Studies On Retaining Properties Of Stem Cells In The Process Of Epidermal Cell Culture

studies on Retaining Properties of stem cens in the Process ofEpidermal cen cultureSince the cultUre method ofepidermal cells in vbo was fOunded in l975, this tecboque hasbeen developed significanly and used in skin tissue engineering widely However, poorproliferative potential of epidermal cells obtained from cultUre in vbo limitS itS amer use. MoStcells in cultUred sheet in vitrO had differentiated and lost most proliferaive ability Proliferativepotential of cultUre epidermal cells depends on the percentage and biological state of Stem cells incultUre cells. Therefore, the key to obtaln a bioengineering sldn in vbo deStined to the coverageof massive fiJll-thickness burn wound is how to preserve high proliferative potential of epidermalceI1s, that is to say characteristics of stem cells. So we should direCtly demonStrate the presenceand evaluaion of the percentage of Stem cells durin cultivation. Epidermal stem cells can beidentified by remarkers on cell surfaCe. But no solo of present remarkers can distingUish stemcells from tranSit amplifying cells. Although several indicators such as vitality grOwth curve andcolony forming efficiency have been used. the whversally accePted criteria for epidermal Stemcells is absent. On research of controlling stem cell fate, recent StUdy reported that high integrinp l (ITG Pl, CD29 )exPression and adhesiveness were essential for maintaining epidermal cells inthe Stem cell compartInent. However, whether high integrin 0l exPression can dedifferenateePidermal cell still remains unclearThs research aiIned at establishing sbople, feasible and inexPensive method of epidermalM cell determinallon by exPression of integrin Pl sutal in sbo of ceIls and Prolifhative' cell nucleus antigen reCNA) using flow cytOmetry On basis of this research, we will improveculthe technique of epidermal cell in order to maintain the proliferatve potential and controldifferentiation of Stem cell in cultUre. Brieny, we will oPtimbe the condition of separaion(working concentration and digestive time of different enZyInes) and cultUre (concentration ofadditives such as epidermal cell growth factr, insulin, cholera toxin and hydroconisone) tOdriprove percentage of Stem ceIIs in epidermal cells. Thery a recombinant adenovch vectorcontalning human integhn Pl subunit will be consbocted and mfected into human primnyepidermal cells tO inveedgate effeCts on their biolOgical State.ElPeriment oue EstablishInent of a method to identify epidermal stem cellsObjectives: To establish a simple and feasible method to identify epidermal stem ce11s.Methods: Epidermal cells isolated from adult human fOreskin were enriched fOr epidermalstem cells with adherence aPproach for different time (l0 min or 10 h). The sorted cells weretested for colonogenic ability (colony-forming efficiency CFE). Doub1e staining fOr CD29 andPW and single staining of bromodeoxyUridine perdU) were perfOrmed on cells for fiowcytometry and inununocytochemistry respechvely The above mentioned indices were analyZed' for correlation.ReSultS:(l) Approalmate1y 9.84% of epidermal ce1ls isolated adhered in 10 min. After cultUred for 7d, the adherent cells fOrmed lmp colonies (cell nurnber more than l00) with a CFE of 28.75%.Flow cytOmetIy showed that the percentage of CD29-positive rpCNA inegative (CD29+NCNA--)ceIls was 67.33%, and BrdU- positive rerdU+)ceIls accounted for 58.57%. Less of freshlyisolated cells (2.95 t l .23 %) showed a S cell cycle profile.(2) About 52.8% of epidermal cells isolated adhered in l0 h. After culturd for 7 d. theadherent cells fOrmed small colonies (ce1l number less than 50) with a CFE of 7.82%. FlowcytOmeny showed that the percentage of CD29+/PCNA-- cells was l4.29%, and BrdU+ cellsWd fOr 9.84%. More cel1s (16.65 l6.24o/o) showed a S cell cyc1e profile.(3) Correlation analysis showed that the PerCentag of CD29+lPCNA--cells corrlated wellwith bOth Brdtr cells and cell cycle profile.Conclusi...