Basic Biological Investigation After Sorting Epidermal Stem Cells On Basis Of Adhering To Type Ⅳ Collagen

Advances in tissue engineering of skin are needed for clinical applications (as in wound healing and gene therapy) for cutaneous and systemic diseases. Seed cells are a key factor. Studies of tissue-engineered skin have shown that epidermal stem cells may provide a superior source of multipotent stem cells for tissue engineering .So, how to sort ESC quickly becomes a prevailing topic. In our study, we sort the ESC according to the difference which KC adhere to collagen IV with different time and investigate their biological characteristics, and provide laboratory evidence.The isolated human's foreskin keratinocytes were inoculated on the six-well dish coated I,IV collagen, after incubated 10,20,30,60min in 370C CO₂ incubation-box, removed the non-adhering cells ,measured the adhering force of four time by centrifuging, counted the cells and measured the size of diameter with a CASY at 10,20,30,60min ,and measured the expression of integrin-β1 with FCM, identified with integrin-β1,involurin,CK10 and CK19 by immunofluorescence and immunocytochemical. Morphological observation was carried out with the aid of contrast phase microscope. Our results indicated that with the elapse of time, more and more cells adhered, and the numbers of those adhering within 20 min was significantly larger than those within 10min, (p<0.05). CFE in 20min is significance than 30,60min, no significance to 10min. Adhering force is significance within 10min than those within 20,30,60min, no significance between 20 and 30min, and there is no significance between IV collagen and I collagen also. Morphologically the epidermal stem cells were brighter,smaller rounder with high nuclear to cytoplasm ratio after the adhesion. all adhered cells were positive to integrant-β1,some adhered cells positive to CK19, all negative to involurin, little parts positive to CK10, and FCM indicated that the cells were 100% positive to integrin-β1 in four time. Conclusion: keratinocytes adhering to IV collagen within 20min have the characteristics of stem cell, and may be used for the further investigation of the ESC.