大鼠触须毛囊干细胞的分离培养和鉴定

背景：毛囊干细胞在体内膀胱及周围组织环境作用下有可能向尿路上皮细胞和平滑肌细胞分化,成为目前研究较新的干细胞之一。 目的：建立一种高效简单的分离培养和鉴定毛囊干细胞的方法。 方法：取大鼠触须部皮肤组织,体式显微镜下分离出毛囊组织,中性蛋白酶Ⅱ与胰蛋白酶和乙二胺四乙酸混合液“两步酶法”消化;所得细胞悬液中添加含体积分数10%胎牛血清的角质细胞无血清培养基,Ⅳ型胶原差速贴壁法筛选毛囊干细胞,20 min以内贴壁的细胞作为实验组,未贴壁的细胞作为对照组;待筛选后的细胞生长至70%~80%汇合后,进行传代培养。 结果与结论：筛选后的细胞形态均匀一致,折光性强,呈典型的“铺路石状”。透射电镜显示细胞处于原始状态。流式细胞仪检测实验组CD34和β1整合素的表达分别为(39.52±19.57)%和(93.46±4.73)%,对照组相应为(19.20±11.53)%和(63.57± 14.42)%,两组间差异有显著性意义(P < 0.05)。结果表明联用显微分离技术、二步酶法及差速贴壁法筛选可以获得纯度较高的毛囊干细胞。CD34和β1整合素表达是较理想的鉴定方法。

Isolation,culture and identification of rat hair follic stem cells

BACKGROUND:Hair follicle stem cells (HFSCs) based on in vivo role of the bladder and surrounding tissue environment are likely to differentiate into urothelial cells and smooth muscle cells, which become a new stem cell research. OBJECTIVE:To investigate an effective and simple method of isolation, culture and identification of rat HFSCs. METHODS:After sterling, the skin of the rat’s beard was cut off, the vibrissa follicles were dissected under a stereocroscope. The dermal sheath was moved by incubated in Dispase Ⅱ firstly, second step with a mixture of trypsin and EDTA. The cells suspension was cultured in 10% fetal bovine serum keratinocyte free serum medium and selected by rapid adhering on collagen Ⅳ. The cultured cells were passaged, when cells were confluent with 70%-80%. Selected cell microstruture was observed under optics microscope and electron microscopy respectively. Cells were characterized by flow cytomitry with antibody against CD34 and β1-integrin. RESULTS AND CONCLUSION: The selected HFSCs were uniform in shape, more refraction, cobblestone-morph by optics microscope. The form was primitive, small ratio of nucleus and cytoplasm, obvious nucleolus, organelle of developmental immaturity by transmission electron microscope. In adherent cells (experiment groups), CD34 and β1-integrin expression rate was (39.52±19.57)% and (93.46±4.73)%, respectively. In no-adherent cells (control groups) was corresponding (19.20±11.53)% and (63.57±14.42)% respectively. There was a significant difference between two groups (P < 0.05). Highly purified HFSCs can be obtained by dissection under a stereocroscope and two-step enzyme digestion combining with rapid adhering on collagen Ⅳ. CD34 combined with β1-integrin are an ideal marker to identify HFSCs.