《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (8): 2288-2295.doi: 10.16431/j.cnki.1671-7236.2019.08.012

• 生理生化 • 上一篇    下一篇

脂多糖对体外培养奶牛子宫内膜上皮细胞子宫容受性因子表达的影响

冯妍, 倪和民, 郭勇, 盛熙晖, 齐晓龙, 邢凯, 王相国   

  1. 北京农学院动物科学技术学院, 北京 102206
  • 收稿日期:2019-03-06 出版日期:2019-08-20 发布日期:2019-08-17
  • 通讯作者: 王相国 E-mail:xianguo731@163.com
  • 作者简介:冯妍(1994-),女,北京人,硕士,研究方向:动物繁殖机理及技术,E-mail:932641666@qq.com
  • 基金资助:

    国家自然科学基金青年项目(31802263);国家重点研发计划项目(2017YFD0502200);北京市教委科技计划一般项目(KM201810020007);现代农业产业技术体系北京市奶牛创新团队(5075299027)

Effect of Lipopolysaccharide on Expression of Uterine Receptive Factors in Dairy Cow Endometrial Epithelial Cells Cultured in vitro

FENG Yan, NI Hemin, GUO Yong, SHENG Xihui, QI Xiaolong, XING Kai, WANG Xiangguo   

  1. Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China
  • Received:2019-03-06 Online:2019-08-20 Published:2019-08-17

摘要:

试验旨在通过脂多糖(lipopolysaccharide,LPS)诱导奶牛子宫内膜上皮细胞构建子宫内膜炎体外感染模型,研究子宫内膜炎对奶牛子宫容受性因子大分子转膜黏蛋白-糖蛋白1(MUC-1)、高度保守的分泌型WNT家族亚型糖蛋白(Wnt-7a)、β受体1(IFNAR1)、IFNAR2、Integrin αvβ3 mRNA及蛋白相对表达量的影响,进而阐明子宫内膜炎引起奶牛屡配不孕和繁殖率低下的机制。采用组织块法分离培养奶牛子宫内膜上皮细胞,免疫荧光方法鉴定细胞纯度,不同浓度LPS刺激子宫内膜上皮细胞,在不同时间点用CCK8测定细胞存活率,ELISA检测炎性因子白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、IL-6、IL-8的分泌变化,实时荧光定量PCR和Western blotting分别检测子宫内膜感染模型中子宫容受性因子MUC-1、Wnt-7a、IFNAR1、IFNAR2、Integrin αvβ3 mRNA及蛋白的表达变化。结果显示,通过组织块法纯化获得的奶牛子宫上皮细胞数量较多,经免疫荧光角蛋白染色证实纯度较高。采用CCK8测定细胞存活率发现,与对照组相比,浓度为0、5、10、50μg/mL的LPS作用6、12、24、48 h后,细胞活力无显著变化(P>0.05);浓度为100μg/mL的LPS作用24 h时,细胞存活率显著低于对照组(P<0.05),细胞培养上清中的IL-1β、TNF-α、IL-6、IL-8含量均显著高于对照组及低浓度组(P<0.05),引起细胞的炎症反应。与对照组相比,模型组MUC-1 mRNA及蛋白的相对表达量均极显著增加(P<0.01);Wnt-7a mRNA及蛋白的相对表达量均极显著下降(P<0.01);Integrin αvβ3、IFNAR1和IFNAR2 mRNA相对表达量均极显著下降(P<0.01),蛋白相对表达量均显著下降(P<0.05)。结果表明,浓度为100μg/mL的LPS作用24 h可成功构建子宫内膜炎体外感染模型,子宫内膜炎对奶牛子宫容受性因子MUC-1、Wnt-7a、IFNAR1、IFNAR2、Integrin αvβ3 mRNA及蛋白表达的影响可能是引起奶牛屡配不孕和繁殖率低下的重要原因。

关键词: 奶牛; 子宫内膜上皮细胞; 脂多糖(LPS); 子宫容受性因子

Abstract:

The aim of this study was to establish an in vitro infection model of endometritis by inducing endometrial epithelial cells of dairy cows with lipopolysaccharide (LPS) and study the effects of endometritis on the relative expression of uterine receptive factors MUC-1, Wnt-7a, IFNAR1, IFNAR2, Integrin αvβ3 mRNA and protein in dairy cows.The mechanism of repeated sterility and low reproductive rate in dairy cows caused by endometritis were further clarified.Endometrial epithelial cells of dairy cows were isolated and cultured by tissue block method;The purity of the cells was determined by immunofluorescence;Endometrial epithelial cells were stimulated by LPS at different concentrations;The cell survival rate was measured by CCK8 at different time points;The secretion of inflammatory factors interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6 and IL-8 were detected by ELISA.Real-time PCR and Western blotting were used to detect the mRNA and protein expression of uterine receptive factors MUC-1, Wnt-7a, IFNAR1, IFNAR2, Integrin αvβ3 in endometrial infection model.The results showed that the number of bovine uterine epithelial cells purified by tissue block method was more, and the purity was higher by immunofluorescence keratin staining.CCK8 assay showed that compared with control group, LPS at 0, 5, 10, 50 μg/mL had no significant change in cell viability after 6, 12, 24 and 48 h (P>0.05).When LPS concentration was 100 μg/mL for 24 h, the cell survival rate was significantly lower than that of control group (P<0.05).The contents of IL-1β, TNF-α, IL-6 and IL-8 in cell culture supernatant were significantly higher than those in control and low concentration groups (P<0.05).Compared with control group, the relative expression of MUC-1 mRNA and protein in the model group extremely significantly increased (P<0.01), Wnt-7a mRNA and protein extremely significantly decreased (P<0.01).The mRNA expression of Integrin αvβ3, IFNAR1 and IFNAR2 extremely significantly decreased (P<0.01), and their protein expression were significantly decreased (P<0.05).The results showed that LPS with concentration of 100 μg/mL for 24 h could successfully construct the infection model of endometritis in vitro.The effects of endometritis on the expression of MUC-1, Wnt-7a, IFNAR1, IFNAR2, Integrin αvβ3 mRNA and protein might be the important reasons for repeated sterility and low reproductive rate of dairy cows.

Key words: dairy cow; endometrial epithelial cells; lipopolysaccharide (LPS); uterine receptive factor

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