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

代谢表型胰腺切除术患者，定义为活体供体（LD），是一个新的和可再生的胰岛研究来源。由于传统的胰岛酶分离可以改变其基因表达，我们通常通过激光捕获显微解剖（LCM）从我们的生物库中速冻手术组织中获得最高质量的转录组学样本，这是世界上最大的此类生物库。根据我们之前对LCM大块胰岛的NGS转录组学和蛋白质组学分析，我们建议采用非靶向方法分析血糖正常（10）、糖耐量受损（10）或2型糖尿病（10）的LD 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起源。生成的数据将与来自这些LD的其他数据集集成，包括临床数据、LCM大块和单个胰岛细胞的蛋白质组学（正在进行中）以及LCM胰岛和血浆样本的散弹枪脂质组学。因此，这一雄心勃勃的、前所未有的项目的完成，将产生一个新的胰岛细胞类型的基因表达图谱，沿着从健康到T2D的连续体原位，从而成为该领域的参考研究。

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.