Regulation of intestinal development by a Lactobacillus GG-derived protein

 描述（由申请人提供）：肠道微生物群与宿主之间的共生关系在肠道发育中起着重要作用。中断此 关系导致传染病和免疫介导的疾病。这强调了确定益生菌对儿童肠道发育和疾病预防的重要性。然而，关于益生菌作用机制的知识存在重大差距。益生菌在营养和临床应用中的功效仍有待进一步研究。 令人信服地建立。p40是本课题组分离克隆的一种LGG衍生蛋白，通过刺激表皮生长因子受体（EGFR）配体的释放激活肠上皮细胞中EGFR及其下游靶点PI 3 K/Akt。p40激活EGFR是抑制肠上皮细胞中精氨酸诱导的细胞凋亡、保护屏障功能和增强粘蛋白产生所必需的。我们已经产生了果胶/玉米醇溶蛋白水凝胶珠系统，以特异性地将p40递送到小鼠结肠中。通过使用这种递送系统，p40已经以EGFR依赖性方式被播种以预防和治疗小鼠中的实验性结肠炎。由于EGFR的激活在发育中起着生理作用，我们扩大了工作范围，以研究p40对肠道发育的影响。基于我们的初步研究结果，LGG定植促进肠道功能成熟，并降低对成年小鼠肠道损伤和结肠炎的易感性，提出了三个综合的具体目标，以提高对p40在调节肠道发育和p40作用机制中的功能性质的认识。在具体目标1中，将确定p40在肠功能成熟中的作用。我们将使用新生小鼠，人类胎儿肠道器官培养，人类和小鼠肠上皮细胞，以确定p40在肠上皮细胞增殖，分化，迁移，消化和吸收能力和上皮紧密连接形成的作用。在特定目标2中，将确定肠上皮细胞中参与p40调节的细胞反应以促进肠功能成熟的转录靶点。我们将确定p40调控的肠上皮基因在新生小鼠和人胎儿肠道器官培养，并验证预测的单个候选p40调控基因的功能，以调节上皮细胞增殖，分化，迁移，转运蛋白，和紧密连接形成使用在体外和离体测定。在具体目标3中，将阐明促进肠功能成熟所需的肠上皮细胞中p40刺激的信号通路。我们将使用肠上皮细胞特异性EGFR和PI 3 K缺陷以及人胎儿肠器官培养物和肠样组织中EGFR和Akt药理学抑制的小鼠模型，以确定p40刺激的上皮反应和p40调节的EGFR和Akt依赖性转录靶点功能成熟对EGFR和Akt激活的需求。这些研究将为营养和临床应用提供新的信息 p40对儿童健康的影响

 DESCRIPTION (provided by applicant): The symbiotic relationship between intestinal microbiota and the host plays an important role in the intestinal development. Interruption of this relationship leads to infectious and immune-mediated diseases. This underscores the importance of identifying the effects of probiotics on intestinal development and disease prevention in children. However, there are critical gaps in knowledge about mechanisms of probiotic action. The efficacy of probiotics for nutritional and clinical application remains to be convincingly established. p40, a LGG-derived protein isolated and cloned by our group, activates epidermal growth factor receptor (EGFR) and its downstream target, PI3K/Akt in intestinal epithelial cells through stimulation of EGFR ligand release. Activation of EGFR by p40 is required for inhibition of cytokine-induced apoptosis, preservation of barrier function, and enhancement of mucin production in intestinal epithelial cells. We have generated a pectin/zein hydrogel bead system to specifically delivered p40 to the colon in mouse. By using this delivery system p40 has been sown to prevent and treat experimental colitis in mice in an EGFR-dependent manner. Since activation of EGFR plays a physiological role in development, we have expanded the scope of our work to investigate the effects of p40 on intestinal development. Based on our preliminary results that LGG colonization promotes functional maturation of the intestine and decreases susceptibility to intestinal injury and colitis in adult mice, three integrated specific aims are proposed to enhance knowledge of the functional nature of p40 in regulation of intestinal development and mechanisms of the action of p40. In Specific Aim 1, the roles of p40 in intestinal functional maturation will be determined. We will use neonatal mice, human fetal intestinal organ culture, and human and mouse enteroids to determine the roles of p40 in intestinal epithelial cell proliferation, differentiation, migration,and digestive and absorptive capacities and epithelial tight junction formation. In Specific Aim 2, transcriptional targets in intestinal epithelial cells involved in p40-regulated cellular responsesto promote intestinal functional maturation will be defined. We will determine p40-regulated intestinal epithelial genes in neonatal mice and in human fetal intestinal organ culture, and verif the function of individual candidate p40-regulated genes predicted to regulate epithelial proliferation, differentiation, migration, transporters, and tight junction formation using in vitr and ex vivo assays. In Specific Aim 3, the p40- stimulated signaling pathway in intestinal epithelial cells that is required for promotion of intestinal functional maturation will be elucidaed. We will use mouse models with intestinal epithelial cell-specific deficiency of EGFR and PI3K and pharmacologic inhibition of EGFR and Akt in human fetal intestinal organ culture and enteroids to determine the requirement of EGFR and Akt activation for p40-stimulated epithelial responses and p40-regulated EGFR and Akt-dependent transcriptional targets for functional maturation. These studies will contribute new information for nutritional and clinical applications of p40 for health in children.