小鼠毛乳头细胞体外分离培养方法的优化和评价

Optimization and evaluation of the method of the isolation and cultivation of mouse dermal papilla cells in vitro

目的 :探索小鼠毛乳头细胞(DPCs)体外分离培养的改良新方法。方法 :选用雌性C57BL/6小鼠,以精细显微解剖获取毛乳头部位,联合胶原酶消化,并添加10 ng/mL碱性成纤维细胞生长因子(bFGF)以优化培养体系,建立体外分离培养小鼠DPCs的改良新方法;以光镜下观察、细胞计数、碱性磷酸酶(ALP)特异性染色及荧光定量PCR鉴定DPCs的ALP mRNA表达水平,与传统方法比较进行相关评价。结果 :与传统方法比较,改良方法的毛乳头基本全部贴壁,细胞可更快迁出及生长融合,所获得的细胞数量是传统方法的5.85倍;经传代后传统方法的低代次DPCs有聚集性生长现象,随代次增加,细胞形态逐渐变大衰老,增殖明显迟缓,聚集性生长愈发不明显,至第5代聚集性现象消失。而改良方法的DPCs经传代后,第1、2和5代形态仍呈长梭形及多角形的特点,至第5代仍保有聚集性生长的特点;且具有更强的ALP活性,尤其是高代次(第5代)细胞更为明显,而传统方法的高代次细胞则几乎无ALP活性;改良方法的ALP mRNA表达水平与传统方法相比也明显上调,尤其是第5代细胞更加明显。结论 :改良方法可获取更多数量、并具有较好功能的小鼠DPCs,为开展毛囊相关研究提供可靠的细胞来源。

Objective:To explore an efficient and rapid method of the isolation and cultivation of mouse dermal papilla cells(DPCs)in vitro.Methods:The dermal papilla of six-week-old female C57BL/6 mice was harvested by microdissection and digested with collagenase.DPCs were cultured and passaged in medium containing 10ng/mL basic fibroblast growth factor(bFGF)in vitro.DPCs isolated with modified methods were evaluated and compared with the DPCs with traditional methods through morphological observation,cell counting,alkaline phosphatase(ALP)specific staing and ALP mRNA expression.Results:Compared with cells by the traditional method,almost all dermal papilla by the modified method adhered,migrated,and grew faster into cell confluence.The number of cells obtained was 5.85 times that of the traditional method.In DPCs by the traditional method,aggregative growth was noted after subculture at low passage numbers.After several passages,the cell morphology gradually became larger and degenerated,and less aggregative in pattern,even disappeared(5th passage).However,the morphology of the DPCs of the modified method still showed the characteristics of long spindle shape and aggregative pattern in the 1st,2nd,and 5th passage.The results also showed that the DPCs of the modified method had high ALP activity,especially in the higher passage cells(5th passage).While nearly no ALP activity was detected in DPCs by traditional method at high passage.The expression level of ALP mRNA of the DPCs of the modified method was significantly up-regulated compared with that in the traditional method,especially in the cells at the 5th passage.Conclusion:The modified method is a simple and efficient method for isolating mouse DPCs.It can provide more reliable cell source for the research of hair follicle.