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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。
Objective To establish a model of primary culture and continuous culture of keratinocytes and fibroblasts in human skin and provide sufficient seed cells for tissue engineered skin. Methods The prepuce of 5 ~ 15-year-old children were used as material. After digestion with trypsin, the two kinds of cells were separated and cultured in K SFM and DMEM (10% serum) Formed cells, 10 ~ 40 generations of fibroblasts) were observed with an inverted microscope for general observation, transmission electron microscopy ultrastructure observation, and take pictures recorded at the same time immunocytochemistry identification. Results After digestion, a large number of cells were obtained and passaged after 2 weeks of culture. Keratinocytes were typical paving stone under light microscope and polygonal under transmission electron microscope, which was consistent with keratinocyte characteristics. Fibroblasts were spindle-shaped and radial Or spiral arrangement, electron microscopy confirmed that fibroblasts. Immunocytochemistry detection: keratinocyte anti-human keratin (AE1 / AE3) monoclonal antibody (mouseanti cytokeratin) staining; anti-fibroblasts type Ⅰ, type Ⅲ collagen monoclonal antibody (mouseanti TypeⅠ, Mouse anti vimentin positive staining. Conclusion Separation, culturing and passage of keratinocytes and fibroblasts in vitro with a simple, convenient and economical method can simultaneously obtain a sufficient number of seed cells of tissue engineered skin.