Next generation sequencing of laser capture microdissected single islet cells from metabolically phenotyped living donors

对来自代谢表型活体供体的激光捕获显微切割的单个胰岛细胞进行下一代测序

• 批准号: 514060695
• 负责人: Professor Michele Solimena
• 金额: --
• 依托单位: Paul-Langerhans-Institut Dresden (PLID) des Helmholtz-Zentrums München am Universitätsklinikum Carl Gustav Carus der TU Dresden
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/514060695?language=en
• 关键词: Next; generation; sequencing; laser; capture

Metabolically phenotyped pancreatectomised patients, defined as living donors (LD), are a novel and reproducible source of pancreatic islets for research. As conventional enzymatic isolation of islets can alter their gene expression, we routinely obtain highest-quality samples for transcriptomics by laser capture microdissection (LCM) from snap-frozen surgical tissues in our biobank, which is the largest of this type worldwide. Following our previous NGS transcriptomics and proteomic analyses on LCM bulk islets, we propose here to profile with a non-targeted approach gene expression in LCM single islet cells from LD with normoglycemia (10), impaired glucose tolerance (10) or type 2 diabetes (10). This shall enable us to unequivocally identify the cell type origin of gene expression changes found through bulk islet NGS which correlate with progressive deterioration of hyperglycemia. Generated data will be integrated with other datasets from these LD, including clinical data, proteomics of LCM bulk and single islet cells (ongoing) as well as shotgun lipidomics of LCM islets and plasma samples. Thus, the accomplishment of this ambitious, unprecedented project, shall generate a novel atlas of gene expression in islet cell types in situ along the continuum from health to T2D and thus be a study of reference in the field.

代谢型胰腺切除患者被定义为活体供体(LD)，是一种用于研究的新的和可重复的胰岛来源。由于传统的酶分离胰岛可以改变它们的基因表达，我们经常通过激光捕获显微解剖(LCM)从我们的生物库中快速冷冻的手术组织中获得最高质量的转录样本，这是世界上此类组织中最大的。根据我们之前对LCM块状胰岛进行的NGS转录和蛋白质组学分析，我们建议用非靶向方法分析来自正常血糖(10例)、糖耐量受损(10例)或2型糖尿病(10例)的LCM单个胰岛细胞的基因表达。这将使我们能够明确地确定通过大块胰岛NGS发现的与高血糖进行性恶化相关的基因表达变化的细胞类型来源。生成的数据将与来自这些LD的其他数据集整合，包括临床数据、LCM大宗细胞和单个胰岛细胞的蛋白质组学(正在进行)以及LCM胰岛和血浆样本的鸟枪式脂肪组学。因此，这一雄心勃勃、史无前例的计划的完成，将产生一个新的图谱，沿着从健康到T2D的连续体原位研究胰岛细胞类型的基因表达，从而成为该领域的参考研究。