不同代次角质形成细胞构建组织工程皮肤的比较分析-comparative analysis of different generations of keratinocytes constructing tissue engineering skin.docxVIP

Comparative Study on tissue-engineered skin reformed by different passages of keratinocytesAbstractThe skin is the first defense barrier of human body, and the most vulnerable to a variety of injuries resulting in skin defects.The manufacture of tissue engineering skin products will provide the new method for the treatment of skin damage. Constructing epidermal structure by keratinocytes can provide favourable physical barrier for the outer layer of the skin. However, keratinocytes cultured in-vitro are easy to appear the differentiation, limitedly amplification ability and long cultivation cycle. Studies on the proliferation and differentiation of keratinocytes and its passage cells onto the tissue-engineered skin will provide a theory basis in order to find better seed cells.Objective：In the three-dimensional culture condition, the ability of proliferation and differentiation of the different passages keratinocytes will be compared on the surface of dermal equivalent;To observe the effect of exogenou type Ⅳ collagen on the reformation of basemembrane and the change of epidermal morphology;To understand the change of keratin and flaggrin in the tissue-engineered skin reconstituted by different passages keratinocytes.Method:The keratinocytes were obtained from young foreskin after circumcision digested by enzyme, were selected by the quickly adhesion of type Ⅳ collagen, then cultured and passaged in keratinocyte serum free medium. The growth of 1-to5-passage keratinocytes in the two-dimensional condition was observed under an inverted microscope. The tissue-engineered skin was reconstructed by these passages of keratinocytes. Two groups were divided, including with type Ⅳ collagen group and without the exogenetic type Ⅳ collagengroup between the epidermal and the dermal equivalent. The morphology of the tissue-engineered skin and the reformation of basement membrane were observed by electronmicroscopy, HE and PAS staining.The 2nd passage of ker