NEUTROPHILS IN ISCHEMIA REPERFUSION INJURY IN SHOCK

休克缺血再灌注损伤中的中性粒细胞

• 批准号: 2444729
• 负责人: ROBERT K WINN
• 金额: $ 17.47万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444729
• 关键词: CD antigens; Primates; body temperature; cardiopulmonary resuscitation; enzyme linked immunosorbent assay; gene expression; hemorrhagic shock; human subject; hypothermia; immunocytochemistry; in situ hybridization; ischemia; laboratory rabbit; leukocyte activation /transformation; leukocyte adhesion molecules; messenger RNA; monoclonal antibody; neutrophil; northern blottings; phagocytosis; phorbols; reperfusion; selectins; tissue /cell culture; vascular endothelium

A significant portion of the injury that occurs following ischemia and reperfusion has been established to occur during the reperfusion period. This reperfusion injury is leukocyte mediated and blocking leukocyte adherence with monoclonal antibodies provides significant protection from injury. Likewise, mild hypotheremia (1-4 degrees C) during ischemia and reperfusion results in a significant reduction in injury. Ischemia-reperfusion is seen in a variety of clinical disorders including myocardial infarction, stroke, organ transplantation, hemorrhagic shock and traumatic injuries. Clearly, this broad spectrum of clinical disorders that may have a component of reperfusion injury places it as a major cause of mortality and morbidity in the United States. Prolonged ischemia in these disorders results in significant injury due to tissue hypoxia and rapid return of perfusion will reduce this type of injury. However, reperfusion of previously ischemic tissue can initiate an inflammatory response that results in significant additional tissue injury. Blocking leukocytes adhesion has been shown experimentally to significantly reduce portion of this reperfusion injury. Likewise, mild hypothermia has been shown to reduce the injury that occurs in ischemia and reperfusion. The hypotheses of this project are: 1) The expression and/or activation of leukocyte adhesion molecules on endothelial cells or on neutrophils (pMNs) is dependent on small changes in temperature (1 to 4 degrees C). Adhesion molecule function is reduced during mild hypothermia, thus providing protection from ischemia and reperfusion injury. and 2) Significant tissue protection results from mild hypothermia during the immediate post reperfusion period (4-8 hours). Therefore, maintaining patients slightly hypothermic during this period may provide protection from injury and be of significant therapeutic benefit. The specific aims are: Aim 1 A: To determine the stimulated expression of adhesion molecules CD62P, CD62E, CD106, and CD54 as well as expression of mRNA by cultured HUVECs using ELISA and Northern blot analysis at 33, 35, 37, and 39 degrees C. Aim 1 B: By understanding the mechanism of hypothermia-mediated inhibition of gene expression in endothelial cells, it will be possible to reverse or activate the effects of hypothermia in vivo - Aim 2: To measure indicators of PMN function and activation following stimulation of leukocytes with PMA, heat killed bacteria, and C5a. Temperature will be set at 33 degrees C, 35 degrees C, 37 degrees C and 39 degrees C. Aim 3: To determine the effect of mild hypothermia on leukocyte rolling and adherence under flow conditions at temperatures of 33, 35, 37, and 39 degrees C. Aim 4: To determine the quantitative changes in expression of CD62P, CD62E and CD54 and their mRNA in rabbit ear ischemia- reperfusion at ambient temperatures of 22 degrees C and 24 degrees C. Aim 5: To determine the timing of hypothermia that is effective in preventing tissue injury following ischemia and reperfusion. Aim 6: To determine the protective effects of reduced temperature following hemorrhagic shock and resuscitation. The experiments of this project will use cell and molecular biology techniques and will be completed by using in vivo and in vitro experiments.

很大一部分损伤发生在缺血和 已确定在再灌注期间发生再灌注。 这种再灌注损伤是白细胞介导的并且阻断白细胞 与单克隆抗体的粘附提供了显着的保护 免受伤害。 同样，轻度低体温（1-4 摄氏度） 缺血和再灌注导致损伤显着减少。 缺血再灌注见于多种临床疾病 包括心肌梗塞、中风、器官移植、 失血性休克和外伤。显然，这个广谱 可能与再灌注损伤有关的临床疾病 将其列为美国死亡率和发病率的主要原因 国家。这些疾病的长期缺血会导致显着的 组织缺氧和灌注快速恢复造成的损伤将减少 这种类型的伤害。然而，先前缺血组织的再灌注 可以引发炎症反应，导致显着 额外的组织损伤。已证明可阻断白细胞粘附 通过实验显着减少再灌注的部分 受伤。同样，轻度低温已被证明可以减少伤害 发生在缺血和再灌注时。本项目的假设 是：1）白细胞粘附分子的表达和/或激活 内皮细胞或中性粒细胞 (pMN) 取决于小 温度变化（1 至 4 摄氏度）。粘附分子功能 在轻度低温期间减少，从而提供保护 缺血和再灌注损伤。 2) 显着的组织保护 再灌注后立即发生轻度低温的结果 期间（4-8小时）。因此，保持患者轻微低温 在此期间可以提供免受伤害的保护并且是 显着的治疗效益。具体目标是： 目标 1 A： 确定粘附分子 CD62P、CD62E 的刺激表达， 使用培养的 HUVEC 检测 CD106 和 CD54 以及 mRNA 的表达 在 33、35、37 和 39 摄氏度下进行 ELISA 和 Northern 印迹分析。 1 B：通过了解低温介导的抑制机制 内皮细胞基因表达的改变，将有可能逆转 或激活体内低温的影响 - 目标 2：测量 PMN 功能和刺激后激活的指标 含有 PMA、热灭活细菌和 C5a 的白细胞。温度将为 设置为 33 摄氏度、35 摄氏度、37 摄氏度和 39 摄氏度。 目标 3：确定亚低温对白细胞滚动和 在温度为 33、35、37 和 39 的流动条件下的粘附性 目标 4：确定表达的定量变化 兔耳缺血中 CD62P、CD62E 和 CD54 及其 mRNA 的变化 在22℃和24℃环境温度下进行再灌注。 目标 5：确定有效的低温治疗时间 防止缺血和再灌注后的组织损伤。目标 6： 确定以下降低温度的保护作用 失血性休克和复苏。本项目的实验 将使用细胞和分子生物学技术，并将由 使用体内和体外实验。