MOLECULAR PATHOGENESIS OF BURN SHOCK

这项建议的目标是确定分子机制， 其中肿瘤坏死因子-α（TNF）和其他细胞因子诱导心脏 烧伤休克时的功能障碍以前的研究表明，TNF是 烧伤后心脏细胞分泌的， 烧伤后TNF显著改善心肌抑制。 本文提出的实验将阐明分子机制 导致心肌抑制，从而提供生物学基础 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， 参与钙转运和其他蛋白质的基因表达 对正常的收缩和放松至关重要。这个假设 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 内质网钙ATP酶（SERCA），钙外排通道（ryanodine 受体）和受磷蛋白。此外， 基因表达将通过基于PCR的方法系统地表征。 烧伤前后心肌细胞cDNA的差异显示 诱导介质。到目前为止，实验表明， 心肌细胞对细胞因子诱导诱导型一氧化氮的表达 合成酶（iNOS）。通过使用启动子-报告子构建体，我们将 表征细胞内信号传导和转录激活 需要在心肌细胞中诱导iNOS。的归纳与表达 烧伤后心肌细胞中的iNOS将通过 北方和PCR分析以及肌细胞亚硝酸盐测定 体外分泌。 烧伤后心脏TNF的促分泌作用 也是未知的。确定内毒素是否是TNF的刺激物 分泌，我们将测量门静脉中的内毒素， 烧伤豚鼠的全身静脉血浆;然后我们将中和 持续输注杀菌增强蛋白（BPI） 在体内烧伤后，并确定是否心功能不全 提高了最后，建立一个模型来研究影响 的TNF和二级介质对心脏在体内，并建立一个 模型中，针对细胞因子的疗法可以测试，我们将推动 肌红蛋白启动子在转基因小鼠中表达小鼠TNF cDNA 小鼠该模型将在分子、生理和 从解剖学上讲在这些研究结束时，我们将更好地 了解烧伤休克的分子发病机制， 开发治疗这种疾病的新方法

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.