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目的毛囊干细胞在体外培养下可向软骨、骨骼、脂肪等细胞分化。文中研究毛囊干细胞的培养方法及其成脂和成骨分化的能力。方法采用显微分离技术联合两步酶消化法获得毛囊干细胞,差速贴壁法对毛囊干细胞进行提纯,利用体积分数10%胎牛血清的角质细胞无血清培养基进行培养,应用流式技术对干细胞进行CD34、β1整合素、CK15检测。第3代毛囊干细胞分别使用成骨及成脂诱导液进行培养。结果培养后的毛囊干细胞形态均匀一致,折光性强,呈典型的“铺路石”状,流式细胞仪检测CD34、β1整合素、CK15分别表达为59.6%、97.2%、61.1%,第3代细胞生长曲线显示细胞生长情况良好。经成骨诱导21 d后茜素红染色可见钙结节形成,成脂诱导14 d后油红O染色后可见脂滴形成。结论培养的毛囊干细胞具有很强的生长活性,并具有多向分化的潜能。
Purpose The hair follicle stem cells can differentiate into cartilage, bone, fat and other cells in vitro. This article studies the method of culturing hair follicle stem cells and its ability of adipogenic and osteogenic differentiation. Methods Hair follicle stem cells were obtained by microscopic separation combined with two-step enzymatic digestion. Hair follicle stem cells were purified by differential adhesion method. Cultured in serum-free keratinocytes with 10% fetal bovine serum, flow cytometry Stem cells for CD34, β1 integrin, CK15 detection. The third generation of hair follicle stem cells were cultured using osteogenic and adipogenic inducing fluid. Results The shape of hair follicle stem cells after culturing was uniform and highly refraction, showing a typical “paving stone” shape. The expressions of CD34, β1 integrin and CK15 were 59.6%, 97.2% and 61.1% respectively by flow cytometry The 3rd generation cell growth curve shows that the cell growth is good. After 21 days of osteogenic induction, alizarin red staining showed the formation of calcium nodules. After adipogenic induction for 14 days, lipid droplets formed after oil red O staining. Conclusion The cultured hair follicle stem cells have strong growth activity and have the potential of multidirectional differentiation.