论文部分内容阅读
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 epidermal cell dedifferentiation in vivo and to explore the signaling pathways involved in dedifferentiation of epidermal cells. Methods The foreskin was removed by subcutaneous tissue and the epidermis was separated by neutral protease. The type Ⅳ collagen was repeatedly applied and the epidermis was pulled to remove the basal cell layer. The transplanted epidermal cells were transplanted into the full-thickness skin of nude mice. The skin grafts were removed on day 3, 5, and 7, respectively. Immunohistochemical staining, flow cytometry, Western blot and RT-PCR were used to detect the changes Phenotypic changes and ERK MAPK pathway signal molecules expression changes. Results The dendritic stem cell markers CK19 and β1 integrin staining in basal cell layer were negative. At 5 days after transplantation, the surviving epidermis CK19 and β1 integrin staining showed multi-layer distribution rather than monolayer in normal epidermis. The results of flow cytometry showed that the positive rate of CK19 positive cells and β1 integrin positive cells increased from 0.00% before transplantation to 7.54% and 5.24% after transplanted epidermal cells, while CK10 positive cells decreased from 99.62% to 86.56%. Western blot and PCR also confirmed that CK19 and β1 integrin protein levels and mRNA levels were significantly increased after transplantation. ERK and upstream and downstream signaling molecules (p-Raf, p-MEK1 / 2, p-ERK1 / 2 and c-myc) increased expression. There was no significant difference in the expression of t-MEK1 / 2 and t-ERK1 / 2 before and after transplantation (P> 0.05). Conclusion Epidermal cells in the stage of terminal differentiation may reverse the state of stem cells, which may be related to the ERK MAPK signaling pathway.