特异性敲除小鼠肠神经系统多巴胺D2受体促进肠道转运OA
Specific knockout of dopamine D2 receptor in the enteric nervous system promotes intestinal transit in mice
目的 构建特异性敲除肠神经系统(ENS)多巴胺 D2 受体(D2R)的小鼠模型,阐明其促进肠道转运的机制.方法 利用 Cre-LoxP 系统构建特异性敲除 ENS D2R(D2RNmu-CKO)小鼠及其对照组(D2Rflox/flox)小鼠,免疫荧光染色、DAB 染色、RT-qPCR 和 Western blot 等检测 D2R 和乙酰胆碱转移酶(ChAT)的表达以及 ELISA 技术检测乙酰胆碱(ACh)的含量;利用活体小动物 X 线成像技术检测小鼠肠道转运情况.结果 免疫荧光染色证实野生型小鼠的十二指肠肌间神经丛(MP)中 D2R 与神经元标志物(PGP 9.5)以及胆碱能神经元标志物(ChAT)3 者共存,说明 D2R表达于小鼠十二指肠 ENS 的胆碱能神经元.鼠尾鉴定结果显示成功构建基因型为 Nmu-Cre+/+D2R-flox+/+或 Nmu-Cre+/-D2R-flox+/+的 D2RNmu-CKO小鼠以及基因型为 Nmu-Cre-/-D2R-flox+/+的对照组 D2Rflox/flox 小鼠模型.与对照组D2Rflox/flox小鼠相比,D2 RNmu-CKO小鼠十二指肠肌层 D2R mRNA(n=6,P＜0.001)以及蛋白表达(n=8,P＜0.01)水平显著降低,而 ChAT mRNA(n=6,P＜0.001)以及蛋白表达(n=6,P＜0.05)水平明显升高,且 ACh 含量也显著升高(n=8,P＜0.01).DAB 染色进一步显示 D2RNmu-CKO小鼠十二指肠 MP 中 ChAT 阳性神经元数量明显升高(n=6,P＜0.01).肠道转运实验表明 D2RNmu-CKO小鼠肠道转运速度显著加快,全肠转运时间显著缩短(n=5,P＜0.05).结论 本研究成功构建特异性敲除 ENS D2R 小鼠模型,并阐明 ENS D2R 缺失通过增加 ACh 的释放进而促进肠道转运,为特异性干预 ENS D2R 治疗功能性消化不良、慢传输型便秘等肠动力障碍疾病的新策略提供理论依据.
Objective To establish a mouse model with specific knockout of dopamine D2 receptor(D2R)in the enteric nervous system(ENS)and elucidate its mechanism in promoting intestinal motility.Methods The Cre-LoxP system was employed to generate ENS-specific D2R knockout mice(D2RNmu-CKO)and control mice(D2Rflox/flox).Expression levels of D2R and choline acetyltransferase(ChAT)were detected using immunofluores-cence staining,DAB staining,RT-qPCR,and Western blot.Acetylcholine(ACh)content was measured by ELISA.Intestinal transit was assessed in vivo using X-ray imaging in live small animals.Results Immunofluo-rescence staining confirmed the co-localization of D2R with the neuronal marker PGP 9.5 as well as the cholin-ergic neuronal marker ChAT in the duodenal myenteric plexus(MP)of wild-type mice,indicating that D2R is expressed in cholinergic neurons of the duodenal ENS.Genotyping confirmed successful establishment of D2RNmu-CKO mice(genotypes:Nmu-Cre+/+D2R-flox+/+or Nmu-Cre+/-D2R-flox+/+)and control D2Rflox/flox mice(genotype:Nmu-Cre-/-D2R-flox+/+).Compared with control mice,D2RNmu-CKO mice exhibited significantly re-duced mRNA(n=6,P＜0.001)and protein(n=8,P＜0.01)expression of D2R in the duodenal muscle layer,while mRNA(n=6,P＜0.001)and protein(n=6,P＜0.05)expression of ChAT were markedly in-creased.The content of ACh was also significantly elevated(n=8,P＜0.01).DAB staining further revealed a significant increase in the number of ChAT-positive neurons in the duodenal MP of D2RNmu-CKO mice(n=6,P＜0.01).Intestinal transit assays demonstrated accelerated intestinal transit and significantly shortened whole-gut transit time in D2RNmu-CKO mice(n=5,P＜0.05).Conclusions This study successfully established an ENS-specific D2R knockout mouse model and revealed that loss of ENS D2R promotes intestinal transit by enhancing ACh release.These findings provide a theoretical basis for novel strategies targeting ENS D2R to treat gastrointestinal motility disorders such as functional dyspepsia and slow-transit constipation.
汪国庆;朱愔喆;王益嘉;冯小燕
首都医科大学 基础医学院 生理学与病理生理学系,北京 100069首都医科大学 基础医学院 生理学与病理生理学系,北京 100069首都医科大学 基础医学院 生理学与病理生理学系,北京 100069首都医科大学 基础医学院 生理学与病理生理学系,北京 100069
医药卫生
肠神经系统多巴胺D2受体乙酰胆碱肠道转运
enteric nervous systemdopamine D2 receptoracetylcholineintestinal transit
《基础医学与临床》 2026 (6)
765-771,7
北京市自然科学基金(7222010)国家自然科学基金(32071126)
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