CD9/ADAM17调节表皮细胞移行表型的作用与机制研究

Keratinocyte switching from resting to migratory phenotype is an essentical feature of epidermal migration during wound reepithelialization, in which the release of epithelial growth factor (EGF) family of growth factors derived from keratinocyte plays a crucial role. We found that CD9 functions as a negative regulator of the migratory phenotype of keratinocyte, the underlying mechanisms however remain unclear. We hypothesize here that CD9 correlates to migratory phenotype of keratinocyte by negatively regulating the sheddase activity of ADAM17, a membrane-anchored protease responsible for the release of EGF family growth factors by controlling the ectodomain shedding of the precusor of EGF family growth factors. To test it, we would set up experiments to :1) investigate the expression, location and direct interaction of CD9 with ADAM17 in both cultured keratinocyte and mice epidermis ; 2) determine the negative role of CD9 in sheddase activity of ADAM17 and its cause relationship with the migratory phenotype of keratinocyte; 3) elucidate the exact EGF family growth factor and its downstream signaling accounting for the regulatory role of CD9/ADAM17 in the migratory phenotype of keratinocyte; 4) reveal the structure domain in CD9 that mediates the interaction with ADAM17 in keratinocyte functionally; 5) provide in vivo evidence showing a crucial role played by CD9/ADAM17 interaction in maintaining the migratory phenotype of keratinocyte in skin wound using the CD9 trangenic mice. These studies, taken together, would enable us a better understanding of how CD9 participates in epidermal migration and involves in physiological or pathophysiological wound healing, which ae therefore theoretical and clinic significant.

创面再上皮化有赖于创缘表皮细胞移行表型的转换, 而表皮细胞来源的EGF家族分子在其中发挥重要作用。我们前期研究发现CD9是负调节表皮细胞移行表型的功能分子，但机制尚不清楚。结合相关进展，我们推测CD9负调节ADAM17裂解释放EGF家族分子的活性是其调节表皮细胞移行表型的重要机制。研究拟首先观察表皮细胞CD9与ADAM17的表达定位与相互作用；继而明确CD9对ADAM17活性的负调节作用以其与表皮细胞移行表型变化的因果关系，阐明对CD9/ADAM17调节表皮细胞移行表型起关键作用的EGF分子及其信号机制，揭示CD9调节ADAM17裂解释放EGF家族分子活性的功能结构域。最后，采用CD9转基因小鼠在体证明CD9 /ADAM17调节创面表皮细胞移行表型的重要作用和意义，研究对深入揭示CD9参与创面再上皮的作用机理，深化对创面愈合乃至创面慢性不愈发生机制的认识，有重要的理论意义和潜在的临床价值。

创面再上皮化有赖于创缘表皮细胞移行表型的转换, 而表皮细胞来源的EGF家族分子在其中发挥重要作用。我们前期研究发现CD9是负调节表皮细胞移行表型的功能分子，但机制尚不清楚。我们推测CD9负调节ADAM17裂解释放EGF家族分子的活性是其调节表皮细胞移行表型的重要机制。研究首先采用离体细胞培养和小鼠皮肤全层缺损创面模型，发现CD9与ADAM17在表皮细胞膜上具有共定位现象和相互作用关系。通过采用酵母双杂交系统，我们进一步证实，CD9的大胞外套环（LEL）与ADAM17的解聚素区是其相互作用的关键结构域。在此基础上，我们通过调控CD9基因表达水平，发现CD9并不影响ADAM17成熟蛋白的表达水平，但对ADAM17脱落酶活性具有重要的负调节作用。采用细胞划痕实验发现，ADAM17脱落酶活性降低导致其底物HB-EGF的裂解释放减少，并进而使HB-EGF/EGFR/ERK途径受到抑制是CD9负调控表皮细胞迁移和创面愈合的重要分子信号机制。研究对深入揭示CD9参与创面再上皮的作用机理，深化对创面愈合乃至创面慢性不愈发生机制的认识，具有重要的理论意义。靶向调控CD9/ADAM17轴有可能成为促进表皮迁移和创面愈合的新型策略。