成年鼠被毛毛乳头细胞分离方法的建立与评价

目的 寻求一种简单高效的分离培养小鼠被毛毛乳头细胞的方法.方法 取5周龄C57小鼠背部皮肤,刮去毛发后予0.2％Ⅰ型胶原酶37℃消化2h,刮下毛球部,经过2次Ficoll密度梯度离心后,得到纯净的毛乳头进行培养.检测毛乳头贴壁率、毛乳头细胞特异性标记表达及其体内毛囊重建能力.结果 该分离方法能显著降低工作强度,减少污染机会,获得的毛乳头贴壁快、细胞迁出快.qRT-PCR、细胞免疫荧光实验结果均证实体外培养的小鼠被毛毛乳头细胞可表达与毛囊诱导能力相关的特异性标记物ALP、β-catenin及Versican,且与触须毛乳头细胞相比差异无统计学意义(P＞0.05).体外培养的小鼠被毛毛乳头细胞具有诱导毛囊再生的能力.结论 胶原酶消化结合Ficoll梯度离心是一种简单、有效地分离获取小鼠背部毛乳头的方法.

Establishment and evaluation of separation for adult mousepelage dermal papilla

Objective To seek a simple and efficient method for isolating and culturing mouse pelage dermal papilla cells (DPCs).Methods Mouse dorsal skin was cut off from 5-week-old C57BL/6J mice,and then the hair shafts were shaved,and digested in 0.2％ type Ⅰ collagenase at 37 ℃ for 2 h.The hair bulbs were obtained by scraping,and collected by Ficoll gradient centrifugation for 2 times.The pure dermal papilla (DP) were obtained and cultured in vitro.The adherent rate and specific markers of DPCs were detected,and the hair follicle reconstruction was observed in vivo.Results Our method was simple and easy,and reduced the risk of contamination significantly.The adherence of obtained pelage DP and the cell emigration from DP were quite fast.qRT-PCR and cell immunofluorescence assay confirmed that the cultured pelage DPCs expressed the specific markers associated with hair follicle-inductive ability,such as ALP,β-catenin and Versican,and the expression levels had no significant differences with those of vibrissa DPCs (P ＞ 0.05).The obtained pelage DPCs showed the ability to induce hair follicle regeneration after being transplanted in the back of nude mice.Conclusion Collagenase digestion combined with Ficoll gradient centrifugation is a simple and effective separation method to obtain mouse pelage DP.