毛囊来源的表皮干细胞在不同培养条件下细胞生长情况的比较及效果评价

Comparison of growth status of epithelial stem cells derived from plucked hair follicle between different culture conditions

目的:探讨毛囊来源的表皮干细胞(ESCs)培养条件,阐明成纤维细胞在ESCs培养中的作用。方法:以拔出的毛发为细胞来源,通过细胞培养的方式,比较在无包被、鼠尾胶原包被、Ⅳ型胶原包被和3T3成纤维细胞作为饲养层细胞条件下细胞生长情况。并通过RT-PCR和免疫荧光等方法对培养出的细胞进行细胞角蛋白(CK)15的检测。结果:在无包被、鼠尾胶原包被、Ⅳ型胶原包被和3T3成纤维细胞作为饲养层细胞条件下,以3T3成纤维细胞作为饲养层对细胞的生长最为有利;同时,对不同毛囊节段(毛球、中间节段、隆突和峡部)的培养表明,毛囊隆突部位的细胞增殖能力最强。RT-PCR及免疫荧光结果表明,应用此种方法从毛囊中分离培养的细胞表达表皮干细胞的特异性标志CK15。结论:以3T3成纤维细胞作为饲养层细胞,可以从拔出毛发根部的隆突部位培养出ESCs,此培养条件对细胞的生长最为有利。

Objective To explore the method of obtaining and expanding epithelial stem cells （ESCs） from plucked hair follicle in vitro and clarify that fibroblasts are necessary for ESCs growth. Methods A amount of ESCs from plucked hair were obtained, the epithelial outgrowth of human plucked hair follicle was studied and compared between different culture conditions including no coating well, rat tail-collagen coating well, collagen IV coating well and 3T3 fibroblasts feeder. The characteristics of these cells were detected by RT-PCR and immunofluorescent staining for CK15. Results After compared between different culture conditions including no coating well, rat tailcollagen coating well, collagen IV coating well and 3T3 fibroblasts feeder, the results indicated that the plucked hair fragment B3 （bulge area of hair follicle） placing on 3T3 fibroblasts feeder was the optimum condition. RT-PCR and immunofluorescent method confirmed that these cells expressed CK15 which was the special marker of ESCs. Conclusion ESCs can be obtained from bulge area of hair follicle which grows on the 3T3 fibroblasts feeder and this method is the optimum condition for ESCs growth after compared between different conditions.