胰蛋白酶差速脱壁法分离纯化人表皮干细胞

Isolation and purification of human epidermal stem cells by differential detachment with trypsin

背景:表皮干细胞对于修复皮肤缺损具有重要意义,但如何方便、快捷地对所获取的表皮干细胞进行纯化培养一直没有比较好的技术方法。目的:拟应用胰蛋白酶差速脱壁法去除表皮干细胞中混杂的成纤维细胞。设计、时间及地点:细胞学体外观察,于2006-06/2007-06在解放军军事医学科学院输血医学研究所干细胞与再生医学研究室完成。材料:实验所用皮肤来源于20～30岁女性乳房缩小整形术患者,由北京协和医院整形外科提供。方法:无菌条件下切取皮肤,剪除皮下脂肪组织,采用胶原酶/Dispase酶和胰蛋白酶两步法体外分离培养表皮干细胞。待细胞生长达70%～80%融合时,加入胰蛋白酶消化尚未脱壁的细胞,30s～1min后吸出脱壁细胞,同法传代培养至第5代细胞。主要观察指标:第1,5代表皮干细胞表面标志CD29、CD49f、CD71的表达。流式细胞仪检测第5代细胞生长周期。免疫荧光检测第5代表皮干细胞和传代时胰蛋白酶消化30s～1min即脱壁细胞角蛋白19及波形蛋白的表达。透射电镜观察表皮干细胞超微结构。结果:第1代表皮干细胞CD29阳性率94.32%,CD49f阳性率86.24%,CD71呈弱阳性,经过4次胰蛋白酶差速脱壁去除混杂细胞后,第5代表皮干细胞CD29阳性率96.77%,CD49f阳性率96.31,CD71呈弱阳性,82.58%表皮干细胞处于G0～G1期。第5代表皮干细胞角蛋白19呈阳性,波形蛋白呈阴性,而传代时胰蛋白酶消化脱壁的细胞角蛋白19呈阴性,波形蛋白呈阳性,证实为成纤维细胞。表皮干细胞呈克隆样生长,透射电镜下胞质中可见呈束状排列的角蛋白丝。结论:应用胰蛋白酶差速脱壁法能够简单、有效地获取纯化的表皮干细胞。

BACKGROUND： Epidermal stem cells are significant for repairing skin deficiency. However, there is still no good method to conveniently and rapidly obtain purified epidermal stem cells OBJECTIVE： To remove fihroblasts among epidermal stem cells using trypsin differential detachment. DESIGN, TIME AND SETTING： The cytology in vitro observation was performed at the Research Room of Stem Cells and Regenerative Medicine, Beijing Institute of Transfusion Medicine, Academy of Military Medical Sciences of Chinese PLA from June 2006 to June 2007. MATERIALS： Skin collected from 20-30 years female patients undergoing reduction mammoplasty was obtained from Department of Plastic and Aesthetic Surgery, Peking Union Hospital. METHODS： Skin was sterilely incised with removing subcutaneous fatty tissue. Epidermal stem cells were in vitro separated from breast skin by Collagenase/Dispase and trypsin. When covering 70%-80% area of culture flasks, cells were digested with trypsin for 30 seconds to 1 minute and the floated cells were sucked out and threw away. The fifth passage of cells were collected by the same method. MAIN OUTCOME MEASURES： CD29, CD49f and CD71 expression of the first passage and the fifth passage cells were examined respectively. Cell growth cycle at the fifth passage was measured by flow cytometry. Keratin 19 and vimentin of cells which was harvested by exposure to trypsin for the first 30 seconds to 1 minute was examined by using immunofluorescence. Ultrastructures of the fifth passage ceils were observed using a transmission electron microscope. RESULTS： The first passage cells were CD29 positive （94.32%）, CD49f positive （86.24%） and CD71 relative positive. After four times of trypsin differential detachment, the fifth passage cells were CD29 positive （96.77%）, CD49f positive （96.31%） and CD71 relative positive. 82.58 % cells of the fifth passage were in G0-G1 stage. The fifth passage cells were cytokeratin 19 positive and vimentin negative, while cells which was harvested by exposure to trypsin were cytokeratin 19 negative and vimentin positive, which was verified fibroblasts. Epidermal stem cells showed clone-like growth. Keratin filaments could be observed under the transmission electron microscope. CONCLUSION： More purified epidermal stem cells may be isolated easily and effectively by differential detachment with trypsin.