显微分离培养与免疫磁珠法分离纯化人毛囊干细胞

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 folliclestem 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.

SEPARATION AND PURIFICATION OF HUMAN HAIR FOLLICLE STEM CELLS BY MICROMANIPULATION AND MAGNETIC CELL SORTING

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 expressed K19 strongly. The viability rate of pre-purification cells was 95.0% +/- 0.6% while that of post- purification cells was 94.2% +/- 1.0%. There was no significant difference (P < 0.05). By flow cytometry and immunofluorescence staining examination, the CD200+ cell rate was 8.31% before cell sorting purification while that was 82.31% after cell sorting purification. CONCLUSION: Highly purified and viable HFSCs could be obtained by micromani pulation and magnetic cell sorting assay.