【摘要】 目的　建立人皮肤角质形成细胞 (keratinocyte)和成纤维细胞 (fibroblast)原代培养和体外连续培养的模型 ,为组织工程化皮肤提供充足的种子细胞。方法　以 5～ 15岁小儿包皮为材料 ,胰蛋白酶消化后 ,分离两种细胞 ,分别用无血清表皮培养液 (K SFM)、DMEM (10 %血清 )培养 ,对年轻细胞 (2～ 4代角质形成细胞 ,10～ 4 0代成纤维细胞 )均用倒置显微镜进行大体观察 ,透射电镜进行超微结构观察 ,并拍照记录 ,同时进行免疫细胞化学鉴定。结果　消化后得到大量细胞 ,在培养 2周后开始传代 ,光镜下角质形成细胞呈典型铺路石状 ,透射电镜下呈多边形 ,符合角质形成细胞特征 ;成纤维细胞光镜下呈梭形 ,放射状或漩涡状排列 ,电镜证实是成纤维细胞。免疫细胞化学检测 :角质形成细胞鼠抗人角蛋白 (AE1/AE3)单克隆抗体 (mouseanti cytokeratin)染色阳性 ;成纤维细胞鼠抗人Ⅰ型 ,Ⅲ型胶原单克隆抗体 (mouseanti TypeⅠ ,ⅢCollagen)和鼠抗人波形蛋白单克隆抗体 (mouseanti vi mentin)染色阳性。结论　用简便易行 ,经济实用的方法在体外对角质形成细胞和成纤维细胞进行分离、培养和传代 ,能同时得到足够数量的组织工程皮肤的种子细胞。更多还原

【Abstract】 Objective To establish a method of culturing human keratinocytes and fibroblasts so as to provide sufficient seeding cells for skin tissue engineering.Methods Keratinocytes and fibroblasts were dissociated and harvested by trypsin which isolated from the normal prepuce by circumcision (5～15 years old).The performance of cells’ growth were observed with light microscope and transmission electron microscope.The cells’ certain antigens were detected with immunohistochemical method.Results Histologically, the keratinocytes showed positive staining in cytokeratin antibody.The antigens of collagen of typeⅠ,typeⅢ and vimentin were detected positive in fibroblasts.Conclusion Simply and suitably, this method can supply tissue engineering skin with enough seeding cells.更多还原