大鼠胎儿表皮干细胞的分离鉴定和体内分化

目的探索一种简单而又高效的分离大鼠胎儿表皮干细胞方法,以及观察其在体内环境中的分化潜能。方法采集ED14、ED16、ED18及ED20不同发育阶段的SD大鼠胎儿皮肤,HE染色观察其结构。鼠尾胶原快速黏附法筛选胎鼠表皮细胞,在无血清培养基(K-SFM)中进行培养。免疫组织化学方法检测CK-19和1β-整合素表达情况。同时,也对细胞周期和细胞增殖能力进行检验。经荧光染料(PKH26)标记过的胎鼠表皮干细胞与多孔凝胶海绵结合,然后植入大鼠肾被膜下进行诱导分化。结果在ED18之前皮肤表皮和真皮尚未充分完成分化。使用快速黏附法能够有效的从ED18胎鼠皮肤分离到表皮干细胞。获得的细胞CK19和1β整合素呈阳性表达,细胞周期分析显示,细胞呈慢周期性,移植体在肾被膜下分化并展现典型的表皮样结构。结论表皮干细胞存在于早期发育皮肤中,但在ED18才能使用快速黏附法分离到表皮干细胞。此时的胎鼠表皮干细胞具有向表皮分化的能力。

ISOLATION AND IDENTIFICATION OF RAT FETAL EPIDERMAI STEM CELL AND ITS DIFFERENTIATION IN VIVO

Objective To explore a feasible and efficient method for isolating rat fetal epidermal stem cells (rfESCs) , and to observe the differentiation potential of rfESCs in vivo. Methods The different development stages of SD rat fetal skin, ED14，ED16, ED18 and ED20, were collected and the structures were analyzed by HE staining. The rfESCs were isolated by rapidly adhering to rat tail collagen (RA-RTC) and were cultured in keratinocyte serum free medium(K-SFM). The expression of β1 integrin and cytokeratin 19 in rfESCs were detected by immunhistochemical staining. Meanwhile, the cell cycle and proliferative capacity were also determined. To induce its differentiation, these cells were grafted onto porous gelatin sponge after labelled with a fluorescent dye (PKH26), and then transferred into rat sub renal capsule. Results Epidermis and dermis were incompletely differentiated until ED18. The rfESCs were effectively isolated from ED18 rat fetal skin by RA RTC. The expressions of cytokeratin 19 and β1 integrin in rfESCs were positive. Cell cycle analysis indicated a slow cell cycle. rfESCs were differentiated and exhibited epidermal like structure under renal capsule. Conclusion The early