組織透明化と3次元イメージングを用いた腸管形態およびインクレチン産生細胞の解析

京都大学新制・課程博士博士(医学)甲第24517号医博第4959号新制||医||1065(附属図書館)京都大学大学院医学研究科医学専攻(主査)教授 妹尾 浩, 教授 松田 道行, 教授 小濱 和貴学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

An analysis of intestinal morphology and incretin-producing cells using tissue optical clearing and 3-D imaging

Tissue optical clearing permits detailed evaluation of organ three-dimensional (3-D) structure as well as that of individual cells by tissue staining and autofluorescence. In this study, we evaluated intestinal morphology, intestinal epithelial cells (IECs), and enteroendocrine cells, such as incretin-producing cells, in reporter mice by intestinal 3-D imaging. 3-D intestinal imaging of reporter mice using optical tissue clearing enabled us to evaluate both detailed intestinal morphologies and cell numbers, villus length and crypt depth in the same samples. In disease mouse model of lipopolysaccharide (LPS)-injected mice, the results of 3-D imaging using tissue optical clearing in this study was consistent with those of 2-D imaging in previous reports and could added the new data of intestinal morphology. In analysis of incretin-producing cells of reporter mice, we could elucidate the number, the percentage, and the localization of incretin-producing cells in intestine and the difference of those between L cells and K cells. Thus, we established a novel method of intestinal analysis using tissue optical clearing and 3-D imaging. 3-D evaluation of intestine enabled us to clarify not only detailed intestinal morphology but also the precise number and localization of IECs and incretin-producing cells in the same samples

Gene expression of nutrient-sensing molecules in I cells of CCK reporter male mice

Cholecystokinin (CCK) is secreted from enteroendocrine I cells in response to fat, carbohydrate, and protein ingestion. Gene expression of nutrient-sensing molecules in I cells remains unclear, primarily due to the difficulty in distinguishing I cells from intestinal epithelial cells in vivo. In this study, we generated CCK reporter male mice in which the red fluorescence protein tdTomato (Tomato) is produced by activation of the native murine Cck promoter. Fluorescence microscopy revealed the presence of Tomato-positive cells in upper small intestine (SI), lower SI, and colon. Flow cytometer analysis revealed that Tomato-positive cells among epithelial cells of upper SI, lower SI, and colon occurred at the rate of 0.95, 0.54, and 0.06%, respectively. In upper SI and lower SI, expression levels of Cck mRNA were higher in Tomato-positive cells than those in Tomato-negative cells. The fatty acid receptors Gpr120, Gpr40, and Gpr43 and the oleoylethanolamide receptor Gpr119 were highly expressed in Tomato-positive cells isolated from SI, but were not found in Tomato-positive cells from colon. The glucose and fructose transporters Sglt1, Glut2, and Glut5 were expressed in both Tomato-positive cells and -negative cells, but these expression levels tended to be decreased in Tomato-positive cells from upper SI to colon. The peptide transporter Pept1 and receptor Gpr93 were expressed in both Tomato-positive cells and -negative cells, whereas Casr was expressed only in Tomato-positive cells isolated from SI. Thus, this transgenic mouse reveals that I cell number and gene expression in I cells vary according to region in the gastrointestinal tract

Challenging Differential Diagnosis of Hypergastremia and Hyperglucagonemia with Chronic Renal Failure: Report of a Case with Multiple Endocrine Neoplasia Type 1

A 53-year-old woman developed end-stage renal failure during a 15-year clinical course of primary hyperparathyroidism and was referred to our hospital for evaluation of suspected multiple endocrine neoplasia type 1 (MEN1). Genetic testing revealed a novel deletion mutation at codon 467 in exon 10 of the MEN1 gene. Systemic and selective arterial calcium injection (SACI) testing revealed hyperglucagonemia and hypergastrinemia with positive gastrin responses. A pathological examination revealed glucagonoma and a lymph node gastrinoma. The findings in this case indicate the importance of early diagnosis of MEN1 and demonstrate the utility of systemic and SACI testing in renal failure cases