条件性敲除小鼠十二指肠黏膜上皮多巴胺D5受体的类器官培养和鉴定

doi:10.16352/j.issn.1001-6325.2026.04.0478

基础医学与临床 ›› 2026, Vol. 46 ›› Issue (4): 478-483.doi: 10.16352/j.issn.1001-6325.2026.04.0478

条件性敲除小鼠十二指肠黏膜上皮多巴胺D5受体的类器官培养和鉴定

朱愔喆¹, 马雪睿², 汪国庆¹, 王益嘉¹, 冯小燕^1*

1.首都医科大学基础医学院生理学与病理生理学系,北京 100069;
2.首都医科大学附属世纪坛医院 2021级五年临床医学专业,北京 100038

收稿日期:2025-04-30 修回日期:2025-09-24 出版日期:2026-04-05 发布日期:2026-03-24
通讯作者: ^*fengxy@ccmu.edu.cn
基金资助:
国家自然科学基金(32071126);北京市自然科学基金(7222010)

Establishment and characterization of intestinal organoids from dopamine D5 receptor conditional knockout mice in duodenal mucosal epithelium

ZHU Yinzhe¹, MA Xuerui², WANG Guoqing¹, WANG Yijia¹, FENG Xiaoyan^1*

1. Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069;
2. Grade 2021 Clinical Medicine, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China

Received:2025-04-30 Revised:2025-09-24 Online:2026-04-05 Published:2026-03-24
Contact: ^*fengxy@ccmu.edu.cn

摘要/Abstract

摘要： 目的构建条件性敲除小鼠十二指肠黏膜上皮多巴胺D5受体(dopamine D5 receptor, D₅R)的类器官模型。方法采用Villin-Cre^ERT2+/+D₅R-flox^+/+基因型的C57BL/6J小鼠,分离其十二指肠隐窝并进行类器官培养。利用4-羟基他莫昔芬诱导Cre-loxP系统实现D₅R条件性敲除,通过形态学观察、免疫荧光染色、实时定量PCR和Western blot等方法对十二指肠黏膜上皮类器官的结构特征和D₅R敲除效率进行鉴定。结果成功构建条件性敲除十二指肠黏膜上皮D₅R类器官模型。形态学观察显示该类器官具有体内十二指肠黏膜上皮类似的结构特征;免疫荧光染色证实4-羟基他莫昔芬诱导类器官D₅R的表达显著降低,荧光强度由0.904±0.061降低到0.385±0.020(n=6, P＜0.001);实时定量PCR和Western blot结果表明D₅R的mRNA水平由2.483±0.204降低至0.241±0.048以及蛋白表达水平由0.923±0.075降低至0.569±0.032(n=6,P＜0.001)。结论本研究成功构建的条件性敲除十二指肠黏膜上皮D₅R类器官模型可模拟小鼠十二指肠黏膜的生理特性,为揭示D₅R在十二指肠黏膜上皮功能中的调控机制提供了可靠的体外模型。

关键词: 类器官, 十二指肠, 肠黏膜上皮, 多巴胺D5受体

Abstract: Objective To establish a mouse duodenal mucosal epithelial organoid model with conditional knockout of dopamine D5 receptor (D₅R) in intestinal mucosal epithelium. Methods Villin-Cre^ERT2+/+D₅R-flox^+/+ C57BL/6J mice were utilized to isolate duodenal crypts for organoid culture. D₅R conditional knockout (CKO) was induced via the Cre-loxP system by 4-hydroxytamoxifen administration. The structural characteristics of duodenal mucosal epithelial organoids and the knockout efficiency of D₅R were evaluated through morphological observation, immunofluorescence staining, quantitative PCR (qPCR), and Western blot assays. Results A duodenal mucosal epithelial organoid model with conditional D₅R knockout was successfully established. Morphological examination revealed that the organoids exhibited structural features similar to the in vivo duodenal mucosal epithelium. Immunofluorescence staining confirmed a significant reduction in D₅R expression following 4-hydroxytamoxifen induction,with fluorescence intensity decreasing from 0.904±0.061 to 0.385±0.020 (n=6, P＜0.001). qPCR analysis demonstrated a marked decrease in D₅R mRNA levels from 2.483±0.204 to 0.241±0.048, and Western blot showed a significant reduction in protein expression from 0.923±0.075 to 0.569±0.032 (n=6, P＜0.001). Conclusions A conditional D5R knockout organoid model derived from duodenal mucosal epithelium is successfully developed, which faithfully recapitulates the physiological characteristics of duodenal mucosa. This model provides a reliable in vitro platform for elucidating the regulatory mechanisms of D5R in duodenal epithelial functions.

Key words: organoid, duodenum, intestinal mucosal epithelium, dopamine D5 receptor

中图分类号:

R592

朱愔喆, 马雪睿, 汪国庆, 王益嘉, 冯小燕. 条件性敲除小鼠十二指肠黏膜上皮多巴胺D5受体的类器官培养和鉴定[J]. 基础医学与临床, 2026, 46(4): 478-483.

ZHU Yinzhe, MA Xuerui, WANG Guoqing, WANG Yijia, FENG Xiaoyan. Establishment and characterization of intestinal organoids from dopamine D5 receptor conditional knockout mice in duodenal mucosal epithelium[J]. Basic & Clinical Medicine, 2026, 46(4): 478-483.

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条件性敲除小鼠十二指肠黏膜上皮多巴胺D5受体的类器官培养和鉴定

Establishment and characterization of intestinal organoids from dopamine D5 receptor conditional knockout mice in duodenal mucosal epithelium

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