大肠杆菌对藏羊子宫内膜上皮细胞相关炎性因子表达的影响

为了探究大肠杆菌(Escherichia coli)对藏羊子宫内膜上皮细胞(EECs)相关炎性因子表达的影响,本研究采用组织块贴壁法和酶消化法培养藏羊EECs;用不同感染复数(MOI)进行E. coli感染试验,通过实时荧光定量PCR(qRT-PCR)及酶联免疫(ELISA)技术,分析白细胞介素1β(IL-1β)、白细胞介素6(IL-6)、白细胞介素8(IL-8)和肿瘤坏死因子α(TNF-α)等炎性因子的RNA和蛋白表达水平。结果表明,培养液中加入50 ng·mL^-1表皮生长因子(EGF)能成功培养出藏羊EECs,纯度达到98%以上;E. coli感染试验中,当MOI为1:1时,IL-1β、IL-6和IL-8基因和蛋白的表达量与对照组相比差异不显著,而TNF-α表达量显著升高(P<0.05);当MOI为20:1时,IL-8基因和蛋白的表达量显著升高(P<0.05);当MOI为50:1时,IL-1β、IL-6基因和蛋白的表达量显著升高(P<0.05),而TNF-α表达量与对照组差异不显著(P>0.05)。结果表明,酶消化法可成功培养藏羊EECs;不同MOI E.coli感染后,藏羊EECs中IL-1β、IL-6、IL-8和TNF-α等炎性因子的表达量较对照组均显著升高(P<0.05),它们可作为识别子宫内膜炎病理变化程度的指标。本研究结果为预防子宫内膜炎、提高藏羊的生产性能以及生态养殖提供了理论依据。

Effect of Escherichia coli on the Expression of Inflammatory Factors Associated With Endometrial Epithelial Cells in Tibetan Sheep

In order to investigate the effect of E. coli on the expression of inflammatory factors associated with Tibetan sheep endometrial epithelial cells (EECs), EECs of Tibetan sheep were cultured by tissue block attachment and enzymatic digestion; E. coli infection assays were performed with different multiplicity of infection (MOI), and the RNA and protein expression levels of inflammatory factors such as interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8) and tumor necrosis factor α (TNF-α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA).The results showed that EECs were successfully cultured in DMEM with 50 ng·mL^-1epidermal growth factor (EGF), and the purity was 98%. The expression of IL-1β, IL-6 and IL-8 genes and proteins after infection did not change significantly compared with the control when MOI was 1:1(P>0.05), but that of TNF-α increased dramatically (P<0.05); When MOI was 20:1, the expression of IL-8 gene and protein increased (P<0.05), but that of IL-1β, IL-6 and TNF-α didn’t change much(P>0.05); And when MOI was 50:1, the expression of IL-1β, IL-6 genes and proteins increased significantly (P<0.05), while that of TNF-α remained at the same level (P>0.05). It indicated that EECs can be successfully cultured in vitro; the expression levels of IL-1β, IL-6, IL-8, and TNF-α on EECs after being infected by E. coli at different MOI were significantly higher than those of the control (P<0.05). These factors could be taken as indicators to identify the pathological changes of endometritis in Tibetan sheep. That also provides a theoretical basis for preventing endometritis, improving the performance of Tibetan sheep and ecological breeding.