High-Throughput Screen for Beta-Cell Replication

描述(申请人提供)：人类β细胞的有丝分裂指数极低，即使与啮齿动物的β细胞相比也是如此。此外，刺激体外增殖的努力会导致胰岛素基因表达的迅速关闭。调节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，但如果弄清楚，可能会被用来诱导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因子(CDKI)在控制细胞周期进入中起着关键作用。在人类胰岛中，微阵列分析发现只有两个CDKI，p57Kip2和P21Cip1，高水平表达。P57Kip2是成人胰腺中的β细胞特异性表达，在人类患者中，p57Kip2表达的突变丢失会导致β细胞的增殖。因此，p57Kip2是旨在促进β细胞复制的干预措施的极佳靶点。在这个方案中，我们将利用人胰腺内分泌细胞株诱导表达细胞周期蛋白依赖性激酶抑制物p57Kip2的优势，开发一种高通量筛选，以调节细胞周期进入和p57Kip2转录的化合物。小分子HITS将作为研究p57Kip2调控的探针，并可能成为糖尿病治疗药物开发的主要候选者。通过进一步了解控制细胞周期进入人类β细胞的机制，我们应该能够更好地在体内和/或体外诱导β细胞再生。

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.