MODULATION OF NO WITH PULMONARY GENE TRANSFER

通过肺部基因转移调节 NO

• 批准号: 6725425
• 负责人: Loretta G Que
• 金额: $ 12.49万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6725425
• 关键词: apoptosis; arginase; biotechnology; cell proliferation; chemokine; enzyme activity; free radical oxygen; gene therapy; histology; hyperoxia; immunocytochemistry; inflammation; laboratory rabbit; lung injury; nitric oxide; nitric oxide synthase; oxidative stress; superoxide dismutase; transfection; transfection /expression vector; western blottings

Acute lung injury in the intensive care unit is treated with administration of high inspired oxygen tensions which can be toxic to the lung. Pulmonary oxygen toxicity is generally attributed to the excessive production of reactive oxygen intermediates (RO1), e.g., superoxide (O2), hydrogen peroxide (H2O2), and hydroxyl radicals. Recently nitric oxide (NO) is being used to treat hyperoxic lung disease, but its effect is variable and its role remains controversial. We hypothesize that selective modulation of NO/RO1 in different cellular compartments regulates tissue responses to hyperoxia. In this proposal, we will employ state of the art adenoviral gene transfer technique in vitro and in vivo. The transgenes we have selected for these studies are neuronal nitric oxide synthase (nNOS), extracellular superoxide dismutase (ECSOD), and arginases I and II (AI and AII). Overexpression of these four genes are expected to affect NO and reactive oxygen intermediates (RO1) production in different cell compartments: nNOS (yields intracellular NO/yields 02-), ECSOD (yields extracellular NO/yields 02-), and AI and AII (yields intracellular NO/yields O2-). We will determine how these transgenes modulate, oxidative/nitrosative stress, and NO/ROI mediated inflammation, cellular proliferation, and apoptosis. The specific aims of this proposal are 1) characterize the transgene expression of nNOS, ECSOD, AI, and AII in vitro 2) characterize intratracheal adenoviral gene transfer in normal rabbit lung and 3) evaluate how adenoviral mediated transgenes nNOS, ECSOD, AI, and AII modulate effect of NO in hyperoxia. These studies should not only better unravedl the basic mechanisms of hyperoxic lung injury, but also provide a rationale basis for therapeutic gene transfer using NO. Ultimately we are hopeful that these results may also extend to other oxidant injuries in the lung.

重症监护病房的急性肺损伤是用高吸氧张力治疗的，这可能对肺部有毒。肺氧毒性通常归因于活性氧中间体（RO1）的过量产生，如超氧化物（O2）、过氧化氢（H2O2）和羟基自由基。近年来，一氧化氮（NO）被用于治疗高氧性肺病，但其效果不一，其作用仍存在争议。我们假设不同细胞区室中NO/RO1的选择性调节调节了组织对高氧的反应。在这个建议中，我们将采用最先进的腺病毒基因转移技术在体外和体内。我们为这些研究选择的转基因是神经元型一氧化氮合酶（nNOS）、细胞外超氧化物歧化酶（ECSOD）和精氨酸酶I和II （AI和AII）。这四个基因的过表达预计会影响不同细胞区室中NO和活性氧中间体（RO1）的产生：nNOS（产生细胞内NO/产生02-）、ECSOD（产生细胞外NO/产生02-）、AI和AII（产生细胞内NO/产生O2-）。我们将确定这些转基因如何调节氧化/亚硝化应激，以及NO/ROI介导的炎症、细胞增殖和凋亡。本研究的具体目的是：1)表征nNOS、ECSOD、AI和AII在体外的转基因表达；2)表征正常家兔肺气管内腺病毒基因转移；3)评估腺病毒介导的转基因nNOS、ECSOD、AI和AII在高氧条件下对NO的调节作用。这些研究不仅可以更好地揭示高氧性肺损伤的基本机制，还可以为NO治疗性基因转移提供理论依据。最终，我们希望这些结果也可以扩展到肺部的其他氧化损伤。