| In the clinic,it is one of the basic and emergency problems in the surgeon that the skin deficiency caused by all kinds of reasons were treated with suitable method so as to make rapid,effective,supported and good recovery.The tissue engineering give the new way to the artificial skin constructed in vitro.Epidermal stem cell is the enssential cell in the formation of the skin for the construction of the skin tissue engineering and in suitable enviorment,it have the features of multipotential classification including the formation of affiliated organ such as the sweat gland,hair,follicle.Epidermal stem cell is very important seed cell to the construction for the skin tissue engineering but have not the completed report about the continous observation in vitro.Recently,a new kind of biological material, named porcine small intestinal submucosa(SIS), was discovered,which is natural extracellualr matrix without cells structure conducted by the physical and chemical method,and it is similar to the natural skin tissue.Some experts described the characteristic on the SIS and described it was a good extracellullar scaffold with much cellular growth factor such as the TGF,VEGF,PDGF and so on,which is enssential to the recovery and replastication and advantaged to the other cellular matrix.Our study observed the epidermal stem cell about the growth,prolification and classification in order to research the mechanism of the classification of ESC and with the method of the tissue engineering,we discuss the recovery of the skin deficiency treated by the porcine small intestinal submucosa in order to see the change of the epidermal stem cell during the regeneration to provide the experimental datas of the construction of the tissue engineered skin in vitro.Materials and Methods1．Experimental animals and materials:(1),Experimental animals :Wistar rats were randomly provided by the experimental animal department of the china medical university.the porcine jejunium was provided by the shenyang meat manufacturer.(2),Experimental materials :Dulbecco's modified essential medium (DMEM) was purchased from GIBCO/BRL and fetal bovine serum (FBS) was purchased from TDB Biological Production Co.Ltd (Tianjin,China).The trypsin was purchased from DIFCO. Polyclonal rabbit antibody against Integrinβ1, p63 and keratin15 were obtained from the neumaker biological production Co.Ltd.FITC and CY3 were purchased from Wuhan Bosten biological production Co.Ltd (Hubei,China),PBS2．Experimental method :(1) The isolation,culture and the evaluation of ESC of the wistarratsWith heat-cool alternative method and by way of the trysin digestion to the skin tissue mass , the ESC , obtained from skin of wistar rats,were collected and observed continuously under the invert microscope in vitro and isolated by the poly-tubline.the ESC was observed through the lighting,immunohistochemical integrinβ1,keratin 15and p63 and fluorescence staining signal by the FITC and CY3 and scanning electron microscope。(2) The observation of the biological characteristic of the porcine smallintestinal submucosaWith the physical and chemical method ,the SIS was made and observed by the lighting and analysis of the imagination,and evaluated through it emboyed under the skin for the biological compatability.3．The observation of the position and the relocation of the ESC at theedge of the wound repaired by the SISRandomly forty wistar rats were selected and two deficiency whosecolume is 2．54cm~2 were made respectively on the two side of the vertebral colume.The rats were divided into two groups,the experiment SIS (40 wounds)and the blank (wounds).respectively three days,one week,two weeks,and three weeks after operation, the healing result of every group were observed witn the histological method and theImmunohistochemical integrinβ1,keratin 15and p63 staining signal by the FITC and CY3 so as to make acknowledge to the position and relocation of the ESC at the course of the healing of of the wound.Result1．Growth of the ESC and histological observation24 hours later from submerging the digested skin tissue mass in the DMEM,the ESC started to crawl out of the skin mass and A large amount of ESC,whose appearance were round, stepped out of the skin tissue mass with different figures. In cultured three weeks later, the cellular confluence node could been seen,Subgenerated cell is similar to the original cell,after 4-5 days the bottle could be filled up. Observed continuously two weeks,the hair-like structure appeared around the cell confluence largely.The ESC grow with the colonely and CFE is obviously higher than the BMSCS,p<0．05 ,statisticly.Maker of the fluorescence of ESC is correspond to the result of the Immunohistochemical integrinβ1,Keratin15and P63 staining.2．Histological observation of the porcine small intestinal submucosaThe HE staining shows that the spongy and the density of the SIS gradually deepen inside ,the incident of the foraman flew between the 70%-90%, the diameter of the foramen flew around 250um。The collagen are very regulate in the frame with abundent foramen.The result of the emboyed under the subdermic explain the good compatibility without obvious inflammation reaction,advantage of the vesselization and slow degeneration in vivo.At last period ,the SIS could form the dermic-liked structure regulately in the fiber frame.3．The position and the relocation of the ESC at the edge of the woundrepaired by the SISIn two group ,the SIS group healing incident is clearly higher than the blank group.The integrinβ1,keratin 15 and p63 positive cell are promptly out-sticking around the edge of the annualr and spinous layer of the dermal.the more adjacent to the wound,the more were the positive cell.and the amount of the ESC increased with the contract of the wound of the skin.Conclusion1．The ESC amount is not only larger with this method, heat-coolalternative method and by way of the trysin digestion, than other way but easily differenciated and primarily isolated and cultured the hair in vitro ,which make it compatible to construct the tissue engineering skin.2．SIS we made with the method has special characteristic which isobvious frame,much elastic and good biological compactibility with perfect vesselization,which is a terrific cellular scaffold and good material for the healing of the tissue.3．SIS could stimulate ESC to promote the healing of the wound of the skin and the relocation of the ESC at the edge of the wound have the intimate realation with the supplement of he skin and the dermal epithelium...
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