MOLECULAR PATHOGENESIS OF BURN SHOCK

这项提议的目标是通过以下方式确定分子机制 肿瘤坏死因子-α和其他细胞因子诱导心脏 烧伤休克时的功能障碍。以前的工作已经证明，肿瘤坏死因子是 烧伤后心肌细胞分泌，并抑制其活性 烧伤后应用肿瘤坏死因子可明显改善心肌抑制。 这里提出的实验将阐明分子机制。 对心肌抑制负责，从而提供了生物学基础 用于未来的临床治疗。 我们认为烧伤诱导的细胞因子直接作用于心肌细胞改变 钙转运等相关蛋白的基因表达 对正常的收缩和放松至关重要的功能。这个假说 将通过确定细胞因子和其他烧伤的影响进行测试- 诱导产物对肌浆蛋白基因表达的影响 网状钙ATPase(SERCA)，钙外流通道(Ryanodine) 受体)和磷脂蛋白。此外，整个阵列中的更改 基因表达将通过基于聚合酶链式反应的系统表征 烧伤前后心肌细胞基因表达的差异显示 诱导调解人。到目前为止，实验已经表明，暴露在 心肌细胞对细胞因子的诱导表达诱导型一氧化氮 合酶(INOS)。通过使用发起人-记者结构，我们将 表征细胞内信号和转录激活 是诱导心肌细胞诱导型一氧化氮合酶所必需的。基因的诱导和表达 烧伤后心肌细胞中的一氧化氮合酶将通过 Northern和PCR分析，以及心肌细胞亚硝酸盐的测定 体外分泌。烧伤后心脏组织中肿瘤坏死因子的促分泌剂 也是未知的。确定内毒素是否是肿瘤坏死因子的刺激因素 烧伤休克期间的分泌物，我们将检测门静脉和 烧伤豚鼠的全身静脉血浆；然后我们将中和 持续输注杀菌增加蛋白(BPI)引起的内毒素 活体烧伤后，并确定心功能不全 是有改进的。最后，创建一个模型来研究这些影响 肿瘤坏死因子和次级介质在体内心脏的作用，并建立一种 可以测试针对细胞因子的治疗方法的模型，我们将推动 肌红蛋白启动子在转基因系统中表达小鼠肿瘤坏死因子基因 老鼠。这个模型将从分子、生理学和 从解剖学上讲。在这些研究结束后，我们将更好地 了解烧伤休克的分子发病机制，获得洞察力 为这种情况开发新的治疗方法。

The goal of this proposal is to determine the molecular mechanisms by which tumor necrosis factor-alpha (TNF) and other cytokines induce cardiac dysfunction during burn shock. Previous work has demonstrated that TNF is secreted by cardiac cells following burn injury, and that inhibition of TNF following burn injury significantly ameliorates myocardial depression. The experiments proposed here will elucidate the molecular mechanisms responsible for myocardial depression and thereby provide a biologic basis for future clinical therapies. We propose that burn-induced cytokines act directly on myocytes to alter gene expression for proteins involved in calcium transport and other functions vital to normal contractility and relaxation. This hvpothesis will be tested by determining the effects of cytokines and other burn- induced products on the expression of genes encoding sarcoplasmic reticulum calcium ATPase (SERCA), the calcium efflux channel (ryanodine receptor), and phospholamban. In addition, the entire array of changes in gene expression will be systematically characterized by PCR-based differential display of myocyte cDNA before and after exposure to burn- induced mediators. Thus far, experiments have indicated that exposure of myocytes to cytokines induces expression of inducible nitric oxide synthase (iNOS). Through the use of promoter-reporter constructs, we will characterize the intracellular signaling and transcriptional activation required for iNOS induction in myocytes. The induction and expression of iNOS in cardiac cells following burn injury will then be examined by Northern and PCR analysis, as well as by assay of myocyte nitrite secretion in vitro. The secretagogue for TNF in the heart following burns is also unknown. To determine whether endotoxin is the stimulus for TNF secretion during burn shock, we will measure endotoxin in the portal and systemic venous plasma of burned guinea pigs; we will then neutralize endotoxin by continuous infusion of bacteriacidal increasing protein (BPI) following burn injury in vivo, and determine whether cardiac dysfunction is improved. Finally, to create a model in which to study the the effects of TNF and secondary mediators on the heart in vivo, and to establish a model in which therapies against cytokines can be tested, we will drive expression of the mouse TNF cDNA by a myoglobin promoter in transgenic mice. This model will be characterized molecularly, physiologically, and anatomically. At the conclusion of these studies, we will better understand the molecular pathogenesis of burn shock, and gain insights into the development of novel treatments for this condition.