The Role Of Growth Factors And Inflammatory Mediators In

生长因子和炎症介质在疾病中的作用

• 批准号: 6534982
• 负责人: Dori R Germolec
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6534982
• 关键词: apoptosis; arsenic; cell proliferation; cytokine; cytotoxicity; dioxins; endotoxins; environmental exposure; environmental toxicology; fibroblasts; flow cytometry; gene expression; gene mutation; genetically modified animals; glutathione; growth factor; human tissue; inflammation; keratinocyte; laboratory mouse; metal poisoning; microarray technology; northern blottings; oxidative stress; polymerase chain reaction; skin hypersensitivity; tissue /cell culture

The main focus of this project is to investigate the complex cytokine regulatory network involved in arsenic-induced dermatotoxicity. We have completed in vitro studies that evaluate the toxicity of both ArsenicIII and ArsenicV, and their monomethyl and dimethyl metabolites in normal human keratinocyte cultures. We are comparing these results with ongoing studies to examine the relative toxicity of ArsenicIII and ArsenicV, and their monomethyl and dimethyl metabolites in the Tg.AC transgenic mouse model. Microarray studies using NHEK indicate that short-term, non-toxic arsenic-exposure results in the modulation of multiple genes from several classes (e.g., oxidative stress, glutathione metabolism, heat shock/stress response, cell proliferation and DNA damage). Gene expression changes are currently being validated via PCR and Northern analysis. Additional microarray studies in progress are designed to examine similar/dissimilar gene expression profiles in NHEK and HaCaT keratinocytes (an immortalized cell line commonly used in arsenic, oxidative stress, and epidermal research) following arsenic exposure. Pathway mapping, FACs analysis and viability studies also have been performed and will be used to correlate gene expression alterations with physiological effects (e.g., cell cycle arrest, DNA damage, elevated cell proliferatory responses, etc.). The role of glutathione in arsenic-induced toxicity also is being examined and our studies suggest that glutathione may play a role not only in attenuating the toxic effects of arsenic but also in the positive modulation of arsenic-induced proliferation. Arsenic-mediated effects on viability and long-term growth in dermal fibroblasts have been evaluated. Data indicate that viability is affected to the same degree as that seen in human keratinocytes, suggesting a close relationship between arsenic tolerance in both cell types. Dermal fibroblasts cannot tolerate long-term growth in concentrations of arsenic > 5 mM, similar to that of keratinocytes. RNA samples from dermal fibroblasts have been obtained from time- and dose-response experiments and are in the process of being analyzed via RT-PCR and northern blotting. It is hypothesized that arsenic exposure alters the expression/production/inducibility of mitogens such as KGF and TGF in dermal fibroblasts, and these changes are predicted to alter keratinocyte physiology/function (e.g., proliferation, differentiation and cytokine expression). A second component of this project uses in vivo models of endotoxin hypersensitivity to examine the relationship between TNF signaling and apoptosis. We have characterized the kinetics of TCDD-induced hepatic damage in the liver by evaluating serum enzyme levels, quantifying apoptotic cells, and measuring alterations in gene expression at critical time points in B6C3F1 mice exposed to TCDD in the presence or absence of endotoxin. TCDD modulated endotoxin-mediated early expression of Fas, and upregulated expression of caspase 3 (day 10), NFkappaB (day 14), and TNFalpha (days 10 and 14). In addition, combined TCDD/endotoxin altered the kinetics of TCDD-induced hepatoxicity and caspase activity, with peak levels occurring 3-4 days earlier. TNFR1 and TNFR2 gene expression, IkappaBalpha and IkappaBbeta protein expression, and NFkappaB DNA-binding activity did not appear to be modulated by TCDD under the conditions of the study. Previous studies had shown that when rodents are treated with TCDD prior to endotoxin exposure a significant increase in toxicity occurs, and that inhibition of protein synthesis with cycloheximide blocks the TCDD-induced sensitivity to TNF in this model. Overexpression of caspases has been shown to induce apoptosis, and activation of the caspases appears to be necessary for development of the apoptotic phenotype. The protective effects of cycloheximide and alterations in TNFalpha, and NFkappaB gene expression suggest that TCDD treatment may result in alterations in TNF-induced activation of NFkappaB via the TNFalpha/TNFR2/TRAF pathway. The alterations in Fas and Caspase 3 gene expression indicate initiation of apoptosis and suggest involvement of the Fas Ligand/Fas/Caspase pathway.

本项目的主要重点是研究砷诱导的皮肤毒性中复杂的细胞因子调控网络。我们已经完成了体外研究，评估了砷III和砷V的毒性，以及它们在正常人角质形成细胞培养物中的单甲基和二甲基代谢产物。我们正在将这些结果与正在进行的研究进行比较，以检查砷III和砷V及其单甲基和二甲基代谢产物在Tg.AC转基因小鼠模型中的相对毒性。使用NHEK的微阵列研究表明，短期、无毒的砷暴露导致来自几个类别的多个基因的调节（例如，氧化应激、谷胱甘肽代谢、热休克/应激反应、细胞增殖和DNA损伤）。基因表达变化目前正在通过PCR和北方分析进行验证。正在进行的其他微阵列研究旨在研究砷暴露后NHEK和HaCaT角质形成细胞（常用于砷，氧化应激和表皮研究的永生化细胞系）中相似/不相似的基因表达谱。还进行了途径作图、FACs分析和活力研究，并将用于将基因表达改变与生理效应（例如，细胞周期停滞、DNA损伤、升高的细胞增殖反应等）。谷胱甘肽在砷诱导的毒性中的作用也正在研究中，我们的研究表明，谷胱甘肽可能发挥作用，不仅在减弱砷的毒性作用，但也在砷诱导的增殖的积极调制。砷介导的影响，真皮成纤维细胞的活力和长期生长进行了评估。数据表明，活力的影响程度相同，在人类角质形成细胞，这表明砷耐受性在两种细胞类型之间的密切关系。真皮成纤维细胞不能耐受砷浓度> 5 mM的长期生长，与角质形成细胞相似。已从时间和剂量反应实验中获得了真皮成纤维细胞的RNA样品，并正在通过RT-PCR和北方印迹法进行分析。假设砷暴露改变了真皮成纤维细胞中有丝分裂原如KGF和TGF的表达/产生/诱导，并且预测这些变化会改变角质形成细胞的生理学/功能（例如，增殖、分化和细胞因子表达）。该项目的第二部分使用内毒素超敏反应的体内模型来检查TNF信号传导和细胞凋亡之间的关系。我们的特点是TCDD诱导的肝脏损伤的动力学，通过评估血清酶水平，定量凋亡细胞，并在关键时间点在B6 C3 F1小鼠暴露于TCDD在存在或不存在内毒素的情况下，测量基因表达的变化。TCDD调节内毒素介导的Fas早期表达，并上调caspase 3（第10天），NF κ B（第14天）和TNF α（第10和14天）的表达。此外，结合TCDD/内毒素改变了TCDD诱导的肝毒性和caspase活性的动力学，峰值水平出现在3-4天前。TNFR 1和TNFR 2基因表达、IkappaB α和IkappaB β蛋白表达以及NF κ B DNA结合活性在研究条件下似乎不受TCDD调节。先前的研究表明，当啮齿动物在内毒素暴露之前用TCDD处理时，毒性显著增加，并且在该模型中用放线菌酮抑制蛋白质合成阻断了TCDD诱导的对TNF的敏感性。半胱天冬酶的过表达已被证明可诱导细胞凋亡，并且半胱天冬酶的激活似乎是细胞凋亡表型发展所必需的。放线菌酮的保护作用和TNF α和NF κ B基因表达的改变表明，TCDD治疗可能导致TNF诱导的NF κ B活化通过TNF α/TNFR 2/TRAF途径的改变。Fas和Caspase 3基因表达的改变表明细胞凋亡的开始，并表明Fas配体/Fas/Caspase途径的参与。