In Vivo Role of NO in Mucosal Inflammation and Cancer

该项目将表征一氧化氮(NO)及其衍生物种在炎症性肠病(IBD)和癌症小鼠模型中的作用。这些研究将使用129/SvEv重组酶激活基因2(RAG-2)基因敲除(KO)小鼠和C57BL/6 T细胞受体(TCR)Alphabeta KO小鼠。具有129/SvEv背景的RAG-2 KO小鼠在感染肝包虫时会发生严重的IBD和癌症。RAG-2KO小鼠的组织病理损伤、巨噬细胞和中性粒细胞的浸润模式将与DNA和蛋白质的硝化、氧化和卤化的生物标志物相关。这些生物标志物将通过比较幼稚和有效的CD45RB高T细胞RAG-2 KO小鼠的疾病，以及通过N-甲基精氨酸(NMA)调节NO的产生来进一步验证。TCR AlphabetaKO小鼠感染肝吸虫后发展为严重的IBD。与RAG-2KO小鼠一样，TCRbetaKO小鼠的IBD是克罗恩病(CD)的模型，其特征是巨噬细胞激活，以及丰富的干扰素-γ(IFN)和肿瘤坏死因子-α(TNF)。TCRαKO小鼠是溃疡性结肠炎(UC)的模型，疾病的发生依赖于白介素4(K-4)，而不是干扰素或肿瘤坏死因子。TCRα和TCRβKO小鼠的组织病理损害和细胞因子水平将与生物标记物相关。为了进一步确定NO及其衍生物种在IBD中的作用，我们将通过产生iNOS缺陷的TCR Alphabeta KO小鼠来比较诱导型一氧化氮合酶(INOS)的药理抑制和该酶的遗传失活。还将对存在和不存在NO和NO衍生物种时体内产生的体细胞突变进行体外分析。通过使用新的IBD相关癌症模型和UC和CD模型，我们将能够验证DNA和蛋白质的硝化、氧化和卤化的新生物标记物。我们还将更好地了解NO和NO衍生物种在广泛的致癌事件中的作用，包括遗传毒性、细胞增殖、细胞毒性和血管生成。具体目标是： 特定目标#1.确定一氧化氮在IBD和RAG-2KO小鼠癌症中的作用 特定目标#2.研究一氧化氮和氧化应激在TCRα-β-KO小鼠IBD中的作用

This Project will characterize the role of nitric oxide (NO) and NO-derived species in mouse models of inflammatory bowel disease (IBD) and cancer. These studies will use 129/SvEv Recombinase-activating gene 2 (Rag-2) knockout (KO) mice and C57BL/6 T cell receptor (TCR) alphabeta KO mice. Rag-2 KO mice on a 129/SvEv background develop severe IBD and cancer when infected with H. hepaticus. Histopathologic lesions and the pattern of macrophage and neutrophil infiltration will be correlated with biomarkers for nitration, oxidation, and halogenation of DNA and proteins in Rag-2 KO mice. These biomarkers will be further validated by comparing disease in naive and effector CD45RB high T cell-bearing Rag-2 KO mice, and by modulating NO production with N-methylarginine (NMA). TCR alphabeta KO mice develop severe IBD when infected with H. hepaticus. IBD in TCRbeta KO mice, like in Rag-2 KO mice, is a model for Crohn's disease (CD) and features activated macrophages and abundant interferon-gamma (IFN) and tumor necrosis factor-alpha (TNF). TCR alpha KO mice are a model for ulcerative colitis (UC), and are dependent on interleukin-4 (K,-4) but not IFN or TNF for the development of disease. Histopathologic lesions and cytokine levels will be correlated with biomarkers in TCR alpha and TCR beta KO mice. To further characterize the role of NO and NO-derived species in IBD, we will compare pharmacologic inhibition of inducible nitric oxide synthase (iNOS) with genetic inactivation of the enzyme by generating iNOS-deficient TCR alphabeta KO mice. In vitro analysis of somatic mutations arising in vivo in the presence and in the absence of NO and NO-derived species will also be performed. By using a novel IBD associated cancer model and models for UC and CD, we will be able to validate new biomarkers for nitration, oxidation, and halogenation of DNA and proteins. We will also gain a better understanding of the role of NO and NO-derived species across a broad range of carcinogenic events, including genotoxicity, cellular proliferation, cytotoxicity, and angiogenesis. The Specific Aims are: Specific Aim #1. Characterize the role of NO an oxidative stress in IBD and cancer in Rag-2 KO mice Specific Aim #2. Characterize the role of NO and oxidative stress in IBD in TCR alphabeta KO mice