14-3-3σ通过Notch及EGFR/ERK信号通路对表皮角质形成细胞增殖和分化的调控作用

14-3-3σ has a key effect on the regulation of epidermal development, which is manifestated that the mice with inactivation of 14-3-3σ protein (Er/Er mice) has hyperplastic epidermis and lose of terminal differentiation, however, its molcular mechanism is not clear. Our previous research showed that EGFR/ERK signaling activity was increased and Notch signaling activity was decreased in the epidermis of Er/Er mice, so it is supposed that the regulatory effect of 14-3-3σ on epidermal proliferation and differentiation is correlated with Notch or EGFR/ERK signaling pathways. Therefore, in this project, the keratinocytes isolated from Er/Er mice will be cultured and treated with Lentivirus expressing NICD/GFP or inhibitor specific to EGFR signaling pathway, which will recover the activity of the two signaling pathways to normal level in Er/Er KTC. And then the ability of proliferation and differentiation of Er/Er KTC will be observed to explore the regulatory relationship between 14-3-3σ with Notch or EGFR/ERK signaling pathway and regulatory network among the three roles. By this project, it will be clarified that the molcular mechanism of 14-3-3σ regualting proliferation and differentiation of epidermal keratinocytes and provide new theory and researching diections for treatment to cutaneous tumor and construction of tissue engineering skin.

14-3-3σ在表皮的发育中起关键调节作用，表现在14-3-3σ蛋白失活小鼠（Er/Er）的表皮高度增殖，缺乏终末分化，然而其作用的分子机制尚不清楚。我们前期研究发现，Er/Er小鼠表皮中Notch信号通路活性降低而EGFR/ERK信号通路活性增高；故推测14-3-3σ对表皮增殖和分化的调控与Notch和EGFR/ERK信号通路相关。为此，本项目以原代培养的Er/Er小鼠角质形成细胞（Er/Er KTC）为研究对象，利用NICD/GFP慢病毒及EGFR信号通路抑制剂，纠正Er/Er KTC信号通路活性的异常，通过观察其增殖和分化能力的变化，探讨14-3-3σ与Notch、EGFR/ERK信号通路的调控关系以及三者间的作用网络。阐明14-3-3σ调控表皮KTC增殖和分化的分子机制，为更好的治疗皮肤肿瘤以及组织工程化皮肤的建立，提供新的理论基础和研究方向。

14-3-3σ在表皮的发育中起关键调节作用，表现在14-3-3σ蛋白失活小鼠（Er/Er）的表皮高度增殖，缺乏终末分化，然而其作用的分子机制尚不清楚。我们前期研究发现，Er/Er小鼠表皮中Notch信号通路活性降低而EGFR/ERK信号通路活性增高；故推测14-3-3σ对表皮增殖和分化的调控与Notch和EGFR/ERK信号通路相关。为此，本项目以原代培养的Er/Er小鼠角质形成细胞（Er/Er KTC）为研究对象，利用NICD/GFP慢病毒及EGFR信号通路抑制剂，纠正Er/Er KTC信号通路活性的异常，通过观察其增殖和分化能力的变化，探讨14-3-3σ与Notch、EGFR/ERK信号通路的调控关系以及三者间的作用网络。通过研究发现，NICD/GFP慢病毒感染Er/Er KTC，恢复其Notch信号通路活性；或利用EGFR信号通路抑制剂AG1478抑制Er/Er KTC中EGFR信号通路的高活性后，Er/Er KTC的增殖能力均显著下降，与正常野生型角质形成细胞（WT-KTC）相近，且具有终末分化能力。这一结果说明，14-3-3σ通过Notch和EGFR/ERK信号通路调控表皮的增殖和分化。利用IP实验，表达14-3-3σWT的慢病毒及Notch及EGFR信号通路特异性抑制剂，进一步证实：14-3-3σ能够与EGFR直接结合发挥调控作用，且通过EGFR调控Notch信号通路，即存在14-3-3σ—EGFR—Notch这样的调控关系。通过本课题的研究，阐明14-3-3σ通过EGFR—Notch调控表皮KTC增殖和分化的分子机制，为更好的治疗皮肤肿瘤以及组织工程化皮肤的建立，提供新的理论基础和研究方向。