SOX9, Enteroendocrine and Intestinal Epithelial Stem Cells: A Study of Gene Netwo

SOX9、肠内分泌和肠上皮干细胞：基因网络的研究

• 批准号: 7976009
• 负责人: SCOTT T MAGNESS
• 金额: $ 7.4万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7976009
• 关键词: Ablation; Adult; Anatomy; Animal Model; Applications Grants; Biological Assay; Biological Markers; Biology; Candidate Disease Gene; Cell Fractionation; Cell Maintenance; Cell Proliferation; Cell Separation; Cells; Cellular Morphology; Cessation of life; Chromogranin A; Clinical; Coculture Techniques; Data; Development; Disease; Enteroendocrine Cell; Epithelial; Epithelial Cells; Epithelium; Extramural Activities; Fluorescence-Activated Cell Sorting; Funding; Future; GLP-2; Ganciclovir; Gene Expression; Gene Expression Profile; Gene Targeting; Generations; Genes; Grant; Growth; Homeostasis; Hormones; In Vitro; Infection; Initiator Codon; Injection of therapeutic agent; Injury; Intestines; Knock-out; Knockout Mice; Laboratories; Life; Literature; Mesenchymal; Methods; Mitogens; Mitotic; Molecular Profiling; Morphology; Natural regeneration; Organism; Pathway interactions; Peptides; Pharmaceutical Preparations; Population; Process; Proliferating; Property; Proteins; Proteomics; Radiation; Reagent; Reporting; Role; Signal Transduction; Small Intestines; Somatostatin; Speed; Staining method; Stains; Stem cells; Substance P; TK Gene; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Transgenes; Transgenic Mice; United States National Institutes of Health; Validation; Villus; base; cell type; in vivo; injured; insight; intestinal epithelium; irradiation; morphogens; mouse genome; mouse model; novel; public health relevance; regenerative; regenerative therapy; response; self-renewal; stem; stem cell biology; stem cell niche; tissue regeneration; transcription factor; uroguanylin; villin

DESCRIPTION (provided by applicant): The renewal of the intestinal epithelium is driven by a pool of multipotential intestinal epithelial stem cells (IESCs) residing in the base of the crypts. Our preliminary studies indicate that a sub-set of post-mitotic cells in the small intestine crypt epithelium express high levels of the transcription factor, Sox9; these Sox9-expressing cells are consistent with enteroendocrine cells, and may modulate proliferative and differentiation capacity of IESCs through secreted peptides. Preliminary data demonstrates that a subset of Sox9-expressing Substance-P positive cells have a relatively 'long-lived' tenure in the adult IESC 'niche'. We provide further evidence that high Sox9-expressing cells populate the early developing gut, are the first differentiated cell type observed in developing 'mini-guts' (crypt-villus units derived from a single IESC in vitro), and are present in regenerating epithelial micro-colonies following irradiation. These observations suggest that cells marked by high levels of Sox9 are a conserved feature of the proliferating intestinal epithelium. The mechanism by which SOX9 functions in the EE population is unknown. EE cells are known to secrete both growth promoting and inhibitory peptides, so with advances made with NIDDK-K01 funding, we are now poised to study the relationships between EE cells and their influence on IESC proliferation and differentiation capacity. Our central hypothesis for this proposal is that crypt-based enteroendocrine cells (CBEE) are critical for modulating proliferation/self-renewal and differentiation of IESCs, and also for instructing crypt morphology. We extend this hypothesis to predict that Sox9 in the CBEE controls downstream target genes that extrinsically influence IESC and crypt biology. To begin to test these hypotheses we propose the following Specific Aims: 1a) To characterize CBEE cells, and determine their gene expression profile, and 1b) to indentify Sox9-dependent genes in EE cells. 2) To establish the effect of EE cells on IESC proliferation and crypt development using novel co-culture assays. 3) To generate an EE cell ablation transgenic mouse model. Successful completion of this study will: 1) establish a role for the CBEE cells, 2) identify novel EE-derived proteins involved in stem cell maintenance and differentiation, and 3) provide rationale, preliminary data and reagents for further extramural funding. The potential clinical implications of this study are identification of peptides that could be used as therapeutics to protect the IESC during chemotherapeutic or radiation treatments by slowing division rates of IESCs; or conversely identification of pro-growth peptides that could be used to enhance IESC proliferation by speeding the regenerative process following epithelium depletion cause by infection, irradiation or drug treatments. PUBLIC HEALTH RELEVANCE: A pool of epithelial stem cells drives regeneration of the intestinal epithelium during normal and injured states. Understanding the mechanisms that regulate and influence the proliferation and differentiation of the intestinal epithelial stem cell is critical for identifying therapies to enhance tissue regeneration during disease or following injury. There is evidence that a small population of cells that surround the stem cell zone in the intestinal epithelium may be providing both instructional queues for the larger structural anatomy of the intestinal epithelium, and also signals that control proliferation. We hypothesize that this population of cells is a subset of 'long-lived' hormone producing cells called enteroendocrine cells that have a critical role in intestinal stem cell biology. This study will investigate the role of these cells and active gene pathways within this subset of enteroendocrine cells to provide information regarding their role in the stem cell zone. Data gathered from this study will likely provide new mechanisms of action for these cell types and indentify novel targets for regenerative therapies.

描述（由申请人提供）： 肠上皮的更新是由位于隐窝底部的多重肠上皮干细胞（IESC）驱动的。我们的初步研究表明，小肠隐窝上皮中有丝裂细胞的子集表达高水平的转录因子SOX9。这些表达SOX9的细胞与肠内分泌细胞一致，并且可能通过分泌的肽调节IESC的增殖能力和分化能力。初步数据表明，表达Sox9物质-P阳性细胞的子集具有相对“长期寿命”的任期。我们提供了进一步的证据表明，表达高Sox9的细胞填充了早期发育的肠道，是在开发“迷你螺旋”（crypt-Villus单位）中观察到的第一种分化细胞类型（源自单个IESC体外的crypt-Villus单位），并且存在于复生的上皮上皮微效中。这些观察结果表明，以高水平的Sox9标记的细胞是增殖肠上皮的保守特征。 SOX9在EE种群中起作用的机制尚不清楚。众所周知，EE细胞可以分泌生长促进和抑制性肽，因此，随着NIDDK-K01资金的进步，我们现在准备研究EE细胞及其对IESC增殖和分化能力的影响之间的关系。我们对该提案的中心假设是，基于密码的肠肠分泌细胞（CBEE）对于调节IESC的增殖/自我更新和分化以及指导隐式形态至关重要。我们扩展了这一假设，以预测CBEE中的Sox9控制着外在影响IESC和Crypt生物学的下游靶基因。为了开始检验这些假设，我们提出以下特定目的：1a）以表征CBEE细胞并确定其基因表达谱，并确定1B），以缩小EE细胞中的Sox9依赖性基因。 2）使用新型共培养测定法确定EE细胞对IESC增殖和隐窝发育的影响。 3）生成EE细胞消融转基因小鼠模型。这项研究的成功完成将：1）确定CBEE细胞的作用，2）确定与干细胞维持和分化有关的新型EE衍生的蛋白质，以及3）提供基本原理，初步数据和试剂，以进一步进行校外资金。这项研究的潜在临床意义是鉴定肽，这些肽可以用作化学治疗或放射治疗期间通过减慢IESC的分裂速率来保护IESC的治疗疗法。或相反，可以通过通过感染，辐射或药物治疗来加速再生过程来加快再生过程来加速再生过程，以鉴定可用于增强IESC增殖的促肽。 公共卫生相关性： 一群上皮干细胞在正常和受伤状态下驱动肠上皮的再生。了解调节和影响肠上皮干细胞的增殖和分化的机制对于鉴定疗法以增强疾病或损伤后增强组织再生的疗法至关重要。有证据表明，围绕肠上皮的干细胞区域的少数细胞可能正在为肠上皮的较大结构解剖结构提供两个教学队列，并信号控制增殖。我们假设该细胞群是“长寿命”激素产生的细胞的子集，称为肠内分泌细胞，在肠道干细胞生物学中具有关键作用。这项研究将研究这些细胞和活性基因途径在肠道内分泌细胞的这一子集中的作用，以提供有关其在干细胞区域的作用的信息。从这项研究中收集的数据可能会为这些细胞类型提供新的作用机理，并缩减再生疗法的新靶标。