中文摘要：

目的建立人皮肤角质形成细胞体外无血清、无牛垂体提取物及无饲养细胞的原代培养方法。方法应用胰蛋白酶-EDTA冷温两步消化法消化健康青年包皮皮肤,获取角质形成细胞并置于无血清、无牛垂体提取物、成分确定的角质形成细胞培养基中进行原代培养并传代。通过观察细胞形态和采用免疫组织化学方法鉴定及绘制生长曲线,分析、评价细胞质量。结果该方法可稳定获得高纯度、活性好的角质形成细胞。原代角质形成细胞可稳定增殖2周左右,至少可稳定传代增殖5代。经免疫组织化学方法鉴定,99%以上为角质形成细胞。结论冷温两步消化法及体外无血清原代培养技术可为科研工作提供足量高质量的角质形成细胞,是高效经济的获得人角质形成细胞的方法。

英文摘要：

Objective To establish a method for the primary culture in vitro of human skin keratinocytes in a serum-,bovine pituitary extract-,and feeder cell-free medium.Methods The keratinocytes were isolated from the foreskins of healthy young men by two-step digestion with trypsin-EDTA.The primary culture and passage of keratinocytes was established in the serum-and bovine pituitary extract-free medium.The keratinocytes were identified by morphological observation and immunohistochemistry,and the growth curve was plotted.Results Primary keratinocytes with high purity and activity were obtained in 2 weeks and passaged for at least 5 consecutive generations.The results of immunohistochemistry showed that 99% of the cultured cells were keratinocytes.Conclusion Two-step digestion and primary culture in vitro in a serum-and bovine pituitary extract-free medium is an ideal method to obtain enough karatinocytes for research work.