去分化表皮干细胞的来源鉴定和分析

目的：利用染色体特异性分析方法鉴定去分化表皮干细胞的来源。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干细胞。处理后的表皮片经免疫组化鉴定无表皮干细胞后移植到雌性BALB／c裸鼠背部全层皮肤缺损创面，5d后用免疫组化法检测皮片内角蛋白10（CK10）、CK19和β1整合素的表达，然后用Ⅳ型胶原粘贴法分离皮片内去分化来源的表皮干细胞，并采用PCR的方法检测去分化细胞内的Y染色体。结果：未经处理的表皮片基底部细胞呈CK10染色阴性，CK19和β1整合素染色阳性；Ⅳ型胶原处理后的表皮片内细胞全呈CK10染色阳性。而去基底皮片移植后5d，在皮片的基底侧重新出现CK10染色阴性、CK19和β1整合素染色阳性的细胞。Ⅳ型胶原粘贴分离去分化来源的表皮干细胞，结果表明，移植皮片组在10min内约有4．56％的贴壁细胞出现，而未移植皮片组3h内无贴壁细胞出现。PCR分析结果显示在上述贴壁细胞内能检测到Y染色体的特异序列，而宿主来源的组织检测不到。结论：去分化表皮干细胞来源于移植皮片内成熟表皮细胞的去分化，而非宿主体内的干细胞。

Source identification and analysis of dedifferentiation-derived epidermal stem cells

Objective：To identify the source of dedifferentiation-derived epidermal stem cells （DDESCs） by analysis of Y-chromosome specific sequences. Methods： Human foreskin was digested overnight after removal of adipose tissue, and then the epidermis was separated from the dermis. Basal stem cells were eliminated by repeated adhesion to type Ⅳ collagen. Epidermal sheets without basal stem cells were transplanted wounds devoid of epidermis on the back of BALB/c nude mice. After 5 days,cells negative for keratin 10 （CK10） but positive for CK19 and β1 integrin were identified by immunohistochemistry. Then these cells were isolated by rapid adhesion to type Ⅳ collagen, and Y-chromosome specific sequences were detected by polymerase chain reaction. Results：Cells which were positive for CK19 and β1 integrin and negative for CK10 were found in the basal layer of full-thickness epidermal sheets which were not pre-treated. In contrast to full-thickness epidermal sheets, all the cells in ultrathin epidermal sheets pretreated with collagen type Ⅳ exhibited CK10 positive. But 5 days after the ultrathin epidermal sheets were transplanted, cells negative for CK10 but positive for CK19 and β1 integrin appeared in the wound adjacent to the skin grafts. DDESCs were isolated and it was found that there were 4. 56% adhering cells in the grafting group within 10 minutes. Y-chromosome-specific sequences could be detected in the adhering cells. However, in mouse skin tissue, there were no Y-chromosome specific sequences. Conclusion：DDESCs isolated from xenografts are originally from human male donors and are not mouse stem cells coming from the host that have attached to the graft.