脂多糖诱导奶牛子宫内膜上皮细胞炎性损伤的研究

Study on Inflammatory Injury of Endometrial Epithelial Cells Induced by Lipopolysaccharide in Dairy Cows

为了初步探讨大肠杆菌型奶牛子宫内膜炎发生机制,进而为后续筛选出作用于关键信号通路靶点的药物奠定基础。试验通过组织块培养法和传代培养分离纯化得到奶牛子宫上皮细胞(Bovine endometrial epithelial cell,bEEC)。30μg/mL的脂多糖(Lipopolysaccharide,LPS)刺激bEEC 12 h后,收集细胞用于奶牛子宫内膜上皮细胞体外炎性损伤模型的研究。通过荧光定量RT-PCR检测LPS作用于bEEC 2、6、9、12 h后细胞内IL-1β、IL-8、IL-6、TNF-αmRNA表达,然后通过Western-blot检测LPS诱导bEEC NF-кB和MAPKs相关蛋白的表达。结果表明:30μg/mL的LPS作用b EEC细胞2、6、9、12 h可极显著(P<0.01)诱导IL-1β、IL-8、IL-6、TNF-αmRNA表达。LPS可上调MAPKs相关蛋白P38、ERK、JNK的磷酸化水平(P<0.01),促使NF-κB抑制蛋白IκBα的降解(P<0.05)。说明LPS可激活MAPKs和NF-кB信号通路,诱导大量炎症介质的产生,从而对细胞造成炎性损伤。

The study aims to investigate the mechanism of endometrial inflammatory in cows with Escherichia coli,and lay a foundation for screening drugs that act on key signaling pathway targets.In this study,bovine endometrial epithelial cells(bEEC)were obtained and purified from cow uterus with primarily cultured tissue explant and trypsin.The cells by 30μg/mL lipopolysaccharide(LPS)stimulating 12 h was screened out,which was used as the bEEC inflammatory injury model in vitro.IL-1β,IL-8,IL-6 and TNF-αmRNA levels of bEEC induced in 2 h,6 h,9 h and 12 h by LPS were detected with RT-PCR,and NF-κB and MAPKs related proteins in LPS-bEEC cells were detected by Western-blot.The results showed that IL-1β,IL-8,IL-6 and TNF-αmRNA were highly expressed in 2 h,6 h,9 h and 12 h caused by 30μg/mL LPS(P<0.01).LPS could up-regulate the phosphorylation levels of P38,ERK and JNK which were related with MAPKs(P<0.01),and promote the degradation of IκBαwhich was the inhibitory protein of NF-κB(P<0.05).MAPKs and NF-кB signaling pathway of bEEC could be activated by LPS,and then a large number of inflammatory mediators were released,causing inflammation damage of the cells.