人皮肤角质形成细胞的胰蛋白酶消化分离及无血清培养  

Human Keratinocytes Culture in DMEM-SF Through Two Step Digestion with Trypsin

作　　者：丁锐[1] 沈彤[1] 涂登云[1] 魏凌珍[1] 孙美芳[1] 朱启星[1] 

机构地区：[1]安徽医科大学公共卫生学院毒理中心实验室,合肥230032

出　　处：《安徽医科大学学报》2003年第6期415-418,共4页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金项目 (编号 :0 3 0 43 80 1) ;安徽省教育厅自然科学重点科研项目(编号 :2 0 0 3KJ0 3 6ZD)

摘　　要：目的　探索用DMEM SF培养基对经胰蛋白酶两步消化分离的人角质形成细胞进行培养的方法和条件。方法　取临床整形外科手术后剩余皮肤 ,用 0 2 5 %的胰蛋白酶经冷和温两步消化分离后 ,用DMEM SF完全培养基于 5 %CO2 、37℃培养 ,绘制生长曲线 ,并用单克隆抗角蛋白抗体和鼠 IgG免疫组化试剂盒进行细胞鉴定。结果　用胰蛋白酶两步消化分离的角质形成细胞在DMEM SF培养基中可正常生长 2周以上 ,生长曲线显示细胞在第 4天进入对数生长期 ,第 10天进入停滞期。鉴定显示角质形成细胞占 95 %以上。来源于 3个不同个体的皮肤角质形成细胞经等量混合后培养与单独培养相比 ,细胞生长无统计学差异。结论　用胰蛋白酶对人皮肤进行冷和温两步消化分离的角质形成细胞 ,在DMEM SF完全培养基中生长状况良好 ,其他细胞污染少 ,实验成本低廉 ,操作简单。Objective To explore culture methods and conditions of human keratinocytes isolated by trypsin with DMEM serum free medium. Methods Remnant skins of orthopaedics were used. After digestion with 0 25% trypsin through 2 steps, keratinocytes were collected by centrifugation. Keratinocytes were seeded in pre warmed DMEM SF medium and incubated in a 37℃ incubator containing a humidified atmosphere of 5% CO 2 in air. Growth curve of human kerationcytes was described. Monoclonal Anti Pan Cytokeratin and Mouse IgG were used to identify the cells. Results The growth curve displayed that the cells came into logarithmic growth phase at 4th day, and at 10th the cells came into stagnate phase. Cell identification showed that more than 95% cells were keratinocytes. Compared with the culture from the cells pooled of 3 different clonors. No significant difference was found in the cells when were cultured seperately. Conclusion The cells grow very well in DMEM SF medium and there are few other kinds of cells growing together after the digestion with trypsin. It is cheap and easy to operate.

关 键 词：人皮肤角质形成细胞 胰蛋白酶 无血清培养 细胞培养 细胞鉴定 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]