GFP标记成年大鼠表皮神经嵴干细胞的分离培养与形态学特征

Culture and morphologic characteristics of epidermal neural crest stem cell isolated from GFP adult rat

目的建立绿色荧光蛋白(GFP)标记大鼠表皮神经嵴干细胞(EPI-NCSC)的分离培养方法,为EPI-NCSC在体移植研究脊髓损伤修复奠定基础。方法分离成年GFP大鼠胡须毛囊,取毛囊隆突部贴附于胶原包被的培养板上。在培养第4天有大量细胞游出,取出贴附的毛囊隆突部,细胞传代培养。细胞采用SOX10和Nestin免疫细胞化学染色鉴定,并计算细胞纯度。结果 EPI-NCSC在胶原基质上从隆突部游出,细胞为圆形或梭形,呈强绿色荧光。免疫细胞化学染色SOX10和Nestin双阳性,纯度在99%以上。结论本实验方法简便易行,能分离出高纯度的GFP-EPI-NCSC,可作为一种有效的工具细胞用于损伤脊髓修复研究。

Objective To establish methods for isolating and culturing the epidermal neural crest stem cell（EPI-NCSC） from GFP adult rat,and to lay the foundation for tracking transplanted cell of spinal cord injury and repair.Methods We isolated whisker hair follicle,and took out of bulges from collagen capsule,setting several bulges on pre-incubate collagen-coated culture plates.Removed bulges,4 days after onset of EPI-NCSC emigration from the bulge explants,and enrichment cultured the migratory cells.The EPI-NCSC were identified by the immunocytochemistry of SOX10 and Nestin,and its purity was calculated according to the positive rate.Results The EPI-NCSC emigrated from the blunge explants on collagen substratum,showed morphological types of round and spindle with strong green fluorescence.More than 99% cultured cells were identified to be EPI-NCSC,which expressed SOX10 and Nestin.Conclusion This method is simple.The high purity of GFP-EPI-NCSC can be harvested to be used as an effective tool cells,and widely used for researching on the spinal cord injury repairing.