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Pathogenesis of Experimental Necrotizing Enterocolitis

实验性坏死性小肠结肠炎的发病机制

基本信息

批准号：
7107149
负责人：
HENRI R FORD
金额：
$ 37.18万
依托单位：
CHILDREN'S HOSPITAL OF LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-02-01 至 2008-01-31
项目状态：
已结题

项目摘要

Necrotizing enterocolitis (NEC) is the most frequent and the most lethal disease that affects the gastrointestinal tract of the premature infant. The exact etiology of the disease is undefined. The only consistent identifiable epidemiological precursors for NEC are prematurity and enteral alimentation. We have previously shown upregulation of inducible nitric oxide (NO) synthase (NOS-2) mRNA and protein in intestinal segments from infants with acute NEC. NOS-2 colocalized with enterocyte apoptosis and nitrotyrosine immunoreactivity. NOS-2 was downregulated at the time of intestinal stoma closure when the acute inflammation had subsided. We have developed a reproducible model of gut inflammation in neonatal rats thereby simulating the conditions associated with human NEC. We simply formula-feed hypoxic neonatal rats thereby simulating the conditions associated with human NEC. These pups show NOS-2 mRNA upregulation, nitrosative stress, increased enterocyte apoptosis and decreased enterocyte proliferation in the crypts. Intraepithelial lymphocytes (IEL)from hypoxic formula-fed rats secrete more TNF-alpha and IFN- gamma compared to breast-fed rats. In vitro studies suggest that co- culture of IEL with the rat intestinal epithelial cell line IEC-18, in the presence of IL-1beta induces IFN-gamma, TNF-alpha and NOS-2 production which can be abrogated with antibody to IFN-gamma. Furthermore, peroxynitrite (ONOO) induces apoptosis and inhibits proliferation of IEC-6 cells. The data suggest that intestinal necrosis in NEC may be the result of NO-induced imbalance between tissue injury and repair mechanisms. Mucosal injury resulting from perinatal insults leads to bacterial-epithelial interactions, local release of cytokines such as IFN-gamma and TNF-alpha by IEL and lamina propria (LP) lymphocytes. These mediators induce NOS-2 upregulation with production of NO and ONOO by enterocytes or LP macrophages. NO or ONOO in turn promotes further tissue injury (enterocyte apoptosis) and concurrent inhibition of tissue repair mechanisms (enterocyte proliferation) leading to gut barrier failure and NEC. To test our hypothesis, we propose the following Aims: I) To define the mechanism of NOS-2 upregulation in human and experimental rodent NEC. II) To define the mechanisms by which NOS-2 upregulation promotes intestinal injury and alters tissue repair mechanisms in rodent NEC. III) To determine the effects of scavengers of NO or inhibitors of NO production on the development of experimental rodent NEC.

坏死性小肠结肠炎（NEC）是影响早产儿胃肠道的最常见和最致命的疾病。该疾病的确切病因尚不清楚。 NEC 唯一一致可识别的流行病学前兆是早产和肠内营养。我们之前已经发现，患有急性 NEC 的婴儿肠段中诱导型一氧化氮 (NO) 合酶 (NOS-2) mRNA 和蛋白质的上调。 NOS-2 与肠细胞凋亡和硝基酪氨酸免疫反应性共定位。当急性炎症消退时，肠造口关闭时，NOS-2 下调。我们开发了新生大鼠肠道炎症的可重复模型，从而模拟与人类 NEC 相关的条件。我们简单地用配方奶喂养缺氧的新生大鼠，从而模拟与人类 NEC 相关的条件。这些幼崽在隐窝中表现出 NOS-2 mRNA 上调、亚硝化应激、肠上皮细胞凋亡增加和肠上皮细胞增殖减少。与母乳喂养的大鼠相比，缺氧配方喂养的大鼠的上皮内淋巴细胞（IEL）分泌更多的 TNF-α 和 IFN-γ。体外研究表明，在IL-1β存在的情况下，IEL与大鼠肠上皮细胞系IEC-18共培养会诱导IFN-γ、TNF-α和NOS-2的产生，而这些产生可以被IFN-γ抗体消除。此外，过氧亚硝酸盐 (ONOO) 诱导 IEC-6 细胞凋亡并抑制增殖。数据表明，NEC 肠坏死可能是 NO 诱导的组织损伤和修复机制失衡的结果。围产期损伤引起的粘膜损伤会导致细菌-上皮相互作用、IEL 和固有层 (LP) 淋巴细胞局部释放细胞因子，例如 IFN-γ 和 TNF-α。这些介质通过肠上皮细胞或 LP 巨噬细胞产生 NO 和 ONOO 来诱导 NOS-2 上调。 NO 或 ONOO 反过来会促进进一步的组织损伤（肠细胞凋亡）并同时抑制组织修复机制（肠细胞增殖），导致肠道屏障衰竭和 NEC。为了检验我们的假设，我们提出以下目标： I) 定义人类和实验啮齿动物 NEC 中 NOS-2 上调的机制。 II) 明确啮齿动物 NEC 中 NOS-2 上调促进肠道损伤并改变组织修复机制的机制。 III)确定NO清除剂或NO产生抑制剂对实验性啮齿动物NEC发育的影响。