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OA

Objective To establish a mouse duodenal mucosal epithelial organoid model with conditional knockout of dopamine D5 receptor(D5R)in intestinal mucosal epithelium.Methods Villin-CreERT2+/+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,immuno-fluorescence 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 D5 R expression following 4-hydroxytamoxifen induction,with fluores-cence intensity decreasing from 0.904±0.061 to 0.385±0.020(n=6,P<0.001).qPCR analysis demonstrated a marked decrease in D5 R 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.