Enterocyte Apoptosis after Intestinal Resection

小肠切除后肠细胞凋亡

• 批准号: 7007622
• 负责人: BRAD Wayne WARNER
• 金额: $ 33.24万
• 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/7007622
• 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.

在大规模小肠切除（SBR）之后，剩余的肠子通过称为适应的关键过程补偿，其中包括肠肠细胞增殖，绒毛高度，肠子口径和长度的增加。 也许为了平衡这种有丝分裂的反应，还观察到肠细胞凋亡的率提高。 初步研究表明，促凋亡因子BAX的表达增加和生存因子Bcl-W的表达降低参与后凋亡后凋亡的调节。 表皮生长因子（EGF）通过增加肠上皮增殖来增强适应性。 尽管其意义尚不清楚，但EGF还抑制了后肠细胞凋亡。 该项目将测试总体假设，即通过抑制肠细胞凋亡，EGF在大规模小肠切除后增强了肠道适应性。 该项目的长期目标是通过描述肠道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。 我们的具体目的是：1）确定EGF受体刺激和抑制在适应过程中对BAX和BCL-W基因表达以及凋亡的影响。 为了测试EGF受体信号传导调节BAX和BCL-W在适应过程中直接肠细胞凋亡的表达的假设，激光捕获显微镜显微镜（LCM）显微镜将用于建立时间和区域性，区域性，肠肠细胞特异性表达BAX和BCL-W mRNA和BCL-W mRNA和蛋白质沿cragept-villus a s s s s s afters s s s aft sbr aft sbr。 然后，在EGF受体刺激和抑制条件下，将在SBR后LCM提取的肠细胞中确定BAX和BCL-W在肠细胞中的表达。 2）确定EGF减少肠细胞凋亡的机制。 为了测试假设，即通过BAX和BCL-W基因敲除小鼠进行EGF，SBR或假手术对EGF，SBR或假手术调节后凋亡所必需的BAX和BCL-W。 在EGF受体定向的放大或受损适应条件下，跨繁殖实验将允许直接测试不存在BAX或BCL-W表达的影响。 3）通过EGFR信号转导确定BAX和BCL-W表达的机制。 我们将使用肠上皮细胞开发一个体外模型系统，以测试相关的EGFR信号转导途径，以激活BAX和BCL-W表达。 这些研究将大大有助于增强对适应的遗传调节以及凋亡与该过程的相关性的理解。 该信息对于未来开发安全有效的临床疗法至关重要，旨在扩大患有大量肠道损失的患者的这种重要补偿性反应。