去分化来源表皮干细胞的分离、培养和鉴定

目的 分离培养去分化来源的表皮十细胞(dedifferentiation-derived epidermal stem cells,DDESCs),并进一步观察其表型特征.方法 包皮皮片去除皮下脂肪后,中性蛋白酶消化过夜,分离表皮和真皮.分离的表皮片用Ⅳ型胶原反复粘贴并冲洗去除表皮十细胞后,移植到BALB/c裸鼠背部全层皮肤缺损创面,移植5 d后收集皮片制备单细胞悬液,流式检测CK10、CK19和β1整合素阳性细胞白分数,然后用Ⅳ型胶原粘贴法分离皮片内DDESCs,并采用免疫荧光法和荧光定量PCR法分别检测去分化细胞内CK19、β1整合素、Oct4和Nanog的表达.结果 流式检测结果表明,皮片移植5 d后,CK10阳性细胞减少(P＜0.01),CK19和β1整合素阳性细胞增加(P＜0.01).Ⅳ型胶原粘贴分离DDESCs,结果表明,移植皮片组在10 min内约有4.56%的贴壁细胞出现.细胞的表型分析结果表明,DDESCs内CK19、β1整合素、Oct4和Nanog的表达量类似于原始表皮干细胞(P＞0.05),但明显高于成熟表皮细胞(P＜0.01).结论 DDESCs的表型特征与原始表皮干细胞相类似,提示去分化细胞有可能代替原始表皮干细胞应用于创伤部位的修复和再生.

Isolation, culture and phenotypic investigation of dedifferentiation-derived epidermal stem cells

Objective To isolate the dedifferentiation-derived epidermal stem cells (DDESCs)to further investigate their phenotypic characteristics. Methods The sheets of human foreskin were digested overnight after removal of adipose tissues and then the epidermis was separated from the dermis.The epidermis sheets which eliminated basal stem cells by repeated adhesion to type Ⅳ collagen and flushing were transplanted onto the full-thickness skin wounds on the back of BALB/c nude mice. After five days, the sheets were collected and digested into single cells, after which the percentages of positive cells of CK10, CK19 and β1 integrin were detected by flow cytometric analysis. DDESCs were isolated by rapid adhesion to type Ⅳ collagen. The expressions of CK19, β1 integrin, Oct4 and Nanog in the cells were examined using immunofluorescence and quantitative real-time polymerase chain reaction (RTPCR). Results The percentages of positive cells of CK19 and 31 integrin were increased (P ＜0.01 )and those of CK10 in the transplanted sheets decreased ( P ＜0.01 ) five days after transplantation. Isolation of DDESCs by repeated adhesion to type Ⅳ collagen showed 4.56% adhering cells in the transplantation group within 10 minutes. The in vitro phenotypic assays showed that the expressions of CK19, β1 integrin, Oct4 and Nanog in DDESCs were similar to those of original epidermal stem cells ( P ＞0.05 ) but remarkably higher than those in the control group ( P ＜ 0.01 ). Conclusion The phenotypic characteristics of DDESCs cultured in vitro are similar to those of epidermal stem cells, indicating a new approach for wound repair and regeneration.