CDH1基因对小鼠胃类器官生长和E-caherin表达的作用机制  

Mechanism of action of CDH1 gene on gastric organoid growth and E-cadherin expression in mice

作　　者：邵伟 余友杰 夏海娜 郑优优 孙展杭 严皓哲 Wei Shao;You-Jie Yu;Hai-Na Xia;You-You Zheng;Zhan-Hang Sun;Hao-Zhe Yan(Department of Gastroenterology,Putuo District People’s Hospital,Zhoushan 316100,Zhejiang Province,China;Department of Hematology and Oncology,Putuo District People’s Hospital,Zhoushan 316100,Zhejiang Province,China;Department of Pathology,Putuo District People’s Hospital,Zhoushan 316100,Zhejiang Province,China;Institute of Digestive Disease Prevention and Treatment,Putuo District People’s Hospital,Zhoushan 316100,Zhejiang Province,China)

机构地区：[1]舟山市普陀区人民医院消化内科,浙江省舟山市316100 [2]舟山市普陀区人民医院血液肿瘤科,浙江省舟山市316100 [3]舟山市普陀区人民医院病理科,浙江省舟山市316100 [4]舟山市普陀区人民医院浙江舟山消化病防治研究所,浙江省舟山市316100

出　　处：《世界华人消化杂志》2024年第2期141-147,共7页World Chinese Journal of Digestology

基　　金：2021年度舟山市市级公益类计划项目,No.2021C31081.

摘　　要：背景CDH1基因编码的蛋白质E-cadherin在维持上皮组织完整性中发挥重要作用,了解CDH1基因对胃类器官生长和E-cadherin表达的作用机制对于揭示器官发育的分子基础至关重要.因此假设CDH1基因通过调控E-cadherin的表达参与调控胃类器官的发育和生长过程.目的探究钙黏蛋白1(cadherin-1,CDH1)基因对小鼠胃类器官生长和E-钙黏蛋白(E-cadherin)表达的作用机制.方法采用CRISPR/Cas9基因敲除技术获得CDH1^(-/-)C57BL/6品系小鼠10只,及野生型(WT)C57BL/6品系小鼠10只作为实验对照组.分离小鼠胃组织并培养胃类器官,免疫荧光染色观察胃类器官中E-cadherin表达,比较小鼠胃类器官数量和直径,MTT检测胃类器官细胞活力,蛋白质印迹法和RT-PCR法检测胃类器官中E-cadherin蛋白和mRNA表达.结果在光学显微镜下可见小鼠胃组织在培养基中不断生长与分裂,从第0 d开始组织在24 h内形成囊性结构,然后经历快速生长阶段.器官样细胞扩展为大的3D球形结构,到第5 d后逐渐出芽并开始形成胃类器官,提示胃类器官初步构建.免疫荧光染色结果显示,WT C57BL/6小鼠胃类器官中E-cadherin呈阳性表达,进一步提示胃类器官成功构建.和WT C57BL/6小鼠相比,CDH1^(-/-)C57BL/6小鼠胃类器官数量、胃类器官直径、吸光度值及胃类器官中E-cadherin蛋白和E-cadherin mRNA相对表达量均明显减少(P<0.05).结论CDH1基因敲除可抑制胃类器官生长形成及E-cadherin表达.BACKGROUND The E-cadherin protein encoded by the CDH1 gene plays an important role in maintaining epithelial tissue integrity.Understanding the mechanism of the CDH1 gene on gastric organoid growth and E-cadherin expression is crucial for revealing the molecular basis of organ development.Therefore,it was assumed that the CDH1 gene participates in regulating the development and growth of gastric organoids by regulating the expression of E-cadherin.AIM To investigate the mechanism of CDH1 gene on the growth of gastric organoids and E-cadherin expression in mice.METHODS CRISPR/Cas9 gene knockout technique was used to obtain 10 CDH1^(-/-)C57BL/6 mice,and 10 wild-type(WT)C57BL/6 mice were used as controls.Mouse gastric tissue was separated and cultured to obtain gastric organoids to observe the expression of E-cadherin in gastric organoids using immunofluorescence staining,compare the number and diameter of gastric organoids in mice,detect the cell viability in gastric organoids using MTT,and detect the expression of E-cadherin protein and mRNA in gastric organs using Western blot and RT-PCR methods,respectively.RESULTS Under the light microscope,it was observed that the gastric pylorus tissue of mice continued to grow and divide in the culture medium,forming a cystic structure within 24 h from day 0,and then undergoing a rapid growth.Organ like cells expanded into large three-dimensional spherical structures,and after the 5th day,they gradually sprouted and began to form stomach like organs,preliminarily indicating that gastric organoids were obtained.Immunofluorescence staining showed that E-cadherin was positively expressed in the gastric organs of WT C57BL/6 mice,further indicating the successful construction of gastric organoids.Compared with WT C57BL/6 mice,CDH1^(-/-)C57BL/6 mice showed a significant decrease in the number,diameter,absorbance value,and relative expression of E-cadherin protein and mRNA in gastric organoids(P<0.05).CONCLUSION Knockout of the CDH1 gene can inhibit the growth and formation of gast

关 键 词：CDH1 胃类器官 生长 E-CADHERIN 

分 类 号：R735.2[医药卫生—肿瘤]