雌二醇和孕酮对LPS诱导的犬子宫内膜基质细胞TLR4及炎性因子表达的影响  

Effect of Estradiol and Progesterone on the Expression of TLR4 and Inflammatory Cytokines in Canine(Canis lupus familiaris)Endometrial Stromal Cells Induced by LPS

作　　者：倪珺 邱靖淇 李瑾 冯忆秋 刘飞燕 芮荣[1] NI Jun;QIU Jing-Qi;LI Jin;FENG Yi-Qiu;LIU Fei-Yan;RUI Rong(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《农业生物技术学报》2022年第10期1936-1943,共8页Journal of Agricultural Biotechnology

基　　金：南京农业大学SRT计划(202117YX09)。

摘　　要：子宫蓄脓是母犬(Canis lupus familiaris)常见的生殖疾病之一,其主要诱因包括激素分泌异常和细菌感染。子宫内膜是激素应答的靶器官,子宫内膜细胞的异常活化会导致大量炎性因子释放,引起炎症反应,然而在炎症环境下类固醇激素对犬子宫内膜基质细胞的免疫调控作用知之甚少。本研究旨在探讨类固醇激素雌二醇(estradiol,E_(2))和孕酮(progesterone,P_(4))调控体外培养的犬子宫内膜基质细胞(endometrial stromal cells,ESCs)Toll样受体4(Toll like receptor 4,TLR4)及相关炎性因子的分泌规律。临床采集子宫蓄脓犬和健康犬的外周血血清,利用ELISA方法检测血清中E_(2)和P_(4)的浓度;应用胶原酶消化法分离获取健康犬子宫内膜基质细胞并进行免疫荧光鉴定;以检测到的E_(2)(0.2 nmol/L)和P_(4)(0.1μmol/L)作用于细胞,利用噻唑蓝(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT)法测定E_(2)和P_(4)对细胞体外生长的影响;再分别与1μg/mL的细菌脂多糖(lipopolysaccharide,LPS)共同处理细胞,通过qPCR检测LPS诱导6、12和24 h犬子宫内膜基质细胞中TLR4和炎性因子的基因表达。免疫荧光鉴定结果显示,犬子宫内膜基质细胞纯度较高。MTT检测结果显示,P_(4)单独作用及E_(2)和P_(4)联合处理能显著促进细胞的体外增殖。qPCR结果显示,与对照组相比,LPS刺激后TLR4和炎性因子的基因表达量均显著上调(P<0.05);与LPS组相比,P_(4)单独处理与E_(2)和P_(4)联合处理可显著下调LPS刺激引起的TLR4和炎性因子基因过量表达(P<0.05)。上述结果提示E_(2)和P_(4)可能在子宫内膜的免疫调节中发挥重要作用。本研究为阐明子宫蓄脓发生的分子机制及E_(2)和P_(4)对子宫内膜先天性黏膜免疫的影响提供基础资料。Pyometra is a common reproductive disease of the female dogs(Canis lupus familiaris).Hormonal disorder and bacterial infections are main predisposing factors.The endometrium is the target organ for hormone response.A large amount of inflammatory cytokines will produce when endometrial cells are abnormally activated.However,the immunoregulatory effects of steroid hormones on canine endometrial stromal cells(ESCs)in an inflammatory environment are still poorly understood.To study the regulation of estradiol(E_(2))and progesterone(P_(4))on the secretion of Toll like receptor 4(TLR4)and related inflammatory cytokines in canine ESCs in vitro.The peripheral blood serum of pyometra and healthy canine were collected,and the concentration of E_(2)and P_(4)were detected by ELISA;canine endometrial ESCs were isolated by collagenase enzymolysis and identified by immunofluorescence.The detected E_(2)(0.2 nmol/L)and P_(4)(0.1μmol/L)were added to the cell culture medium,then the effects of E_(2)and P_(4)on cell growth in vitro were determined by the method of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT);then the cells were treated with 1μg/mL lipopolysaccharide(LPS),and qPCR was performed to detect the gene expression of TLR4 and inflammatory cytokines in canine ESCs induced by LPS at 6,12,and 24 h.The results showed that the cells isolated were ESCs with a high purity rate after immunofluorescence identification.E_(2)had no significant effect on the ESCs,while P_(4)and combination of E_(2)and P_(4)could significantly stimulate proliferation of ESCs in vitro.Compared with the control group,the gene expression of TLR4 and inflammatory cytokines were significantly up-regulated after LPS stimulation(P<0.05).Compared with the LPS group,P_(4)alone and the combination of two hormones significantly down-regulated the gene expression of TLR4 and inflammatory cytokines caused by LPS stimulation(P<0.05).This experiment proves that E_(2)and P_(4)play an important role in ESCs.This study provides basic data for clari

关 键 词：犬 子宫内膜基质细胞(ESCs) 雌二醇(E_(2)) 孕酮(P_(4)) Toll样受体4(TLR4) 炎性因子 

分 类 号：S857.2[农业科学—临床兽医学]