大鼠毛囊干细胞的纯化培养鉴定及其生物学特性

Purification,cultivation and identification of rat follicle stem cells and biological characteristics

目的采用免疫磁珠法(vario magnetic activated cell sorting,Vario MACS)分离纯化培养大鼠CD34+毛囊干细胞,并研究其生物学特性。方法显微分离获得毛囊bulge区,消化成单细胞悬液并进行CD34抗体标记和磁珠标记,使细胞悬液通过分选柱,分别收集CD34+细胞和CD34-细胞,检测细胞活性后进行细胞培养并绘制细胞生长曲线。扫描电镜观察毛囊干细胞表面形态,透射电镜观察干细胞内部结构形态。免疫细胞化学检测培养细胞中β1-整联蛋白、CD34和α6-整联蛋白的表达。结果用VarioMACS法有效分离并成功培养CD34+毛囊干细胞,分选后的细胞仍具有较好的活性,细胞生长曲线表明,CD34+毛囊干细胞增殖速度快,增殖期长,而CD34-细胞则较快进入平台期。培养的CD34+细胞中干细胞标记物β1-整联蛋白、CD34和α6-整联蛋白的表达均强于CD34-细胞。获得电镜观察毛囊干细胞表面形态学特征及内部结构特征资料。结论VarioMACS法能有效分离获得的CD34+毛囊干细胞,该细胞具有良好的生物学性能。

Objective To enrich and culture rat CD34^＋ follicle stem cells by immunomagnetic beads [ Vario magnetic activated cell sorting （MACS） ], and investigate their biological characteristics. Methods Follicles were digested into cell suspension after obtained by microdissection and digestion. CD34^＋cells and CD34^- cells were isolated after cell suspension was incubated with CD34 antibody and magnetic micro-beads in turn and then flowed though an MACS column. The isolated cells were cultured after viability evaluation. Cell growth curve was plotted. Scanning electron microscopy （SEM） and transmission electron microscopy （TEM） were carried out to observe the morphology and internal structure of follicle stem cells. The isolated cells were cultured and then identified by detecting the expression of β1-integrin, CD34 and α6-integrin by immunohisto- chemical assays. Rosults CD34^＋ follicle cells were isolated effectively by using Vario MACS, and cultured successfully with relative high viability. Cell growth curve showed that CD34^＋cells proliferated quickly and had a longer proliferative phase compared with CD34^- cells. The expressions of β1-integrin, CD34 and α6-integrin in the cultured CD34^＋cells were stronger than in CD34^- cells. SEM and TEM indicated that the CD34^＋ follicle stem cells were round in shape and with large nuclei, clear nucleoli, and increased cytoplasm/nucleus ratio. Conclusion CD34^＋ follicle stem cells with relative high viability can are obtained effectively by using immunomagnetic beads.