P38丝裂原活化蛋白激酶信号通路对角膜缘干细胞调控机制的研究

项目名称： P38丝裂原活化蛋白激酶信号通路对角膜缘干细胞调控机制的研究

项目编号： No.30872810

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 刘祖国

作者单位： 厦门大学

项目金额： 36万元

中文摘要： 本项目以体外培养和扩增的角膜缘干细胞为研究对象，采用抑制剂和逆转录病毒为载体的基因转染方法，从正反两方面验证P38丝裂原活化蛋白信号通路是否通过调控细胞Wnt信号途径中的βatenin，影响下游c-Myc和细胞周期蛋白D的表达，参与p63转录因子对维持上皮组织干细胞高增殖、低分化水平的作用。通过对调控角膜缘干细胞细胞周期蛋白和培养应激诱导的细胞衰老，保持角膜缘干细胞的自我更新能力，维持体外干细胞特性。通过对角膜缘干细胞调控机制研究，以期寻求体外细胞扩增的最佳培养条件。采用体外扩增方法构建组织工程化角膜上皮。以最小损伤，较短体外培养时间以及最接近正常形态和功能的植片,为体外成体干细胞的培养扩增和组织工程化角膜上皮植片运用于临床的治疗研究开辟一条新途径。

中文关键词： P38；上皮干细胞；增殖；分化

英文摘要： Objective： Investigate the effects of p38 signaling pathway on the proliferation of limbal stem cells and skin epithelia stem cells. Methods： Upregulate or downregulate the activation of p38 signaling pathway through the transfection of Lentivirus on limbal stem cells and skin epithelia stem cells. Use western blot, immunofluorescence staining, crystal violet staining MTT analysis to detect the p38 pathway and stem cell MAPKers, to define the effect of p38 pathway on stem cells in vitro, and the crosstalk with Notch signaling pathway. Results： The activation of p38 pathway was powerful in the surface layer while it was weak in the basal layer of human dermal and limbal epithelium. p63, Ki67, keratin 14 and keratin 16 were mainly expressed in the basal layer of dermal and limbal epithelium. The protein MAPKed terminal differentiation, K10 and K12, were expressed in the surface layer of skin and cornea respectively. The down stream of Notch pathway, Hes 1, was strongly expressed in the basal layer while it was weakly expressed in the surface layer of dermal and limbal epithelium. On limbal stem cells and skin epithelial stem cells cultured ex vivo, inhibition of p38 pathway can significantely improve the clone efficiency and colony forming area, and the expression of p63, K14 and K16 was increased, while the expression of Notch1 (Notch recepter) and Hes1 was increased. Conclusion： p38 signaling pathway can affect the proliferation of limbal stem cells and skin epithelial stem cells, leading to accelerated differentiation. Inhibition of p38 pathway, not only can maintain the proliferation of limbal stem cells and skin epithelial stem cells better in vitro, but also can activate Notch pathway to play on the proliferation and differentiation of epithelial stem cells together.

英文关键词： p38; Epithelial stem cells; Proliferation; differentiation