PLATELET ACTIVATING FACTOR AND EPIDERMAL CYTOTOXICITY

该研究的长期目标是阐明甘油酰胆碱衍生介质血小板活化因子（PAF）在角质形成细胞毒性中的作用。最近的研究表明，许多临床相关的细胞毒性药物，包括紫外线B （UVB）辐射，在一定程度上通过诱导氧化应激和细胞因子的产生来损害人角质形成细胞（HK）。中波对HK的严重损伤也可导致程序性细胞死亡（凋亡）。由于PAF是在氧化应激和许多促炎细胞因子的反应中合成的，而HK表达PAF受体（PAF- r），因此使用逆转录病毒介导的PAF-r转导到PAF-r阴性表皮细胞系KB创建的模型系统来评估PAF系统在uvb介导的角质细胞损伤中的参与。表皮PAF-R的表达增加了对uvb诱导的细胞凋亡的敏感性。此外，表皮细胞氧化应激导致产生PAF- r激动活性，质谱研究显示结构上不是PAF。基于抗氧化剂抑制PAF-R活性的能力，以及uvb诱导表达PAF-R的KB细胞凋亡的增强，我们假设HK的氧化损伤导致氧化脂质的产生，氧化脂质的产生可以通过PAF-R激活来调节凋亡。为了验证这一假设，有三个特定的目的：1)评估PAF激动剂/拮抗剂以及野生型或反义PAF- r构建体的过表达对体外uvb诱导的角质细胞凋亡的影响；2)评估PAF-R拮抗剂和野生型或反义PAF-R构建体过表达对uvb诱导的SCID/HK异种移植物体内凋亡的影响；3)质谱分析uvb诱导表皮细胞非paf - PAF-R激动活性的脂质种类的结构特征。该项目将确定PAF系统在HK氧化应激反应中的重要性，并从结构上定义新的脂质过氧化生物活性标记物，这些信息可能导致涉及PAF- r拮抗剂的潜在治疗策略，用于以大量表皮损伤为特征的疾病。

The long-term objective of the proposed research is to elucidate the role of the glycerophosphocholine-derived mediator Platelet- activating Factor (PAF) in keratinocyte cytotoxicity. Recent studies have demonstrated that many clinically relevant cytotoxic agents including ultraviolet B (UVB) radiation damage human keratinocytes (HK) in part by inducing oxidative stress and cytokine production. Severe UVB damage to HK can also result in programmed cell death (apoptosis). Because PAF is synthesized in response to oxidative stress and many pro-inflammatory cytokines, and HK express PAF-receptors (PAF-R), involvement of the PAF system in UVB-mediated keratinocyte damage has been assessed using a model system created by retroviral-mediated transduction of the PAF-R into the PAF-R-negative epidermal cell line KB. Expression of the epidermal PAF-R resulted in an increased susceptibility to UVB-induced apoptosis. In addition, oxidative stress to epidermal cells resulted in the production of a PAF-R agonistic activity which mass spectrometric studies revealed was structurally not PAF. Based on the ability of antioxidants to inhibit this activity as well as the augmentation of UVB-induced apoptosis in PAF-R-expressing KB cells, we hypothesize oxidative damage to HK results in the production of oxidized lipids which can modulate apoptosis through PAF-R activation. Three specific aims will test this hypothesis by 1) Assessing the effects of PAF agonists/antagonists and overexpression of the wild-type or antisense PAF-R constructs on UVB-induced keratinocyte apoptosis in vitro; 2) Assessing the effects of PAF-R antagonists and overexpression of wild-type or antisense PAF-R constructs on UVB-induced apoptosis in SCID/HK xenografts in vivo; and 3) Structural characterization of the lipid species responsible for the UVB-induced non-PAF PAF-R agonistic activity in epidermal cells by mass spectrometry. The project will determine the significance of the PAF system in the oxidative stress response in HK, as well as to structurally define novel biologically active markers of lipid peroxidation, information which could result in potential therapeutic strategies involving PAF-R antagonists for diseases characterized by massive epidermal damage.