外泌体miR-218抑制脂多糖诱发子宫内膜上皮细胞免疫因子释放研究  

Exosomes and miR-218 inhibits lipopolysaccharide-induced release of immune factors from endometrial epithelial cells

作　　者：谢彤彤 付博凡 姚鑫鑫 盛熙晖[1] 郭勇[1] 肖龙菲 倪和民[1] 王相国[1] XIE Tongtong;FU Bofan;YAO Xinxin;SHENG Xihui;GUO Yong;XIAO Longfei;NI Hemin;WANG Xiangguo(College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China)

机构地区：[1]北京农学院动物科学技术学院,北京102206

出　　处：《北京农学院学报》2021年第1期105-111,共7页Journal of Beijing University of Agriculture

基　　金：国家自然科学基金青年项目(31802263);现代农业产业技术体系北京市奶牛创新团队(BAIC05-2017);北京市教育委员会科技计划一般项目(KM201810020007);国家重点研发计划(2017YFD0502200)。

摘　　要：【目的】探究奶牛子宫内膜上皮细胞来源外泌体及其miR-218在减轻大肠杆菌脂多糖诱发免疫反应中的作用。【方法】用不同质量浓度大肠杆菌脂多糖作用于奶牛子宫内膜上皮细胞系24 h后采用细胞计数试剂盒检测细胞活性;酶联免疫吸附测定检测免疫因子IL-1β和TNF-α释放;采用透射电子显微镜和蛋白印记法验证提取外泌体的粒径和标识蛋白CD63表达,荧光定量聚合酶链式反应法检测外泌体中miR-218表达变化;采用激光共聚焦显微镜观察外泌体与奶牛子宫内膜上皮细胞融合;细胞转染mimics 218检测其对大肠杆菌脂多糖诱发奶牛子宫内膜上皮细胞免疫因子释放的影响。【结果】100μg/mL大肠杆菌脂多糖处理奶牛子宫内膜上皮细胞后,IL-1β、TNF-α含量均显著高于对照组(P<0.05);大肠杆菌脂多糖刺激奶牛子宫内膜上皮细胞来源外泌体中miR-218的表达量极显著低于奶牛子宫内膜上皮细胞来源外泌体中miR-218的表达量(P<0.01);正常培养奶牛子宫内膜上皮细胞来源外泌体能够与大肠杆菌脂多糖处理的奶牛子宫内膜上皮细胞融合并与NF-ΚB(p65)蛋白共定位于细胞质中。将奶牛子宫内膜上皮细胞来源外泌体加入到100μg/mL大肠杆菌脂多糖处理奶牛子宫内膜上皮细胞24 h后,显著抑制了大肠杆菌脂多糖引起的奶牛子宫内膜上皮细胞培养上清中的IL-1β和TNF-α释放(P<0.05)。同样的,转染进奶牛子宫内膜上皮细胞内mimics 218与磷酸化的NF-ΚB(p65)核外部分共定位并显著抑制了大肠杆菌脂多糖诱发的奶牛子宫内膜上皮细胞中TNF-α释放(P<0.05),而对IL-1β释放无显著差异。【结论】miR-218能够随着健康子宫内膜上皮细胞分泌外泌体进入到受到大肠杆菌脂多糖刺激奶牛子宫内膜上皮细胞内,并与磷酸化p65核外部分共定位并抑制大肠杆菌脂多糖引起的TNFα的释放。【Objectives】To investigate the role of exosomes and its miR-218 derived from endometrial epithelial cell of dairy cows in reducing lipopolysaccharide-induced immune response.【Methods】Cell count kit 8 was used to detect cell viability after lipopolysaccharide was applied to cow endometrial epithelial cell line for 24 hours.Eenzyme linked immunosorbent assay was used to detect the release of immune factors IL-1βand TNF-α.Transmission electron microscopy and Western Blot were used to verify the particle size of the extracted exosomes and the expression of the marker protein CD63.Detection of miR-218 expression in exosomes by quantitative real-time PCR.Observation of fusion of exosomes and endometrial epithelial cells by laser confocal microscopy.Cells were transfected with mimics 218 to detect its effect on lipopolysaccharide-induced endometrial epithelial cell immune factor release.【Results】When endometrial epithelial cell was treated with 100μg/mL lipopolysaccharide,the release of IL-1βand TNF-αwere significantly higher than those of the control group(P<0.05).MiR-218 expression in lipopolysaccharide treated endometrial epithelial cell-derived exosomes was significantly lower than that in endometrial epithelial cell-derived exosomes(P<0.01).Normally cultured endometrial epithelial cell-derived exosomes can fuse with lipopolysaccharide-treated endometrial epithelial cells and co-localize with NF-KB(p65)protein in the cytoplasm.Adding endometrial epithelial cell-derived exosomes to 100μg/mL lipopolysaccharide treated endometrial epithelial cells for 24 hours co-culture significantly inhibited the release of IL-1βand TNF-αin the culture supernatant of endometrial epithelial cells induced by lipopolysaccharide(P<0.05).Similarly,mimics 218 transfected into endometrial epithelial cells co-localized with the extranuclear part of phosphorylated NF-KB(p65)and significantly inhibited the release of TNF-αin endometrial epithelial cells induced by lipopolysaccharide(P<0.05).There was no significant differe

关 键 词：子宫内膜炎 外泌体 大肠杆菌脂多糖 MIR-218 子宫内膜上皮细胞 

分 类 号：S823.9[农业科学—畜牧学]