双酚A对子宫内膜间充质干/基质细胞干性的影响及人脐带间充质干细胞源性上清对细胞损伤的改善作用  

Effects of bisphenol A on stemness of endometrial mesenchymal stem/stromal cells and improvement effect of human umbilical cord mesenchymal stem cell-derived supernatant on cell injury

作　　者：王爱乔 米旭光 林秀英[2] 付建华[2] 刘磊[2] 王琳 张文琦 邓玲 陈士玲 方艳秋 WANG Aiqiao;MI Xuguang;LIN Xiuying;FU Jianhua;LIU Lei;WANG Lin;ZHANG Wenqi;DENG Ling;CHEN Shiling;FANG Yanqiu(School of Clinical Medical Sciences,Changchun University of Chinese Medicine,Changchun 130021,China;Reproductive Medicine Center,People’s Hospital,Jilin Province,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6期1557-1564,共8页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划项目(20240602029RC,20230204036YY,20230203051SF,YDZJ202102CXJD076);吉林省卫健委卫生健康技术创新项目(2021lc059)。

摘　　要：目的:探讨双酚A(BPA)对子宫内膜间充质干/基质细胞(eMSCs)增殖活性和干性特征的影响,阐明人脐带间充质干细胞源性上清(hUCMSC-Sup)对细胞损伤的改善作用。方法:体外培养eMSCs,以0、200、250、300、350、400μmol·L^(-1)BPA处理。将eMSCs分为对照组(仅培养液培养)、BPA组(含200μmol·L^(-1)BPA的等体积培养液培养)、BPA+hUCMSC-Sup组(含200μmol·L^(-1)BPA及50%体积比hUCMSC-Sup的等体积培养液培养)和BPA+CHIR-99021组(含200μmol·L^(-1)BPA及10μmol·L^(-1)CHIR-99021的等体积培养液培养),使用干细胞成球培养液培养eMSCs干细胞球,其余细胞均使用DMEM/F12完全培养基培养。噻唑蓝(MTT)法检测各组eMSCs存活率,球体形成实验检测各组eMSCs干细胞球数和直径,CCK-8法检测各组eMSCs干细胞球中细胞增殖活性,流式细胞术检测各组eMSCs中CD73+细胞百分率,实时荧光定量PCR(RT-qPCR)法检测各组eMSCs中性别决定区Y框蛋白2(Sox2)、八聚体结合转录因子4(Oct4)和Nanog mRNA表达水平,Western blotting法检测各组eMSCs中β-连环蛋白(β-catenin)蛋白表达水平。结果:MTT法检测,BPA作用24和48 h,与0μmol·L^(-1)BPA组比较,200、250、300、350和400μmol·L^(-1)BPA组eMSCs存活率均明显降低(P<0.01)。药物作用24 h时,与对照组比较,BPA组eMSCs存活率明显降低(P<0.01);药物作用48 h时,与对照组比较,BPA组eMSCs存活率明显降低(P<0.01);与BPA组比较,BPA+hUCMSC-Sup组eMSCs存活率明显升高(P<0.05)。球体形成实验检测,与培养3 d组比较,培养4和5 d组eMSCs干细胞球数和直径均明显增加(P<0.05或P<0.01);与对照组比较,培养48 h时BPA组eMSCs干细胞球数和直径均明显减少(P<0.05或P<0.01)。CCK-8法检测,处理24和48 h时,与对照组比较,BPA组eMSCs干细胞球中细胞增殖活性均明显降低(P<0.01);与BPA组比较,BPA+hUCMSC-Sup组eMSCs干细胞球中细胞增殖活性均明显升高(P<0.01)。流式细胞术检测,与对照组比较,BPA组eMSCs中CD73+细胞百分率Objective:To investigate the effects of bisphenol A(BPA)on the proliferation activity and stemness characteristics of endometrial mesenchymal stem/stromal cells(eMSCs),and to elucidate the improvement effect of human umbilical cord mesenchymal stem cell-derived supernatant(hUCMSC-Sup)on the cell injury.Methods:The eMSCs were cultured in vitro and treated with different concentrations of BPA(0,200,250,300,350,and 400μmol·L^(-1)).The eMSCs were divided into control group(only cultured with culture solution),BPA group(cultured with isovolumetric culture solution including 200μmol·L^(-1)BPA),BPA+hUCMSC-Sup group(cultured with isovolumetric culture solution including 200μmol·L^(-1)BPA and 50%volumetric ratio of hUCMSC-Sup),and BPA+CHIR-99021 group(cultured with isovolumetric culture solution including 200μmol·L^(-1)BPA and 10μmol·L^(-1)CHIR-99021).The survival rates of eMSCs in various groups were detected by methyl thiazolyl tetrazolium(MTT)assay.The numbers and diameters of the spheroids in various groups were detected by spheroids formation assay,the proliferation activities of the cells in eMSCs stem cell spheroids in various groups were detected by CCK-8 assay;the percentage of CD73+cells in eMSCs in various groups were detected by flow cytometry;the expression levels of sex determining region Y-box 2(Sox2),octamer-binding transcription factor 4(Oct4),and Nanog mRNA in the eMSCs in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method,the expression levels ofβ-catenin protein in the eMSCs in various groups were detected by Western blotting method.Results:The MTT results showed that after treated with BPA for 24 and 48 h,compared with 0μmol·L^(-1)BPA group,the survival rates of eMSCs in 200,250,300,350,and 400μmol·L^(-1)BPA groups were significantly decreased(P<0.01).At 24 and 48 h after treatment,compared with control group,the survival rate of the eMSCs in BPA group was significantly decreased(P<0.01);at 48 h after treatment,compared with BPA group,the survival rat

关 键 词：双酚A 子宫内膜间充质干/基质细胞 人脐带间充质干细胞 干细胞球 

分 类 号：Q254[生物学—细胞生物学]