Characterizing the Role of Pancreatic Progenitors in Regeneration

项目摘要 世界卫生组织估计，全球成人糖尿病患病率为9%。1型 和2型糖尿病涉及β细胞质量减少的问题;随后糖尿病的治疗必须涉及 β-细胞替代。理想情况下，治疗将涉及通过内源性β细胞再生诱导再生， 胰腺祖细胞由于这些原因，我们有兴趣解释β细胞新生的过程， 也就是说，胰岛β细胞是如何从胰腺中的祖细胞形成的。与哺乳动物不同， 显示斑马鱼在细胞特异性消融后容易再生其β细胞。我们的目标是找出 斑马鱼β细胞新生能力背后的机制。这样的分子途径可以 在人类中使用的诱导β细胞新生的药物。 我们最近有两个发现，这两个发现对于理解斑马鱼在β- 细胞消融首先，我们确定了β细胞再生的祖细胞来源，即一种称为 第二，我们发现Sox 9 b转录因子活性的降低导致 显著加速了再生。根据这些见解，我们假设1）Sox 9 b作用于细胞 2）Sox 9 b活性的降低改变了CACs的细胞增殖能力; CACs或其后代在再生中的行为;和3）CACs下游基因的鉴定， SOX 9将阐明调节β细胞分化的分子机制。通过测试这些 假设在三个互补但独立的目标，我们希望发现，如果差异， sox 9 b杂合子和野生型之间的再生是由于预先存在的 再生过程中CAC的形态或行为。此外，我们将使用基因组方法， 确定Sox 9同源物的直接下游转录靶点，因为我们预计这些靶点将 是SOX 9 B单倍不足表型的介质。 作为我们初步数据的一部分，我们敲低了人类PANC-1细胞系中的SOX 9水平，PANC-1细胞系是人类胰腺癌的替代物。 胰腺祖细胞，并鉴定了受影响的转录水平。我们也用染色质 通过免疫沉淀和深度测序（ChIP-seq）来鉴定SOX 9在PANC-1基因组中结合的位置。 将这些结果放在一起，使我们能够找到PANC-1中SOX 9转录活性的直接靶点 细胞我们已经鉴定了SOX 9下游的有趣基因，如EpCAM，并鉴定了生物学活性。 SOX 9控制的途径，如纤毛功能和Notch调节。深受鼓舞的是， 我们现在的目标是扩大这一分析，在开发过程中找到SOX 9的目标， 再生 在这项工作结束时，我们希望能更好地了解SOX 9的功能，并发现 缓解人类β细胞缺乏的潜在治疗途径。

Project Summary The World Health Organization estimates that the global prevalence of diabetes in adults is 9%. Both Type 1 and Type 2 diabetes involve the issue of reduced β-cell mass; subsequently a cure for diabetes must involve β-cell replacement. Ideally, a cure would involve inducing regeneration via β-cell neogenesis from endogenous pancreatic progenitors. For these reasons we are interested in explicating the process of β-cell neogenesis, i.e., how β cells are formed from progenitors in the pancreas. Unlike their mammalian counterparts, we have shown that zebrafish readily regenerate their β cells following cell-specific ablation. Our goal is to identify the mechanisms behind the zebrafish’s capacity for β-cell neogenesis. Such molecular pathways could then be pharmacologically exploited in humans to induce β-cell neogenesis. We have recently made two discoveries critical to understanding how zebrafish so easily recover following β- cell ablation. First, we identified the progenitor source for β-cell neogenesis—namely a cell type called the centroacinar cell (CAC); second, we discovered that diminished activity of the Sox9b transcription factor leads to significantly accelerated regeneration. From these insights we hypothesized 1) Sox9b acts cell autonomously to maintain progenitor potency in adult CACs; 2) Diminishing Sox9b activity alters the behavior of CACs or their progeny in regeneration; and 3) The identification of downstream genes of SOX9 will elucidate molecular mechanisms that regulate β-cell differentiation. By testing these hypotheses in three complementary yet independent aims, we expect to discover if the differences in regeneration between sox9b heterozygotes and wildtypes is due either to pre-existing differences in morphology or the behavior of the CACs during regeneration. Furthermore, we will use genomic approaches to identify the direct downstream transcriptional targets of Sox9 homologs because we expect these targets will be the mediators of the sox9b haploinsufficient phenotype. As part of our preliminary data we knocked down SOX9 levels in the human PANC-1 cell line, a surrogate for pancreatic progenitors, and identified affected transcript levels. We have also used chromatin immunoprecipitation and deep sequencing (ChIP-seq) to identify where SOX9 binds in the PANC-1 genome. Putting these results together has allowed us to find direct targets of SOX9 transcriptional activity in PANC-1 cells. We have identified interesting genes downstream of SOX9, such as EpCAM, and identified biological pathways that SOX9 controls, such as cilia function and Notch regulation. Greatly encouraged by our preliminary results we now aim to expand this analysis to find SOX9 targets during development and regeneration. By the end of this proposed work we expect a better understanding of SOX9 function and the discovery of potential therapeutic routes to alleviate β-cell paucity in humans.