摘要
目的 探讨乙醇对小鼠胃腺体中干细胞及上皮细胞标志物表达的影响,揭示胃损伤模型中影响胃干细胞分化的信号通路。方法 雄性C57BL/6J小鼠随机分为正常对照组和乙醇组,乙醇组第1 d(D1)ig给予50%(V/V)乙醇溶液(10 mL·kg^(-1))1次,D2~D9给予含10%(V/V)乙醇饮用水,正常对照组正常饮水。第10 d取胃组织,HE染色观察胃组织病理改变,免疫组织化学观察胃上皮细胞标志物黏蛋白5AC(MUC5AC)、H^(+)/K^(+)ATPase β、胃蛋白酶C(PEP C)表达变化,免疫荧光法观察H^(+)/K^(+)ATPase β、PEP C、胃泌素表达变化。ELISA测定胃组织匀浆上清液中胃泌素、生长抑素和白细胞介素1β(IL-1β)浓度。流式细胞术测定胃腺体中富含亮氨酸重复序列的G蛋白偶联受体5阳性(LGR5^(+))干细胞数量。通过类器官建立成功与否表征胃腺体中干细胞活性变化。RNA测序和生物信息学分析表征乙醇造模引发组织中的炎症信号通路和分化信号通路改变。结果 与正常对照组相比,HE染色结果显示乙醇组胃黏膜层呈现多灶性坏死,黏膜上皮细胞和胃腺细胞出现胞核固缩、溶解、脱落;免疫组织化学结果显示乙醇组胃组织上皮MUC5AC表达减少,H^(+)/K^(+)ATPase β、PEP C表达增多;免疫荧光染色结果显示乙醇组胃组织上皮H^(+)/K^(+)ATPase β、PEP C、胃泌素表达增多;ELISA结果显示乙醇组胃组织胃必素、生长抑素、IL-1β含量显著增高;流式结果显示乙醇组胃体组织LGR5^(+)干细胞数量显著减少;乙醇组未能形成胃类器官。测序及生物信息分析结果表明,炎症通路TNF信号通路、NF-κB信号通路和分化信号Notch通路富集。结论 50%乙醇溶液单次给予伴随10%持续给予8 d可引发小鼠胃腺体结构损伤,干细胞受损,分化细胞代偿性增加。上述改变可能与乙醇引起的炎性通路及Notch信号通路上调有关。
OBJECTIVE To study the effects of ethanol on markers of gastric stem cells and epithe-lial cells,and explore the related signal pathways for stem cells differentiation in a mouse gastric mucous injury model.METHODS Male C57BL/6 mice were randomly divided into normal control and ethanol groups.The mice in the control group were given normal drinking water while those in the ethanol group were gavaged with 10 ml·kg^(-1)50%(V/V)ethanol on day 1,and drinking water containing 10%(V/V)ethanol was given on day 2-9.On the 10th day,stomach tissues were collected.HE staining was used to detect pathological changes in the stomach.Immunohistochemistry(IHC)staining was used to detect changes of such cell markers as mucin 5AC,H^(+)/K^(+)ATPaseβand pepsin C.Immunofluorescence(IF)staining was employed to analyze changes in expression of cell markers such as H^(+)/K^(+)ATPaseβ,pepsin C and gastrin.ELISA assay was used to measure gastrin,somatostatin and interleukin 1β(IL-1β)concentrations in gastric tissue homogenates.Flow cytometry was adopted to measure the number of leucine rich repeat containing G protein-coupled receptor 5 positive(LGR5^(+))stem cells in gastric glands.Organoids was constructed to characterize stem cell activity.RNA sequencing and bioinformatics analysis were conducted to analyze the inflammatory pathways and differentiation signaling pathways during mice gastric mucous injury.RESULTS H&E results showed multifocal necrosis of the mucosal layer appeared in the ethanol group,accompanied by pyknosis,lysis and detachment of mucosal epithelial cells and gastric gland cells.IHC results showed decreased expressions of mucin 5AC and increased expressions of H^(+)/K^(+)ATPaseβand pepsin C.IF results revealed increased expressions of H^(+)/K^(+)ATPaseβ,pepsin C,and gastrin after ethanol treatment.ELISA results demonstrated significant increases in gastrin,somatostatin and IL-1βlevels in gastric tissues of the ethanol group.Flow assay results suggested that the number of LGR5^(+)stem cells significantly decreased in ethanol treated gastric tissues.Stem cells from stomach tissues treated with ethanol did not grow into organoids.RNA sequencing and bioinformatics analyses revealed enrichment of TNF,NF-κB and Notch pathways in the ethanol group.CONCLUSION Administration of 50%ethanol solution on day 1,followed by continuous admin-istration of 10%ethanol solution on day 2-9 can induce histopathological injury to the gastric gland and stem cells,and an increase in epithelial cells.These changes may be related to the up-regulation of inflammatory pathways and Notch signaling pathways triggered by ethanol.
作者
张林丽
田书婷
刘芮希
刘莉
ZHANG Linli;TIAN Shuting;LIU Ruixi;LIU Li(China State Institute of Pharmaceutical Industry,Shanghai 200437,China;Shanghai Professional and Technical Service Center for Biological Material Druggability Evaluation,Shanghai 200437,China;National Key Laboratory of Lead Druggability Research,Shanghai Institute of Pharmaceutical Industry,Shanghai 200437,China;Clinical Medicine School of Xi′an Medical University,Xi′an 710021,China)
出处
《中国药理学与毒理学杂志》
北大核心
2025年第6期444-452,共9页
Chinese Journal of Pharmacology and Toxicology
关键词
干细胞
免疫荧光
类器官
stem cell
immunofluorescence
organoid
