利用CRISPR-Cas9 技术探究肠道ERβ对肠源性TPH1 和5-HT 水平的影响  

Exploring the effect of intestinal ERβ on enterogenic TPH1 and 5-HT levelsusing CRISPR-Cas9 technology

作　　者：呼文强 杨千嬉 贺桂琼[1] 骆世芳 Hu Wenqiang;Yang Qianxi;He Guiqiong;Luo Shifang(Neuroscience Research Center,Chongqing Medical University;Department of Human Anatomy,School of Basic Medical Sciences,Chongqing Medical University)

机构地区：[1]重庆医科大学神经科学研究中心,重庆400016 [2]重庆医科大学基础医学院人体解剖学教研室,重庆400016

出　　处：《重庆医科大学学报》2025年第1期37-43,共7页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:82371203);重庆市基础与前沿研究计划资助项目(编号:CSTB2023NSCQMSX0161)。

摘　　要：目的:探索C57BL/6J小鼠结肠上皮雌激素受体β(estrogen receptorβ,ERβ)缺失对结肠色氨酸羟化酶-1(TPH1,tryptophan hydroxylase-1)和5-羟色胺(5-hydroxytryptamine,5-HT)水平的影响。方法:利用CRISPR-Cas9技术获得Pvillin-Cre^(+/-)小鼠、ERβflox^(+/-)小鼠,通过配种繁殖最终获得肠道ERβ敲除纯合子组(ERβflox-/--Pvillin-Cre^(+/+),ERβCKO,n=5)、杂合子组(ERβflox^(+/-)-Pvillin-Cre^(+/+),ERβCKO^(+/-))(n=5)及同窝野生型(wild type,WT)小鼠。取6个月龄和12个月龄的各品系小鼠作为研究对象。结合苏木素-伊红染色(hematoxylin-eosin staining,HE)、酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)、免疫印迹(Western blot,WB)和免疫组化(immunohistochemistry,IHC)方法对各组小鼠肠道形态、肠道ERβ、5-HT和TPH1水平进行检测。结果:基因鉴定验证肠道ERβCKO小鼠成功构建后,WB显示肠道ERβ水平明显下调(P<0.05)。HE显示肠道ERβ敲除后,肠道结构完整性与肠道黏膜上皮的绒毛发育均异常。ELISA结果显示,2个月龄段ERβCKO小鼠肠道5-HT水平较WT组小鼠下降明显(P<0.05),且呈月龄依赖方式。IHC结果显示,各组小鼠肠道均出现TPH1免疫阳性表达,与WT小鼠相比,ERβCKO小鼠肠道TPH1免疫阳性细胞数量减少(P<0.05),免疫阳性物表达量更低(P<0.05),但TPH1未呈现月龄依赖方式。结论:结肠ERβ下调可引起结肠TPH1蛋白和5-HT水平含量降低,继而引发小鼠肠道功能异常,增加患肠道疾病的风险,这为以ERβ为靶点的肠道相关疾病的发病机制提供了实验室依据。Objective:To investigate the effects of colonic epithelial estrogen receptorβ(ERβ)deletion on colonic tryptophan hydroxylase-1(TPH1)and 5-hydroxytryptamine(5-HT)levels in C57BL/6J mice.Methods:Pvillin-Cre^(+/-)mice and ERβflox^(+/-)mice were obtained using CRISPR-Cas9 technology,and intestinal ERβknockout homozygous(ERβflox-/--Pvillin-Cre^(+/+),ERβCKO,n=5),heterozygous(ERβflox^(+/-)-Pvillin-Cre^(+/+),ERβCKO^(+/-)n=5),and littermate wild-type(WT)mice were ultimately obtained by mating and breeding.Mice of each strain at 6 and 12 months of age were used for the study.Hematoxylin-eosin staining(HE),enzyme-linked immunosorbent assay(ELISA),Western blotting(WB),and immunohistochemistry(IHC)were used for determining the intestinal morphology and measuring intestinal ERβ,5-HT,and TPH1 levels of mice in each group.Results:After the successful construction of intestinal ERβCKO mice as verified by gene identification,WB showed that intestinal ERβlevels were significantly down-regulated(P<0.05).HE showed abnormalities in intestinal structural integrity and intestinal mucosal epithelial villi development after intestinal ERβknockdown.ELISA results showed that intestinal 5-HT levels of ERβCKO mice at 6 and 12 months of age were significantly decreased compared with those of WT mice(P<0.05),and the decrease was in a month-age-dependent manner.IHC results showed that TPH1 was expressed in the intestines of mice of all groups.However,ERβCKO mice showed a reduced number of TPH1 immunoreactive cells(P<0.05)and lower expression of immunoreactive substances(P<0.05)compared with WT mice,but the decline in TPH1 was not in a month-age-dependent manner.Conclusion:Down-regulation of colonic ERβinduces a decrease in colonic TPH1 protein and 5-HT levels,which subsequently triggers abnormal intestinal function and increases the risk of developing intestinal diseases in mice.This study is expected to provide a laboratory basis for the pathogenesis of ERβ-targeted intestinal diseases.

关 键 词：雌激素受体Β 色氨酸羟化酶-1 5-羟色胺 肠道 

分 类 号：R361[医药卫生—病理学]