Small Molecular Regulation of Beta-Cell Differentiation

描述（由申请人提供）： 该合作R 01项目（响应RFA DK-03-015提交）中的初步数据证明了筛选小分子文库以发现促进胚胎干（ES）细胞和人胰管细胞分化的化合物的可行性。在化学图书馆、机器人和自动显微镜方面进行了大量的机构投资，以进行基于细胞的筛选。拟议的研究将建立在这些成功和基础设施上，通过开发高通量筛选技术来发现促进β细胞分化的分子。 每个实验室的互补专业知识创造了强大的协同作用。Mercola实验室领导了该研究所基于细胞的筛查工作的发展，并提供了机器人筛查，ES细胞培养和HIV慢病毒技术的专业知识，而Levine实验室提供了糖尿病知识，培养技术和原代人类胰腺导管组织的操作。 目标1-3将开发和进行促进ES细胞分化为胰腺内胚层前体的分子的试验性筛选。为此，我们将在分化特异性启动子的控制下产生一组具有GFP的ES细胞，以评估10，000种天然产物和已建立的药物的分化促进能力。选择该化合物文库是因为化学多样性和先前在几种细胞类型中产生细胞内信号传导途径调节剂的成功。目的4-5将用成年胰管细胞和永生化细胞系重复筛选，两者都用携带在胰岛素启动子控制下的GFP的HIV-慢病毒载体转导。 通过高通量技术的进步、多种化学文库的可用性以及在表征胰腺分化中不同阶段的基因的控制下容易地将报告蛋白如GFP置于相关细胞中的能力，使得β细胞分化的无偏见的化学方法成为可能。除了提供调控分化的工具外，对“命中”的了解可能揭示与胰腺发育有关的细胞靶点，或用作亲和探针来识别这些蛋白质，这一过程被称为化学遗传学。

DESCRIPTION (provided by applicant): Preliminary data in this Collaborative R01 Project (submitted in response to RFA DK-03-015) demonstrate the feasibility of screening libraries of small molecules to discover compounds that promote the differentiation of embryonic stem (ES) cells and human pancreatic duct cells. A substantial Institutional investment has been made in chemical libraries, robotics and automated microscopy to perform cell-based screens. The proposed research will build on these successes and this infrastructure by developing high throughput screening technology to discover molecules that promote beta-cell differentiation. A strong synergy is created by the complementary expertise of each laboratory. The Mercola laboratory has led development of cell-based screening efforts at the Institute and provides expertise in robotic screening, ES cell culture and HIV lentiviral technology while the Levine laboratory provides knowledge of diabetes, technology for culture and manipulation of primary human pancreatic duct tissue. Aims 1-3 will develop and perform a pilot screen for molecules that promote ES cells to differentiate into pancreatic endodermal precursors. To do this, we will produce a panel of ES cells with GFP under control of differentiation specific promoters to evaluate differentiation-promoting capability of 10,000 natural product and established drugs. This compound library was chosen for chemical diversity and prior success at yielding modulators of intracellular signaling pathways in several cell types. Aims 4-5 will repeat the screens with adult pancreatic duct cells and an immortalized cell line, both transduced with HIV-lentiviral vector carrying GFP under control of the insulin promoter. An unbiased, chemical approach to beta-cell differentiation is made possible by advances in high throughput technologies, availability of diverse chemical libraries and the ability to easily place reporter proteins such as GFP in relevant cells under the control of genes that characterize distinct stages in pancreatic differentiation. In addition to providing tools to regulate differentiation, knowledge of the "hits" might reveal insight into cellular targets that are involved in pancreatic development, or be used as affinity probes to identify such proteins, in a process that is becoming known as chemical genetics