乙硫氨酸通过MAT2A/β-连环蛋白/ZEB1/EMT通路调控小鼠神经管畸形的机制  

Mechanism of ethionine-induced neural tube defects in mice through methionine adenosyltransferase 2A/β-catenin/zinc finger E-box binding homeobox 1/epithelial-mesenchymal transition pathway

作　　者：张丽[1] 张钰璇 卫凯鑫 刘雨榕 张晓娜 孙雨晴 马惠静[3] 曹睿[2] 郑荣华[4] 王秀伟[5] 于保锋[2] Zhang Li;Zhang Yuxuan;Wei Kaixin;Liu Yurong;Zhang Xiaona;Sun Yuqing;Ma Huijing;Cao Rui;Zheng Ronghua;Wang Xiuwei;Yu Baofeng(Department of Hepatobiliary Surgery,the First Hospital of Shanxi Medical University,Taiyuan 030001,China;Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,China;Department of Obstetrics,the First Hospital of Shanxi Medical University,Taiyuan 030001,China;Medical Department of Linfen Vocational and Technical College,Linfen 041000,China;Beijing Key Laboratory of Child Development and Nutrition,the Capital Institute of Pediatrics,Beijing 100020,China)

机构地区：[1]山西医科大学第一医院肝胆外科,太原030001 [2]山西医科大学生物化学与分子生物学教研室,太原030001 [3]山西医科大学第一医院产科,太原030001 [4]临汾职业技术学院医学系,临汾041000 [5]首都儿科研究所儿童发育营养组学北京市重点实验室,北京100020

出　　处：《中华围产医学杂志》2025年第3期233-240,共8页Chinese Journal of Perinatal Medicine

基　　金：国家自然科学基金(82201319);山西省科学技术厅青年基金(20210302123347);山西省卫生健康委员会科研项目(2020090);山西医科大学“百亿工程”校级基金(BYJL063)。

摘　　要：目的探索甲硫氨酸腺苷转移酶2A(methionine adenosyltransferase 2A,MAT2A)/β-连环蛋白/锌指结合同源盒1(zinc finger E-box binding homeobox 1,ZEB1)/上皮间质转化(epithelial-mesenchymal transition,EMT)通路通过细胞内S-腺苷甲硫氨酸调节神经管畸形(neural tube defect,NTD)的分子机制。方法通过S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)代谢障碍抑制剂乙硫氨酸诱导小鼠NTD模型。将80只无特定病原体级C57BL/6小鼠分为正常组(36只)、乙硫氨酸组(46只)和乙硫氨酸+SAM组(44只),在妊娠第7.5天(E7.5)分别腹腔注射磷酸缓冲盐溶液(phosphate buffered saline,PBS)、乙硫氨酸和乙硫氨酸+SAM,并于E10.5处死。收集胚胎脑组织,体视显微镜下观察各组胚胎形态。Autodock软件分析乙硫氨酸和MAT2A的契合作用。利用免疫荧光、免疫组织化学、蛋白免疫印迹、酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和实时荧光定量逆转录聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)等方法测定3组胚胎脑组织MAT2A、β-连环蛋白、ZEB1及EMT相关靶基因的表达水平。采用方差分析分析组间数据。结果(1)MAT2A与乙硫氨酸之间通过共价键结合,具有契合作用,从而加速MAT2A的表达。(2)NTD模型构建成功后,正常胚胎饱满,脑部发育良好,而NTD胚胎发育迟缓,呈现明显的无脑畸形,胚胎神经管未闭合,且左右不对称。(3)乙硫氨酸组小鼠胚胎组织中SAM和S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)的水平明显低于正常组(分别为1737.56±95.64与872.33±205.11,以及89.17±9.50与51.25±9.48);乙硫氨酸+SAM组的SAM水平为(1197.00±222.27),SAH水平为(66.61±12.25),均明显高于乙硫氨酸组(P值均<0.017)。与正常组和乙硫氨酸+SAM组相比,乙硫氨酸组胚胎脑组织中MAT2A mRNA表达明显上调[(1.00±0.00)、(1.59±0.52)和(2.42±0.53),F=49.64,P<0.001;组间两两比较,P值均<0.017]。(4)与正常组相比,乙硫氨酸组CtObjectiveTo explore the molecular mechanism by which the methionine adenosyltransferase 2A(MAT2A)/β-catenin/zinc finger E-box binding homeobox 1(ZEB1)/epithelial-mesenchymal transition(EMT)pathway regulates neural tube defect(NTD)through intracellular S-adenosylmethionine(SAM).MethodsA mouse NTD model was induced using the SAM metabolic disorder inhibitor ethionine.Eighty specific pathogen-free C57BL/6 mice were divided into three groups:a normal group(36 mice),an ethionine group(46 mice),and an ethionine+SAM group(44 mice).Phosphate-buffered saline(PBS),ethionine,and ethionine+SAM were respectively injected intraperitoneally on embryonic day 7.5(E7.5),and the mice were sacrificed on E10.5.Embryonic tissues were collected,and the morphology of embryos in each group was observed under a stereomicroscope.The interaction between ethionine and MAT2A was analyzed using Autodock software.The expression levels of MAT2A,β-catenin,ZEB1,and EMT-related proteins in the brain tissues of embryos from the three groups were measured using immunofluorescence,immunohistochemistry,Western blotting,enzyme-linked immunosorbent assay(ELISA),and real-time quantitative polymerase chain reaction(RT-qPCR).Variance analysis was used for intergroup comparisons.Results(1)Autodock analysis results showed that MAT2A binds to ethionine through covalent bonds,exhibiting a complementary effect,thereby accelerating the expression of MAT2A.(2)After successful construction of the NTD model,normal embryos were plump with well-developed brains.NTD embryos showed delayed development,obvious anencephaly,unclosed neural tubes,and asymmetry.(3)The levels of SAM and SAH in the embryonic tissues of the ethionine group were significantly lower than those in the normal group(1737.56±95.64 vs.872.33±205.11,and 89.17±9.50 vs.51.25±9.48,respectively).The SAM and SAH levels in the ethionine+SAM group was 1197.00±222.27 and 66.61±12.25,significantly higher than those in the ethionine group(P<0.017).Compared with the normal group and the ethionine+SAM grou

关 键 词：神经管畸形 乙硫氨酸 S-腺苷甲硫氨酸 甲硫氨酸腺苷转移酶2A 锌指结合同源盒1 上皮间质转化 

分 类 号：R714.5[医药卫生—妇产科学]