人角膜上皮细胞中胞浆型磷脂酶A2/花生四烯酸信号通路参与表皮生长因子活化NADPH氧化酶的作用及机制研究

Reactive oxygen species (ROS), including hysuperoxide anion（H2O2）,superoxide anion(0˙2ˉ) and hydroxyl radical(oOH)are the production of cell metabolism. ROS are known to be toxic and harmful molecules to cornea, retinal et al. Previously we have firstly demonstrated that epithelial growth factor (EGF) stimulation of corneal epithelical （HCE） cell produces transient ROS increase via NADPH oxidase. The low quantity of intracellular ROS has been shown to be involved in cell proliferation, migration and wound healing signalings as second messenger. However, the mechanisms for the generation of endogenous ROS is not understood. It has been found that cytosolic phospholipase A2 (cPLA2), arachidonic acid (AA)and protein kinase C (PKC) are related to respiratory brust in phagocytic cells. However, the mechanisms of how the action of EGF links to cPLA2, AA, PKC and how activates NADPH oxidase to produce ROS in HCE cells are not well understood. In this study, we are going to use cPLA2 dominant mutation cells and p47phox-siRNA-HCE cells to examined the upstream factors targets of NADPH oxidase by RT-PCR and Western blot studies. And using confocal microscopy, BrdU, cell wound model to analyse the effect on cell proliferation and wound healing.

目前活性氧自由基（ROS）与眼组织的研究都集中在其对角膜、视网膜等的毒性损害。我们前期研究首次发现表皮生长因子（EGF）刺激人角膜上皮（HCE）细胞内NADPH氧化酶产生的微量ROS参与细胞增殖与创伤修复发生。本课题旨在前期研究基础上深入探索NADPH氧化酶活化并产生ROS的分子机制。在吞噬细胞的研究中发现炎症等病理刺激可导致胞浆型磷脂酶A2（cPLA2）、花生四烯酸（AA）及蛋白激酶C（PKC）等因子表达的上调或活化，并被认为与氧化应激发生有关。但上述因子在HCE细胞生长因子信号通路中的作用机制尚不明确。本研究拟采用cPLA2显负性突变细胞株及p47phox-siRNA转染细胞株,通过RT-PCR、Western blot技术研究cPLA2/AA及PKC在EGF受体下游调控NADPH氧化酶活性的分子机制，利用激光共聚焦显微镜、BrdU及细胞创伤模型进一步分析其对细胞增殖及创伤修复的作用。

角膜位于眼球最前端，有可能受到紫外线照射、物理损伤和感染的危害。保持健康的角膜或修复创伤角膜对维护正常视力极为重要。因此了解人角膜上皮（HCE）细胞增殖及创伤修复的作用机制至关重要。表皮生长因子（EGF）可以刺激多种细胞增殖及移行。所以，我们研究了EGF在HCE细胞增殖及移行中的作用及具体机制。我们采用CCK-8试剂盒和创伤修复实验用于研究细胞增殖及移行的研究。 ROS活性由DCFDA荧光染料及流式细胞仪定量检测。Western blot和Q-PCR检查蛋白及RNA水平。结果发现，EGF在生理及病理条件下均可以促进HCE细胞增殖及移行。更为有趣的是，我们发现在HCE中，EGF通过ROS信号通路下调PKC蛋白水平，同时PKC信号通路并没有参与EGF刺激的角膜上皮细胞增殖及移行。ROS、MAPK/ERK和PKC在EGF在生理及病理条件下对保护人角膜上皮细胞增殖及移行作用中的作用各不相同。