| In clinic oral and maxillofacial cancers always happen on the tongue,lip mucosa and salivary glands,but skin cancers on the facial skin are also common diseases.The resection of tumor or skin trauma can cause skin defects.When the skin is injuried deeply,it is always healed by contraction with re-epithelization from the edges and forming extensive scar,which results in limited joint movements and cosmetic defects[1].Currently the conventional method to treat skin injury is to transplate skin from other parts of body skin.This method has a limitation,specially for a large area of skin injury.In tissue engineering field,scientists combine scaffolds,cells and biomolecular signals to create dermal-epidermal composites or skin equivalents,which have been used to treat skin defects resulting from injury,burn or surgery.But this skin equivalents have been not applied for a long time in clinic application because of lack of dermal tissues,subcutaneous tissues and skin appendages.Regenerative medicine aims to create a living,functional tissues which can repair or replace damaged or lost organ resulted from injury,disease,aging or congenital defect.Skin is the largest organ for human body with simple structure and function,so skin regeneration is widely studied in regenerative medicine field.Our group have reconstituted human skin with functional skin appendages,such as hair follicles,sebaceous and sweat glands.The human reconstructed skin asssay not only provides the foundation for studying regenerative medical mechanisms,but also is important for clinical application.In summary,this study aims to improve regeneration efficiency of human skin by optimizing the procedure of isolation of keratinocytes from skin tissues,improving the methods of culturing keratinocytes and dermal fibroblasts before grafting in vitro.Finally we have successfully applied the regenerated human skin model to study the process of skin wound healing.PART ONE:Optimize the method to isolate keratinocytes from human skin tissues to facilitate the human skin regenerationTo obtain a sufficient number of keratinocytes and dermal fibroblasts are the first step for human skin regeneration.The conventional method of primary cells from skin involves a two-step enzymatic digestion.But this method is usually limited by the type of skin specimen.In this study,we developed a new simple,safe and high efficient procurement of cells from human skin,and explore the related mechanisms.1.Y27632 had a different influence on keratinocytes and dermal fibroblasts.Y27632 could promote the growth of primary keratinocytes,but inhibit the growth of primary dermal fibroblasts.And we found in the presence of Y27632 less primary dermal fibroblasts can attach to the cultural dish and exhibit abnormal morphology.2.Y27632 inhibited dermal fibroblasts attach to cell cultural dish.We seeded the same number of dermal fibroblasts to the normal cultural dishes and non-tissue cultural petri dishes.We found that in the presence of Y27632 "attached"dermal fibroblasts were easily uplifted by gentle mechanic force compared to those cells in the absence of Y27632.3.The mechanisms of different influence of Y27632 on keratinocytes and dermal fibroblasts.When we treated keratinocytes and dermal fibroblasts with Y27632,we found Y27632 significantly downregulated the expression of adhesion protein,not.altering the expression of integrin molecules in dermal fibroblasts by RT-PCR,but for keratinocytes Y27632 could enhance the expression of integrin molecules,not affect significantly the expression of adhesion protein by RT-PCR.These conclusions were confirmed by immunofluorescence staining:Y27632 mainly upregulated the expression of a6-integrin for keratinocytes and significantly downregulated the expression of vinclin for dermal fibroblasts.In summary,we could separate keratinocytes and dermal fibroblasts from the mixture of the two cells by adding Y27632.4.Combining Y27632 and G-medium,we created a new method to efficiently isolate high potential keratinocytes from skin tissues.G-medium was found to be the best one to enhance isolation efficiency of keratinocytes by testing five different medium in the same condition.New method by combining Y27632 and G-medium could get more keratinocytes than traditional method in a certain period.When we mixed these keratinocytes and dermal fibroblasts,grafted the mixture on immunodeficient mice,these cells were able to regenerate normal differented human skin.PART TWO:Enhance the efficiency of human skin regeneration by improving the cell culture conditions of keratinocytes and dermal fibroblasts before graftingOur group have successfully regenerated human skin on immumodeficient mice using fetal tissue keratinocytes and fetal scalp tissue dermal cells,but regenerating human skin by adult tissue cells is still a great challenge.In this part we mainly focused on improving the cultural condition of cells prepared for grafting to enhance the regeneration efficiency.1.Keratinocytes cell sheet combining dissociated dermal fibroblasts cultured under 2D attach condition replaced the traditional method to do grafts.Twenty mice were grafted using this method.We found that after 3 months nine mice could regenerate human skin on dorsal skin,six mice having a big size skin with lots of hair.HE histology showed regenerated skins were composed of epidermis,dermis and subcutaneous tissues.Epidermal cells and dermal cells in regenerated skin were mainly origin of human cells by human specific antibody Human Nuclei.2.Keratinocytes cell sheet combining dermal fibroblasts aggregations cultured under 3D suspension condition replaced the traditional method to do grafts.Forty mice were grafted using this method.We found that after 3 months thirty-one mice could regenerate human skin on dorsal skin,twenty-one mice having a big size skin with lots of hair.HE histology showed regenerated skins were composed of epidermis,dermis and subcutaneous tissues.Epidermal cells and dermal cells in regenerated skin were mainly origin of human cells by human specific antibody human nuclei.3.The comparison of the above two methods.By comparison,we found combining keratinocytes cell sheet and dermal fibroblasts aggregations could much efficiently regenerate human skin and form more human hair shafts.The Wnt5a and Versican expression of dermal fibroblasts aggregations cultured under 3D suspension condition was significantly higher than those of dissociated dermal fibroblasts cultured under 2D attach condition.These data suggested that the trichogetic potential ability of dermal fibroblasts aggregations was significantly higher than that of attached dermal fibroblasts and combining keratinocytes cell sheet and dermal fibroblasts aggregations could much efficiently regenerate human skin with hair shafts.PART THREE:Explore one of the application of hRSK model:Establish a hRSK wound healing modelStudying human skin wound healing process,specially dermis remodeling and scar formation has a great significance on clinical therapy.But currently there is still no an ideal animal model to study skin wound healing.The skin structure and wound healing process of mice skin are different from those of human skin,so to some extent the application of mice mould is limited.Therefore,the establishment of a good human skin wound model is important to study wound healing process and improve therapy program in clinic.1.The establishment of human skin wound healing.A 2mm-wide full-thickness wound was created at a 3month-old hRSK and an identical control wound was created on host mice skin.Host mice wounded skin showed complete re-epithelialization at 1 week after injury.But for hRSK,complete re-epithelialization occurred at 2 weeks after injury.At 8 weeks the neo-epidermis of wounded hRSK was stratified and more matrix in dermis was present.Dermal remodeling was still ongoing at this period,which is never reported at other models.2.This part was to test the differentiation status of neo-epidermis.By performing immunofluorescence staining of epidermis differentiation markers(K5,K10,loricrin),they showed the healing neo-epidermis underwent a normal differentiation program.These data further confirmed the wounded hRSK could heal normally.3.This part was to test whether the reparative cells were of human origin.By performing immunofluorescence staining of human-specific pan-cytokeratin and vimentin antibody,we found that the reparative epithelial cells were human and the reparative dermal cells also consisted of human cells.These data were further confirmed by the staining of human nuclei antibody.And immune cells of mouse origin also existed in reparative dermis.These data showed the reparative epidermal and dermal cells were mainly of human origin.4.This model could supply a good in vivo platform to study mechanisms of wound healing.We generated an either green epidermis or green dermis skin by using GFP-labelled epidermal or dermal cells.The wound healing process of skin regenerated by GFP-labeled cells was same to that when we used unlabeled cells.By using immunofluorescence analysis of GFP antibody,we found the reparative cells were of GFP-positive cells origin. |
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