显微分离培养和免疫磁珠法分离纯化人毛囊干细胞.pdfVIP

202 Chinese Journal of Reparative and Reconstructive Surgery, February 2008, Vol. 22, No.2 · · 显微分离培养与免疫磁珠法分离纯化人毛囊干细胞 谭挺 胡志奇 周洪军 【摘  要】 目的 探讨从人毛囊隆突区细胞（bulge cells ，BCs ）获取高纯度有活性的人毛囊干细胞（hair follicle stem cells，HFSCs ）的方法及条件。 方法 取自愿捐献的成人头皮标本，显微分离培养获得BCs ，应用免疫磁珠纯化 BCs 中CD200 阳性的HFSCs 。苔盼蓝染色比较纯化前后细胞活性；流式细胞仪检测细胞纯化效率；免疫荧光检测纯化前 后细胞CD200 的表达情况。 结果 显微分离培养获得的人毛囊BCs ，培养6 d 后细胞生长融合，呈铺路石样。所得细 胞角蛋白19 组织化学染色阳性。CD200 免疫磁珠分选法纯化细胞后，苔盼蓝染色显示纯化前细胞活力为95.0% ± 0.6%， 纯化后为94.2% ± 1.0%，差异无统计学意义（P 0.05 ）。流式细胞仪及免疫荧光检测显示纯化前CD200 阳性细胞的纯度 为8.31%，纯化后CD200 阳性细胞纯度为82.31%，纯化后CD200 阳性细胞回收率为65.39 ％。 结论 联用显微分离培 养与免疫磁珠法，可获得高纯度HFSCs ，且细胞活性不受影响。 【关键词】 毛囊干细胞 免疫磁珠细胞分选 显微分离培养 纯化 中图分类号： R329.28 Q813 文献标志码：A SEPARATION AND PURIFICATION OF HUMAN HAIR FOLLICLE STEM CELLS BY MICROMANIPULATION AND MAGNETIC CELL SORTING/TAN Ting, HU Zhiqi, ZHOU Hongjun. Department of plastic surgery, Nanfang Hospital, Southern Medical University, Guangzhou Guangdong, 510515, P.R.China.Corresponding author: HU Zhiqi, E-mail: doctorhzq@ 【Abstract 】 Objective  To improve the method of obtaining purified and viable human hair follicle stem cells (HFSCs) from bulge cells (BCs). Methods  Firstly, the BCs were isolated from human hair follicles by microdissection. Secondly, the CD200+ HFSCs were selected from BCs using magnetic cell sorting method. The viability of these purified HFSCs was detected under light microscope. The purification rate was analyzed by flow cytometry. The pre- and post-purification cells were compared by immunofluorescence staining. Results  The adherent BCs displayed a typical cobblestone morphology on day 6. The BCs ex- pressed K19 strongly. The viability rate of pre-purification cells was 95.0% ± 0.6% while that of post- purification cells was 94.2% ±