High-Throughput Screen for Beta-Cell Replication

描述（由申请人提供）：人类β细胞具有极低的有丝分裂指数，即使与啮齿动物β细胞相比也是如此。此外，在体外刺激增殖的努力导致胰岛素基因表达的快速关闭。介导生长和维持完全分化状态之间反向关系的分子机制尚不清楚，但如果澄清，可以利用诱导β细胞复制用于糖尿病治疗。细胞周期蛋白依赖性激酶抑制剂（CDKIs）在控制细胞周期进入中起关键作用。在人类胰岛中，微阵列分析发现只有两个CDKIs， p57Kip2和P21Cip1高水平表达。P57Kip2在成人胰腺中特异于β细胞，人类患者中P57Kip2表达的突变缺失导致β细胞增生。因此，p57Kip2是旨在促进β细胞复制的干预措施的一个极好的靶点。在这项提议中，我们将利用诱导表达周期蛋白依赖性激酶抑制剂p57Kip2的人类胰腺内分泌细胞系来开发高通量筛选调节细胞周期进入和p57Kip2转录的化合物。小分子命中将作为研究p57Kip2调控的探针，并可能作为开发糖尿病治疗药物的主要候选物。通过进一步了解控制人类β细胞细胞周期进入的机制，我们应该在体内和/或体外诱导β细胞再生方面处于更好的位置。

DESCRIPTION (provided by applicant): Human beta-cells have an extremely low mitotic index, even compared with rodent beta-cells. Furthermore, efforts to stimulate proliferation in vitro result in the rapid shut off of insulin gene expression. The molecular mechanism that mediates the inverse relationship between growth and maintenance of a fully differentiated state is poorly understood, but if clarified could be exploited to induce beta-cell replication for diabetes therapy. Cyclin-dependent kinase inhibitors (CDKIs) play key roles in controlling cell cycle entry. In the human islet, microarray analysis has found that only two CDKIs, p57Kip2 and P21Cip1, are expressed at high levels. P57Kip2 is specific to beta-cells in the adult human pancreas and mutational loss of p57Kip2 expression in human patients results in beta-cell hyperplasia. Thus, p57Kip2 is an excellent target for interventions designed to promote beta-cell replication. In this proposal, we will take advantage of human pancreatic endocrine cell lines that inducibly express the cyclin dependent kinase inhibitor p57Kip2 to develop a high-throughput screen for compounds that modulate cell cycle entry and p57Kip2 transcription. Small molecule hits will serve as probes to study p57Kip2 regulation and potentially as lead candidates for development of diabetes therapeutics. By furthering our understanding of the mechanisms that control cell cycle entry in human beta-cells, we should be in an improved position to induce beta-cell regeneration in vivo and/or in vitro.