Preservation of mucosal barrier in surgical diseases

描述(申请人提供)：外科危重疾病患者，如热损伤、严重创伤、休克和脓毒症，通常合并胃肠粘膜损伤，可导致全身感染、感染性休克和多器官衰竭。重建是修复受损肠壁的一个重要过程。以前的研究表明，危重患者的肠道上皮重建会延迟。我们最近的工作揭示了乳脂球-EGF因子8(MFG-E8)是一种巨噬细胞来源的肠上皮修复因子。MFG-E8通过依赖PKC5的方式促进肠上皮损伤愈合。在脓毒症中，肠道损伤与MFG-E8的下调有关，MFG-E8的下调有助于恢复正常。因此，在本提案中，我们将检验一个中心假设，即在生理状态下，MFG-E8通过典型的信号通路维持肠上皮细胞的迁移，而在脓毒症条件下，炎症介质下调MFG-E8的表达，从而损害肠上皮细胞的恢复。在具体目标1中，我们将确定MFG-E8促进肠上皮修复的分子和信号机制。我们的初步数据表明，MFG-E8激活了几个与PKC5相关的细胞内信号分子。因此，我们将利用分子生物学和基因敲除的方法研究这些分子在MFG-E8诱导的肠上皮细胞迁移中的作用。在具体目标2中，我们将重点了解脓毒症时肠道MFG-E8基因表达下调和MFG-E8依赖的肠道重建受损的原因。我们的初步数据表明，脓毒症衍生的炎症介质模拟了脓毒症对肠道MFG-E8基因表达和修复的影响。因此，我们将验证这一假设，即该介体抑制肠道MFG-E8基因的表达，从而导致肠道重建的损害。在特定的目标3中，我们将研究败血症诱导性炎症刺激抑制巨噬细胞MFG-E8表达的分子机制。我们的初步数据表明，炎症刺激对MFG-E8基因表达的抑制参与了影响MFG-E8启动子和MFG-E8的3‘-非翻译区(3’-UTR)的活性。为了跟进这些发现，我们将鉴定MFG-E8基因启动子及其结合蛋白中的结合基序，并研究它们在炎症过程中抑制MFG-E8基因表达的作用。将采用分析基因启动子功能的标准方法。此外，我们将使用microRNA和定点突变技术来研究3‘-UTR和microRNAs在炎症刺激调节MFG-E8基因表达中的作用。这些特定目的的实现和我们的假设的证实将提供新的机制信息，使我们能够理解MFG-E8在肠道中的关键作用及其生理/病理意义。我们的研究最终将导致开发新的策略来管理和治疗危重患者的肠道损伤。

DESCRIPTION (provided by applicant): Surgical patients with critical disorders such as thermal injury, sever trauma, shock, and sepsis are commonly complicated with the gastrointestinal mucosal injury, which can lead to the development of systemic infection, septic shock, and multiple organ failure. Restitution is an important process for repair of disrupted intestinal lining. Previous studies have suggested that intestinal epithelial restitution is delayed in critically ill individuals. Our recent work has revealed that milk fat globule-EGF factor 8 (MFG-E8) is a macrophage-derived intestinal epithelial restitution factor. MFG-E8 promotes intestinal epithelial wound-healing via a PKC5 dependent manner. In sepsis, intestinal injury is associated with downregulation of MFG-E8, which contributes to the impairment of restitution. In this proposal, therefore, we will test a central hypothesis that under the physiological state, MFG-E8 maintains intestinal epithelial cell migration via a typical signal pathway, whereas under the septic condition, inflammatory mediators down-regulate MFG-E8 expression, which in turn impairs intestinal epithelial cell restitution. In Specific Aim 1, we will define the molecular and signaling mechanism through which MFG-E8 enhances intestinal epithelial restitution. Our preliminary data suggest that MFG-E8 activates several PKC5-associated intracellular signal molecules. Therefore, we will investigate the role of these molecules in MFG-E8-induced intestinal epithelial cell migration using molecular biology and gene knockdown approaches. In Specific Aim 2, we will focus on understanding the cause of down-regulation of intestinal MFG-E8 gene expression and impairment of MFG-E8-dependent intestinal restitution in sepsis. Our preliminary data suggest that a sepsis-derived inflammatory mediator mimics septic effect on intestinal MFG-E8 gene expression and restitution. Thus, we will test the hypothesis that this mediator inhibits intestinal MFG-E8 gene expression, which subsequently results in impairment of intestinal restitution. In Specific Aim 3, we will investigate the molecular mechanism underlying the inhibition of MFG-E8 expression in macrophages by septic insult-derived inflammatory stimulus. Our preliminary data show that repression of MFG-E8 gene expression by an inflammatory stimulus is involved in affecting the activities of promoter and mRNA 3'-untranslated region (3'-UTR) of MFG-E8. To follow up on these findings, we will characterize binding motifs in the MFG-E8 gene promoter and their binding proteins, and study their role in suppression of MFG-E8 gene expression during inflammation. The standard approach for analysis of the gene promoter function will be applied. In addition, we will use the microRNA and site-directed mutagenesis technologies to study the role of 3'-UTR and microRNAs in regulation of MFG-E8 gene expression by an inflammatory stimulus. Achievement of these specific aims and confirmation of our hypotheses will provide the novel mechanistic information that allows us to understand the crucial role of MFG-E8 in the gut and its physio/pathological implications. Our studies will ultimately lead to development of novel strategies for management and treatment of intestinal injury in critically ill patients.