CD9/E-cadherin调节表皮细胞分化的作用及机制研究

Wound reepithelialization is a critical point in the process of wound healing. The regulation of keratinocyte differentiation is existing throughout the reepithelialization. In the early stage of wound healing, the new epidermis is in the low differentiation status and high motility, while in the late stage the new epidermis will differentiate to promote epidermal differentiation gradient formation. Our previous research revealed that CD9 expression in the migrating epidermis is downregulated following wounding and then increases, reaching levels comparable to that observed in normal skin epidermis when reepithelialization is complete. Mealwile, we also identified that CD9 regulates keratinocyte differentiation, but the mechanism remains unclear. E-cadherin mediated cell-cell junction plays an important role in keratinocyte differentiation. We hypothesize here that "CD9 interacts with E-cadherin interaction and recruits E-cadherin to the plasma membrane so as to promote keratinocyte terminal differentiation". To test it, we would set up experiments to further confirm the effect of CD9 on keratinocyte differentiation in both cultured keratinocyte and mice epidermis; and then investigate the expression, location and direct interaction of CD9 with E-cadherin; and reveal the structure domain in CD9 that mediates the interaction with E-cadherin in the regulation of keratinocyte differentiation; finally, to test the role of E-cadherin in the regulation of CD9-mediated keratinocyte differentiation. These studies, taken together, would enable us a better understanding of how CD9 participates in wound reepithelialization and be involved in physiological wound healing, which has important theoretical significance and potential application value.

创面再上皮化是创面愈合的关键环节，表皮细胞分化调控贯穿于创面再上皮化过程，主要表现为早期新生表皮维持低分化状态高运动性而后期则需重塑表皮分化梯度。我们前期研究在明确“创面愈合早期新生表皮CD9下调促进细胞迁移，后期又恢复正常水平”的基础上发现CD9可调控表皮细胞分化，但机制尚不清楚。E-cadherin介导的细胞间粘附连接可促进表皮细胞分化，结合相关进展，我们提出“CD9同E-cadherin相互作用，使E-cadherin向表皮细胞膜上募集从而促进表皮细胞分化”的科学假设。研究拟首先从动物和细胞水平进一步明确CD9对表皮细胞分化的影响；继而检测CD9同E-cadherin相互作用及其调节表皮细胞分化的关键结构域；最后明确E-cadherin在CD9调节的表皮细胞分化中的作用。研究对深入揭示CD9调节表皮细胞分化的作用机理，深化对创面愈合发生机制的认识，有重要的理论意义和潜在的临床价值。

创面再上皮化是创面愈合的关键环节，表皮细胞分化调控贯穿于创面再上皮化过程。我们在前期研究明确CD9参与创面愈合调控的基础上，发现小鼠皮肤创面愈合重塑期CD9同分化标记物cytokeratin 10表达变化呈同向性改变,同时分化成熟的表皮细胞中CD9 的表达明显增加，提示CD9可调控表皮细胞分化。进一步建立CD9稳定高/低表达的表皮细胞模型，使用Western blot检测分化标记物(cytokeratin 1、cytokeratin 10、 loricrin 和filaggrin)的表达变化，观察CD9对表皮细胞直径及铺展面积的影响并使用Image J软件分析活细胞工作站动态拍摄的单个细胞运动性，发现CD9高表达增加表皮细胞直径，并上调表达分化标记物，同时下调表皮细胞运动性；而CD9低表达则可抑制表皮细胞分化并上调细胞运动性。继而观察CD9调控后表皮细胞中E-cadherin、β-catenin、P120-catenin复合体在表皮细胞中的表达分布变化，并检测PI3K/Akt激活情况，发现CD9可促进表皮细胞E-cadherin复合体向细胞膜上募集，激活下游PI3K/Akt信号通路，而CD9 低表达则可抑制这一过程。采用E-cadherin-siRNA下调CD9调控的表皮细胞中E-cadherin或使用抑制剂LY294002或Wortmannin抑制PI3K/Akt信号激活，进一步证实E-cadherin/PI3K/Akt参与CD9介导的表皮细胞分化及运动性之间转化的调节。而使用JNK信号通路抑制剂SP600125处理 CD9下调表达的表皮细胞，证实JNK通路参与调节CD9介导的E-cadherin向细胞膜上的募集。此外，利用体外三维培养皮肤及CD9基因敲除小鼠皮肤创面切片染色进一步证实下调或敲除CD9抑制表皮细胞E-cadherin向细胞膜上的募集及分化。本研究证实了“CD9通过抑制JNK信号通路，促进E-cadherin复合体向表皮细胞膜上募集并激活下游PI3K/Akt信号通路，从而促进表皮细胞分化并抑制细胞运动性”，另外还证实生物强度电场通过AMPK信号通路可下调表皮细胞中CD9的表达并介导细胞方向性迁移，进一步丰富了CD9对创面再上皮化调节作用及机制的理论体系，深化对创面愈合机制的认识，有重要的理论意义和潜在的临床价值。