Small Molecular Regulation of Beta-Cell Differentiation

描述(由申请人提供)： 这个Collaborative R01项目(根据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细胞(ES)和人胰腺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这一基础设施的基础上，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效应。Mercola实验室领导了该研究所基于细胞的筛查工作的发展，并提供机器人筛查、ES细胞培养和艾滋病毒慢病毒技术方面的专业知识，而Levine实验室则提供糖尿病知识、原发人类胰管组织的培养和操作技术。 AIMS 1-3将开发并进行促进ES细胞分化为胰腺内胚层前体的分子的中试筛选。为此，我们将在分化特异性启动子的控制下，生产一组带有GFP的ES细胞，以评估10,000种天然产品和已建立的药物的促分化能力。选择这个化合物文库是因为化学多样性和先前在几种细胞类型中获得细胞内信号通路调节剂的成功。AIMS 4-5将重复对成人胰腺管细胞和永生化细胞系的筛选，这两种细胞都是在胰岛素启动子控制下用携带GFP的HIV慢病毒载体转导的。 高通量技术的进步、多种化学库的可获得性以及在胰腺分化不同阶段的基因控制下，能够轻松地将报告蛋白(如GFP)放置在相关细胞中，使无偏见的化学方法成为可能。除了提供调节分化的工具外，对HITS的了解可能会揭示与胰腺发育有关的细胞靶点，或者被用作亲和力探针来识别这些蛋白质，这一过程正成为众所周知的化学遗传学。

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