Enterocyte Apoptosis after Intestinal Resection

小肠切除后肠细胞凋亡

• 批准号: 6845964
• 负责人: BRAD Wayne WARNER
• 金额: $ 6.85万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6845964
• 关键词: Bax gene /protein; RNase protection assay; apoptosis; biological signal transduction; cell proliferation; epidermal growth factor; gastrointestinal epithelium; gene expression; gene targeting; genetic regulation; genetically modified animals; growth factor receptors; immunocytochemistry; intestinal villi; laboratory mouse; laser capture microdissection; messenger RNA; mitogen activated protein kinase; phosphatidylinositol 3 kinase; polymerase chain reaction; proliferating cell nuclear antigen; receptor expression; short bowel syndrome; small intestines; terminal nick end labeling; western blottings

After massive small bowel resection (SBR), the remaining intestine compensates by a critical process termed adaptation, which includes increases in enterocyte proliferation, villus height, bowel caliber and length. Perhaps to counterbalance this mitogenic response, increased rates of enterocyte apoptosis are also observed. Preliminary studies suggest that increased expression of the pro-apoptosis factor bax and reduced expression of the prosurvival factor bcl-w are involved in the regulation of postresection apoptosis. Epidermal growth factor (EGF) enhances adaptation by increasing enterocyte proliferation. While its significance is not known, EGF also inhibits postresection enterocyte apoptosis. This project will test that overall hypothesis that EGF enhances intestinal adaptation after massive small bowel resection by inhibiting enterocyte apoptosis. The long-term goal of this project is to optimize therapy for patients with the short bowel syndrome by delineating the significance of enterocyte apoptosis during intestinal adaptation. Our specific aims are: 1) Determine the effect of EGF receptor stimulation and inhibition on bax and bcl-w gene expression and apoptosis during adaptation. To test the hypothesis that EGF receptor signaling regulates the expression of bax and bcl-w to direct enterocyte apoptosis during adaptation, laser capture microdissection (LCM) microscopy will be used to establish a temporal and regional, enterocyte-specific expression of bax and bcl-w mRNA and protein along the crypt- villus axis after SBR. The expression of bax and bcl-w in enterocytes will then be determined in LCM extracted enterocytes after SBR during conditions of EGF receptor stimulation and inhibition. 2) Determine the mechanism for reduced enterocyte apoptosis by EGF. To test the hypothesis that bax and bcl-w are required for the regulation of postresection apoptosis by EGF, SBR or sham operations will be performed in bax and bcl-w knockout mice. Crossbreeding experiments will permit direct testing of the effect of absent bax or bcl-w expression during conditions of EGF receptor-directed amplified or impaired adaptation. 3) Determine the mechanism for Bax and Bcl-w expression by EGFR signal transduction. We will develop an in vitro model system using intestinal epithelial cells to test relevant EGFR signal transduction pathways for the activation of bax and bcl-w expression. These studies will substantially contribute toward an enhanced understanding of the genetic regulation of adaptation and the relevance of apoptosis to this process. This information is vital for the future development of safe and effective clinical therapy designed to amplify this important compensatory response in patients suffering massive intestinal loss.

在大量小肠切除(SBR)后，剩余的肠道通过一个称为适应的关键过程进行补偿，该过程包括增加肠细胞的增殖、绒毛高度、肠管口径和长度。也许为了抵消这种有丝分裂反应，还观察到肠细胞凋亡率的增加。初步研究表明，促凋亡因子bax的表达增加和促生存因子bclw的表达减少参与了术后细胞凋亡的调控。表皮生长因子(EGF)通过促进肠道细胞的增殖来增强适应能力。虽然其意义尚不清楚，但EGF也可抑制术后肠细胞的凋亡。该项目将检验这一总体假设，即EGF通过抑制肠细胞凋亡来增强大量小肠切除后的肠道适应。该项目的长期目标是通过阐明肠适应过程中肠细胞凋亡的重要性来优化短肠综合征患者的治疗。我们的具体目标是：1)确定EGF受体的刺激和抑制对适应过程中bax和bclw基因表达和细胞凋亡的影响。为了验证在适应过程中EGF受体信号调节bax和bclw的表达从而引导肠细胞凋亡的假设，将使用激光捕获显微解剖(LCM)显微镜建立SBR后肠绒毛轴上Bax和bclw基因和蛋白的暂时和区域性的肠细胞特异性表达。在EGF受体刺激和抑制的条件下，检测SBR后LCM提取的肠上皮细胞中bax和bclw的表达。2)探讨EGF减少肠上皮细胞凋亡的机制。为了验证BAX和BCL-W基因敲除小鼠需要通过EGF、SBR或SHAM手术来调节切除后的细胞凋亡这一假设。杂交实验将允许直接测试在EGF受体引导的放大或受损适应条件下缺乏bax或bclw表达的影响。3)通过EGFR信号转导途径探讨Bax、Bclw表达的机制。我们将建立一个使用肠上皮细胞的体外模型系统，以检测相关的EGFR信号转导通路对bax和bclw表达的激活。这些研究将大大有助于加深对适应的遗传调节以及细胞凋亡与这一过程的相关性的理解。这一信息对于安全有效的临床治疗的未来发展至关重要，该治疗旨在放大遭受大量肠道丢失的患者的这一重要的代偿反应。