Primary Study On Application Of Epidermal Stem Cells In Tissue-engineered Skin Construction

Objective:Type Ⅳcollagen adhesion method has been used to select epidermal stem cells(ESC) that are then cultured and evaluated for being used as seed cells to be transplanted into composite chitosan tissue-engineered skin so as to cater an ideal experiment model for studying differentiation potential and differentiative sign changes of ESC. In the meantime, we have managed to find out the tissue-engineered skin with stronger biological performance in order to provide biological repair substance with better biological performance for clinical application. Green fluorescent protein(GFP) has been used to mark the seed cells by means of gene transfer technique, as makes a foundation for further study of life trace of tissue-engineered skin seed cells and for further modification of seed cells. Methods:The ESC digested and differentiated by enzyme were selected by means of type Ⅳcollagen adhesion and cultured in keratinocyte serum free medium(K-SFM) to observe cell growth status and cloning formation.ESC relative specific labeling molecule β1 integrin and monoclonal antibody CK19 were selected and evaluated by immunohistochemistry. Evaluation of the melanocytes(MC) in the selected cells and the tissue-engineered skin using the selected cells as seed cells: specific expression of HMB45 and S-100 was first chosen as marker, and then, the existence and content of MC evaluated by using immunohistochemistry.The cultured seed cells were transfected with GFP respectively for observing success rate of transfection. Thereafter, the transfected cells were used as seed cells to construct tissue-engineered skin and the expression of GFP investigated during culture of tissue-engineered skins. Tissue structure specificity of the tissue-engineered skin using the selected ESC as seed cells were compared with which using original cells as seed cells. Results:1. Extracellular matrix could preliminarily select ESC that formed significant cloning during culturing in vitro. In 15 minutes group, positive stain accounted for 28.50% in β1 integrin and CK19 respectively.2. The cells selected by fast adhesion method contained many MC and could continue to grow and proliferate in the K-SFM culture medium. After proliferative culture for 14 days, Lots of MC could still be seen in the constructed tissue-engineered skin. 3.The seed keratinocytes and fibroblasts were successfully transfected with GFP, with positive rate of 28.40% and 65.50% respectively. Expression of GFP still could be seen in the constructed tissue-engineered skin.4.Compared with the controlled skin, the constructed tissue-engineered skin using selected ESC grew better, with deeper color of epidermis,which organization were more similar with normal epidermis. Conclusion:1.Preliminary selection of ESC can be done by using type Ⅳcollagen adhesion method,which can get high proportion of epidermal seed cells that have good proliferation ability. 2.MCs in sorted cells can produce normal pigmentation after transplantation with the tissue engineered skin. 3.GFP can track the skin seed cells well, which is an effective mean to study the revolution of those cells after transplantation.4.Tissue engineered skin constructed wirh the sorted cells is closer to the normal skin, which pigmentation is thicker and epidermal structure is better. This project has offered reliable experimental evidence for clinic application of the sorted cells in tissue engineered skin.