Effect Of CTGF On The Mechanism Of Scarless Wound Healing In Human Fetal Skin

Background:In 1971,scholars first discovered the phenomenon of no scar formation after human fetal trauma,and put forward the concept of"scarless wound healing".Worldwide,there has been a wave of research into the mechanism of scarless wound healing,and the hope is to replicate the process in humans to avoid scar formation by studying its specific mechanisms.Connective tissue growth factor?CTGF?in recent years as a more and more important profibrotic factor protein,mainly by fibroblasts,hepatic stellate cells,vascular smooth muscle cells and interstitial cells synthesize and secrete.It is rich in cysteine and is secreted in physiological state.Studies have shown that it has a strong and specific role in promoting fibrosis and is considered to be a direct downstream effector factor of TGF-?1.But the research about CTGF is confined to the effect in extracellular matrix and the role of the expression level in the disease tissues,there have been few reports on CTGF scarless healing in embryonic skin,while CTGF's effect in the mechanism of scarless wound healing is still unclear.Objective:To investigate the mechanism of CTGF in the scarless wound healing of human fetal skin,and through the establishment of human embryonic scarless wound healing model,the expression level of CTGF in the model was observed,and the relationship between the expression and the scar free healing of the embryo was determined.At the same time,to observe the influence of CTGF expression in human fetal skin scarless wound healing and provide experimental basis for the elucidation of CTGF in fetal scarless healing mechanism,to provide new ideas for the study of scar.Methods:20 nude mice were randomly divided into two groups:human fetal scarless healing model group and modified scar animal model group,10 mice in each group.Fetal skin of20²4w and human full thickness skin grafts were transplanted subcutaneously into the back of the nude mice.After the graft was alive,a full-thickness incision was performed to make the wound surface.The grafts were taken from two groups of nude mice before and1^st,3^rd,7^th,14^th,120^th days after the wound.The degree of fibrosis samples were detected by picrosirius polarization method,and the application of image analysis technique and SABC immunohistochemical method to detect CTGF protein expression in tissue samples.The differences of hyaluronic acid?HA?and homeobox genes between the 2 groups were compared.In vitro culture of human fetal scarless healing tissue fibroblasts,were randomly divided into ASODN treatment group?AT?,liposome control group?LC?and control group?C?.In group AT,ASODN was used for full phosphorothioate modification,FITC was fluorescently labeled and transfected by DOSPER liposome,The LC group was treated with CTGF free DOSPER liposomes,whereas the C group did not do any ASODN treatment.The phase distribution characteristics of CTGF and ASODN in 3 groups of cells were observed by fluorescence microscope.The synthesis of collagen was detected by H3-proline incorporation method,and the proliferative activity was detected by MTT colorimetric assay.The expression level of CTGF m RNA and the expression level of CTGF protein were detected by RT-PCR and immunocytochemical staining SABC assay.Results:1.All rats in the two groups were alive.In human fetal scarless healing model group,120d after the wound was made,there was no shrinkage of the skin,the surface was rosy and the hair was growing.The volume was not reduced.There was no significant difference between the grafts and the surrounding normal skin under the microscope.In modified scar animal model group,120 days after the trauma of manufacturing,the visible part of the scar becomes soft,with varying degrees of contracture,and was significantly higher than that of leather,microscopic observation into the dermis thickening and dermal papillary layer and reticular layer is unclear,collagen fiber dense,disordered vortex or nodular arrangement,microvessels and mitotic cells increased,no significant difference with adult hypertrophic scar tissue.2.Human fetal scarless wound healing model group type III collagen fibers were widely distributed,type I collagen fibers decreased,CTGF protein content and positive particles distribution density was low,the content of HA and its receptor CD44 expression level significantly increased significantly,developmental biology related homeobox genes?PRX-2,HOXB13,HOX2.2 and HOX2.3?were expressed in the positive the epidermis and dermis.In the modified scar model group,type I collagen fibers were widely distributed in the dermis,CTGF protein content was higher,the positive particle distribution density was high,the content of HA was significant,and the expression level of receptor CD44 was low.3.After transfection of HSF cells,CTGF ASODN first enters the cytoplasm and then transfers to the nucleus and finally disappears.After transfection,the proliferation ability of HSF cells and the rate of H3 proline incorporation decreased,and the expression level of CTGF protein and the expression of m RNA were down regulated.Conclusions:1.After transplanting the skin of the pregnant 20²4w human fetus back to the back of the nude mouse,the skin can survive and maintain the basic characteristics of the growth and development of human fetal skin.At the same time,after the grafts were wound,histological observation was performed.The wound did not scar until 120d after healing.The model can be used as an ideal model for the study of scarless wound healing in human fetal skin.2.In human fetal skin,the expression of CTGF in scar tissue is lower than that in adult skin,This suggests that in the process of healing,CTGF m RNA is a high level of transcription and translation,the CTGF protein stimulates scar tissue Fb value and ECM?by the collagen deposition?,extensive fibrosis and scar tissue and hyperplasia.3.The human fetal skin scarless healing tissue expression of HA and CD44 levels are higher,the combination of the complex formation of HA-CD44 make Fb to trigger the synthesis of HA,change cytoskeleton behavior in fetal scarless wound healing,and plays an important role.4.Developmental biology related homeobox genes?PRX-2,HOXB13,HOX2.2,and HOX2.3?are positively expressed in epidermis and dermis,in the process of adul t scar healing,only PRX-2 and HOXB13 were expressed in epidermis,and only HOXB13 was expressed in dermis.The difference in expression is related to the different processes of healing after trauma.5.CTGF can promote the synthesis of collagen and proliferation of Fb in scar healing.CTGF ASODN can significantly reduce the CTGF and m RNA in HSF,and inhibit the expression of CTGF protein,Thereby reducing collagen synthesis in HSF cells.Therefore,CTGF ASODN can be an effective way to treat scar healing by inhibiting the expression of CTGF.