表皮细胞去分化的初步实验研究

Preliminary investigation of dedifferentiation of epidermal cells

目的:进一步研究表皮细胞去分化,为创伤修复提供治疗途径。方法:包皮皮片去除脂肪细胞后,用蛋白水解酶消化分离表皮,分离的表皮片用Ⅳ型胶原反复粘连并冲洗以去除表皮干细胞。处理后的表皮片用DAPI标记后移植到全层皮肤缺损的BALB/c裸鼠,并局部应用重组人表皮细胞生长因子(rhEGF)和碱性成纤维细胞生长因子(bFGF),7d后用免疫组织化学法和流式细胞仪法检测移植存活皮片的表型改变。结果:用Ⅳ型胶原反复粘连并冲洗的包皮皮片CK19和β1整合素染色阴性;移植后7d,部分皮片柔软、红润,部分皮片干硬呈黑色,皮片存活率为58·3%;存活皮片CK19和β1整合素染色呈多层分布,而不是正常表皮中的单层分布;流式细胞仪检测结果示:处理后的包皮皮片(移植前)α6+CD71-细胞占0·02%,α6+CD71+占0·03%;移植后7dα6+CD71-细胞占1·43%,α6+CD71+占2·82%,移植前与移植后α6+CD71-和α6+CD71+细胞比例有显著差异(P<0·05)。结论:细胞去分化参与创伤组织的修复,去分化源性干细胞可能成为一个新的干细胞来源。

Objective: To attempt to provide an novel approach to enhance wound repair by studying the dedifferentiation of epidermis. Methods: The epidermis of human foreskin was isolated from dermis by digestion with protease, and the cells in the stratum basale of the epidermis together with stem cells were eliminated by repeated adhesion to collagen type IV and flushing, then transplanted the treated epidermis onto the fresh full-thickness skin wounds measuring about 1 cm in diameter on the back of athymic BALB/c nude mice, decermined rhEGF and bFGF were applied topieally twice a day. The changes in phenotype of the xenografts was decermined on the 7d with immunohis to chemic method and flow cqtometry.Results: Immunohistochemical examination of the survival xenografts showed that some cells were positive for both DAPI and either human CK19 or β_1 integrin in spinous and granular layers on day 7 after transplantation. Furthermore, there was a significant increase in the percentages of both α6~ + CD71~ - and α6~ + CD71~ + populations in human epidermal sheet grafts after transplantation when compared with those before xenotransplantation (P<0.05), as determined by two-color flow cytometry. Conclusions: The dedifferentiation of epidermal cells provides an novel effective therapeutic way for wound repair.