PLATELET ACTIVATING FACTOR AND EPIDERMAL CYTOTOXICITY

这项研究的长期目标是阐明甘油磷胆碱衍生的介体-血小板激活因子(PAF)在角质形成细胞细胞毒性中的作用。最近的研究表明，包括中波紫外线(UVB)在内的许多临床上相关的细胞毒剂对角质形成细胞(HK)的损伤部分是通过诱导氧化应激和细胞因子的产生来实现的。严重的中波紫外线对香港的损伤也会导致细胞程序性死亡(细胞凋亡)。由于PAF是在氧化应激和许多促炎细胞因子的作用下合成的，并且HK表达PAF受体(PAF-R)，因此通过逆转录病毒介导将PAF-R转导到PAF-R阴性的表皮细胞系KB中建立的模型系统，评估了PAF-R系统在UVB介导的角质形成细胞损伤中的作用。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，对表皮细胞的氧化应激导致PAF-R激动性活性的产生，质谱学研究表明，该活性在结构上不是PAF。基于抗氧化剂抑制这种活性的能力以及UVB诱导表达PAF-R的KB细胞凋亡的增加，我们假设对HK的氧化损伤导致氧化脂质的产生，而氧化脂质可以通过激活PAF-R来调节细胞凋亡。三个特定的目标将通过以下三个具体目标来验证这一假说：1)评估PAF-R激动剂/拮抗剂以及野生型或反义PAF-R构建体在体外对UVB诱导的角质形成细胞凋亡的影响；2)评估PAF-R拮抗剂以及野生型或反义PAF-R构建体在体内对UVB诱导的SCID/HK异种移植瘤细胞凋亡的影响；以及3)通过质谱仪表征导致UVB诱导的表皮细胞非PAF PAF-R激动化活性的脂类。该项目将确定PAF系统在香港氧化应激反应中的意义，并从结构上定义新的具有生物活性的脂质过氧化标志物，这些信息可能导致使用PAF-R拮抗剂治疗以大规模表皮损伤为特征的疾病的潜在治疗策略。

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.