Wnt/β-catenin信号通路抑制剂MSAB对人子宫内膜基质细胞纤维化反应的影响  

Effect of Wnt/β-catenin signaling pathway inhibitor MSAB on fibrogenic responses of human endometrial stromal cells

作　　者：王飞娜 米旭光 林秀英[2] 付建华[2] 刘磊[2] 于歆悦 臧欢欢 刘霖君 陈士玲 方艳秋 WANG Feina;MI Xuguang;LIN Xiuying;FU Jianhua;LIU Lei;YU Xinyue;ZANG Huanhuan;LIU Linjun;CHEN Shiling;FANG Yanqiu(School of Clinical Medicine,Changchun University of Chinese Medicine,Changchun 130021,China;Reproductive Medicine Center,Jilin Provincial People’s Hospital,Changchun 130021,China)

机构地区：[1]长春中医药大学临床医学院,吉林长春130021 [2]吉林省人民医院生殖医学中心,吉林长春130021

出　　处：《吉林大学学报（医学版）》2024年第5期1266-1274,共9页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划项目(20230204036YY,20230203051SF,YDZJ202102CXJD076);吉林省卫健委卫生健康技术创新项目(2021lc059,2021lc061)。

摘　　要：目的:探讨Wnt/β-连环蛋白(β-catenin)信号通路抑制剂3-(4-甲基苯基磺酰胺基)苯甲酸甲酯(MSAB)对人子宫内膜基质细胞(HESCs)纤维化反应的影响,为MSAB应用于宫腔粘连(IUA)靶向治疗提供依据。方法:体外培养正常HESCs,分为对照组和转化生长因子β1(TGF-β1)组;体外培养IUA患者粘连部分的HESCs,作为IUA组。采用Western blotting法检测TGF-β1作用不同时间(0、 12、 24、 48和60h)后各组细胞中纤维化标志蛋白Ⅰ型胶原α1(COL1A1)蛋白表达水平。采用MTT实验检测各组细胞增殖活性。采用Western blotting法检测对照组和IUA组细胞中细胞COL1A1、间质标志蛋白[N-钙黏蛋白和α-平滑肌肌动蛋白(α-SMA)]以及Wnt/β-catenin信号通路相关蛋白β-catenin表达水平。根据MSAB浓度,将正常HESCs分为0(对照组)、 0.25、 0.50、 0.75和1.00μmol·L^(-1) MSAB组,MTT实验检测各组细胞存活率。MSAB作用后,将正常HESCs分为对照组(正常HESCs)、 TGF-β1组(10μg·L^(-1) TGF-β1诱导正常HESCs 24 h后撤药,更换为完全养基并继续培养24 h)和MSAB组(10μg·L^(-1) TGF-β1诱导正常HESCs 24 h后撤药,更换为含0.75μmol·L^(-1) MSAB的完全培养基并继续培养24 h)。采用实时荧光定量PCR (RT-qPCR)法检测各组细胞中上皮-间质转化(EMT)相关转录因子Snail、Slug、Smuc、 ZEB1和ZEB2和COL1A1mRNA表达水平,采用Westernblotting法检测各组细胞中COL1A1蛋白、 N-钙黏蛋白、 α-SMA蛋白、 β-catenin和c-myc蛋白表达水平。结果:与对照组(TGF-β1作用0 h)比较,TGF-β1组作用12、24、48和60 h时HESCs中COL1A1蛋白表达水平升高(P<0.05或P<0.01)。与对照组比较,IUA组和TGF-β1组HESCs增殖活性差异无统计学意义(P>0.05)。与对照组比较,IUA组HESCs中COL1A1、β-连环蛋白、N-钙黏蛋白和α-SMA蛋白表达水平升高(P<0.05或P<0.01)。与对照组比较,0.75和1.00μmol·L^(-1) MSAB组细胞存活率降低(P<0.05或P<0.01)。与对照组比较,TGF-β1组细胞中Snail、Slug和CObjective:To discuss the effect of Wnt/β-catenin signaling pathway inhibitor methyl 3-{[(4-methyl-phenyl)sulfonyl]amino}benzoate(MSAB)on the fibrogenic response of the human endometrial stromal cells(HESCs),and to provide the foundation for the application of MSAB in the target therapy of intrauteriue adhesion(IUA).Methods:The normal HESCs were cultured in vitro and divided into two groups:control group and transforming growth factorβ1(TGF-β1)group;the HESCs from the adhesion part of the IUA patients were cultured in vitro,regarded as IUA group.Western blotting method was used to detect the expression levels of fibrotic marker protein typeⅠcollagenα1(COL1A1)in the cells in various groups at different time points(0,12,24,48,and 60 h)after treated with TGF-β1.MTT assay was used to detect the proliferation activities of the cells in various groups.Western blotting method was used to detect the expression levels of the fibrotic marker protein COL1A1,stromal marker proteins such as N-cadherin andα-smooth muscle actin(α-SMA),and Wnt/β-catenin signaling pathway-related proteinβ-catenin in the cells in control and IUA groups.Based on the MSAB concentrations,the normal HESCs were divided into 0(control),0.25,0.50,0.75,and 1.00μmol·L^(-1) MSAB groups,and MTT assay was used to detect the survival rates of the cells in various groups.After treated with MSAB,the normal HESCs were divided into control group(normal HESCs),TGF-β1 group(10μg·L^(-1) TGF-β1 induced normal HESCs for 24 h then the drug was withdrawn,replaced with complete culture medium,and the cells continued to be cultured for 24 h),and MSAB group(10μg·L^(-1) TGF-β1 induced normal HESCs for 24 h then the drug was withdrawn,replaced with a complete medium containing 0.75μmol·L^(-1) MSAB and the cells continued to be cultured for 24 h).Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of epithelial-mesenchymal transition(EMT)-related transcription factors Snail,Slug,Smuc,ZEB1,and ZEB2,and COL1A1 mRNA

关 键 词：子宫内膜纤维化 转化生长因子Β1 上皮-间质转化 ASHERMAN综合征 宫腔粘连 

分 类 号：R711[医药卫生—妇产科学]