摘要
目的在体诱导表皮细胞去分化,探索与表皮细胞去分化有关的信号通路。方法包皮皮片去除皮下组织,用中性蛋白酶分离表皮,Ⅳ型胶原反复粘贴并牵拉表皮去除基底细胞层。处理后表皮片移植于裸鼠全层皮肤下,分别于3、5、7d取出移植皮片,采用免疫组织化学染色、流式细胞仪检测、Western blot和RT-PCR等方法检测皮片移植前后表型的改变和ERK MAPK通路各信号分子表达的变化情况。结果去基底细胞层处理的表皮片干细胞标志物CK19和β1整合素染色阴性;移植后5d,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显示,表皮片移植后CK19阳性细胞和β1整合素阳性细胞由移植前的0.00%分别增加到7.54%和5.24%,而CK10阳性细胞由99.62%下降为86.56%。Western blot和PCR检测也证实CK19和β1整合素蛋白水平和mRNA水平表达在移植后明显增强。ERK及上下游信号分子(p-Raf、p-MEK1/2、p-ERK1/2和c-myc)表达增强。t-MEK1/2和t-ERK1/2移植前后表达无显著差异(P>0.05)。结论处于终末分化阶段的表皮细胞可以向干细胞状态逆转,这个过程可能与ERK MAPK信号通路有关。
Objective To induce the dedifferentiation of human epidermal cells in vivo for the signaling pathways involved in the process. Methods The epidermis of human foreskin which was obtained with informed consent and approval of the Ethics Committee of our hospital was isolated from dermis by digestion with protease, and the cells in the stratum basale of the epidermis as well as stem cells were eliminated by repeated adhesion to collagen type Ⅳ. After being labeled with DAPI, the treated uhrathin epidermal sheets were transplanted under the fresh full-thickness skin wounds about 1 cm in diameter on the back of athymic BALB/c nude mice. The transplanted sheets were harvested from the recipient at day 3, 5 and 7 after xenotransplantation. Immunohistochemical assay, Western blot analysis and RT-PCR were used to determine the changes of cell phenotypes and ERK signaling pathway of the xenografts. Results CK19, 131 integrin, 1363 and Oct4 positive ceils were found in a single layer in stratum basal of the full-thickness epidermis, but were undetectable in ultrathin epidermal sheets before grafting. Most of the ultrathin epidermal sheets grew well in a soft and ruddy appearance after transplantation and the total survival rate was 86.3%. Immunological examinations showed that a certain number of cells were positive to CK19, β1 integrin, p63 and Oct4 in spinous and granular layers after transplantation and that ERK/MAPK pathway was involved in the reversion process. Conclusion The differentiating epidermal cells could reverse to stem cell-like cells, and the process might be associated with the regulation of ERK/MAPK signaling pathway.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第21期1998-2002,共5页
Journal of Third Military Medical University
基金
国家重点基础研究发展计划(973计划)(2005CB522603)
国家自然科学基金(30500194)~~
关键词
去分化
异种移植
表皮细胞
表皮干细胞
胞外信号调节激酶
dedifferentiation
xenotransplantation
epidermal cells
epidermal stem cells
extracellular signal regulated kinase
