Masson trichrome staining and immunohistochemical staining showed that the skin epithelium of miPSc-KCs group was significantly higher than that of PBS group.The scar area ratio of miPSc-KCs group PBS group was significantly decreased;miPSc-KCs group of mice after the skin wound healing device than the PBS group significantly increased.5.On the 14th day after operation,the green fluorescent expression was observed in the epithelium of the wound.(D)Conclusion1.miPSc was successfully induced into keratinocytes.2.miPSc-KCs transplantation can migrate to the mouse skin wound,participate in and promote re-epithelialization,thereby promoting wound healing.3.miPSc-KCs transplantation can promote the formation of skin appendages after skin healing in mice,and inhibit scar formation.Part two Effects of miPSc-KCs conditioned medium(miPSc-KCs-CM)on the expression of collagen in mouse fibroblasts(A)PurposeThe effect of miPSc-KCs-CM on the expression of collagen in mouse fibroblasts was investigated at the level of in vitro.(B)Methods1.miPSc-KCs-CM was harvested after 48 hours of culture of miPSc-KCs.MiPSc-KCs-CM or KBM medium were used to culture mouse dermal fibroblasts.After 48 h,the expression of collagen? and collagen? mRNA in miPSc-KCs-CM group and control group were detected by real time-PCR.2.ELISA method to detect the concentration of TNF-? in miPSc-KCs-CM collected at different time points.3.TNF-? neutralizing antibody was added to miPSc-KCs-CM,and mouse skin fibroblasts were cultured for 48 hours.The expression level of collagen? and collagen? mRNA in mouse skin fibroblasts was determined by real time-PCR.4.miPSc-KCs-CM,miPSc-KCs-CM+TNF-? neutralizing antibody and KBM medium were used to culture mouse dermal fibroblasts for 48 hours to extract fibroblast mRNA,real time-PCR The expression levels of collagen??collagen?mRNA in mouse skin fibroblasts were determined.(C)The results1.miPSc-KCs-CM significantly reduced the expression of collagen? and collagen?mRNA in mouse skin fibroblasts.2.The expression of TNF-? in miPSc-KCs-CM reached the highest value at 24 hours.3.TNF-? neutralizing antibody partially compensates the inhibitory effect of miPSc-KCs-CM on the expression of collagen? and collagen? mRNA in fibroblasts.(D)Conclusion1.miPSc-KCs-CM can inhibit the expression of collagen?,collagen?.2.miPSc-KCs-CM contains TNF-? and plays a role in inhibiting the expression of collagen?,collagen?.