奶牛子宫内膜炎早期诊断及益生菌抗大肠杆菌诱导的子宫内膜上皮细胞炎性损伤作用机理

发布时间：2018-01-06 23:26

本文关键词：奶牛子宫内膜炎早期诊断及益生菌抗大肠杆菌诱导的子宫内膜上皮细胞炎性损伤作用机理　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：背景与目的:大肠杆菌(Escherichia coli,E.coli)是引起奶牛子宫内膜炎的主要致病菌之一,该病给奶牛养殖业带来了严重的经济损失,探究早期奶牛子宫内膜炎的诊断指标,做到早发现、早治疗显得尤为重要。益生菌作为潜在的抗生素替代品,治疗和预防奶牛子宫内膜炎的机制还不甚明确。因此,本试验通过研究鼠李糖乳杆菌GR-1(Lactobacill rhamnosus GR-1,L.rhamnosus GR-1)抗E.coli致奶牛子宫内膜上皮细胞炎性损伤的作用机制,为其临床应用提供理论依据。方法:根据对产后200头荷斯坦奶牛第21 d阴道内分泌物性状和子宫内容物细胞学检查,选取21头奶牛分为三组:临床型子宫内膜炎组(CE,n = 7);亚临床型子宫内膜炎组(SE,n = 7)和健康对照组(HC,n = 7)。分别于产后1d、7 d、14 d、21 d和30 d采集血清,使用全自动生化分析仪测定血清中酶活性及离子浓度变化;使用ELISA方法检测血清中炎性细胞因子和急性期蛋白的变化。利用原代奶牛子宫内膜上皮细胞(BEECs),评估L.rhamnosus GR-1抗E.coli致BEECs损伤的作用机制。试验分为四个组:对照组(CONT);大肠杆菌组(ECOL);提前3h加入鼠李糖乳杆菌GR-1组(LRGR);提前3 h加入鼠李糖乳杆菌GR-1后再加入大肠杆菌组(LRGR+ECOL)。E coli添加浓度为 1×105 CFU/mL,L.rhamnosus GR-1 添加浓度为 1×107 CFU/mL。通过倒置显微镜、扫描电镜、透射电镜观察BEECs形态学和超微结构变化;利用流式细胞术检测BEECs的凋亡情况;Western Blot方法检测细胞紧密连接蛋白及信号通路蛋白表达;Real-time PCR方法检测细胞相关受体及炎性因子表达。结果:产后7 d,CE组血清中AST的水平显著高于HC组;产后14 d、21 d和30 d,CE组血清中CK的水平显著高于HC组,且产后21d,SE组血清中CK的水平显著高于HC组。产后21d和30d,CE组血清中TNF-α的水平显著高于HE组和SE组;产后7d、14d、21d和30d,CE组血清中Hp的水平显著高于HC组,且在产后7d、14d和21d,SE组血清中Hp的水平显著高于HC组。L.rhamnosus GR-1减弱了E.coli诱导的细胞结构损伤,显著降低了细胞凋亡率。L.thamnosusGR-1显著降低了E.coli感染细胞导致的模式识别受体(TLR2,TLR4,NOD1和NOD2),炎性体(NLRP3,ASC 和 Caspase-1),TLR4 下游衔接分子(MyD88 和 TICAM2),负调控因子TOLLIP,转录调控因子(NF-κB和IRF3),炎性细胞因子(TNF-α,IL-1β,IL-6,IL-8,IL-10,IL-18和IFNβ)等免疫反应分子mRNA表达水平的增加。L.rhanmsosus GR-1显著提高了E.coli导致的紧密连接蛋白claudin-1/2、Occludin及ZO-1表达减少,显著降低了 NF-κB2和p65的表达。结论:奶牛血清中TNF-α和Hp水平可以作为临床型子宫内膜炎的辅助诊断指标。与其他诊断指标相比,奶牛血清中Hp对炎症的敏感性更高可以作为亚临床型子宫内膜炎的辅助诊断指标。L.rhamnosus GR-1通过衰减MyD88依赖性和非依赖性信号传递途径的信号转导,以及加强NLRs和TLRs之间的相互作用,抑制E.colE诱导的奶牛子宫内膜上皮细胞NF-κB信号通路激活,进而减少了炎性细胞因子的释放,同时缓解了E.coli对细胞紧密连接的破坏,减少了细胞的凋亡率,从而减轻了E coli诱导的奶牛子宫内膜上皮细胞的炎性损伤作用。
[Abstract]:Background and objective: Escherichia coli (Escherichia coli E.coli) is one of the main pathogenic bacteria of cow endometritis, the disease has brought serious economic loss to the dairy industry, explore the early diagnosis index of cow endometritis, early detection, early treatment is very important. Probiotics as potential alternatives to antibiotics, the mechanism for treatment of cow endometritis and prevention is not clear yet. Therefore, the research of Lactobacillus rhamnosus GR-1 through this test (Lactobacill rhamnosus GR-1, L.rhamnosus GR-1) against E.coli induced mechanism of cow endometrial epithelial cells inflammatory injury and provide theoretical basis for its clinical application. Methods: Based on the 200 postpartum Holstein cows twenty-first vaginal D in the secretion and uterine contents of cytology, selected 21 cows were divided into three groups: clinical endometritis group (CE, n = 7); Subclinical Type of endometritis group (SE, n = 7) and control group (HC, n = 7) respectively. In postpartum 1D, 7 d, 14 d, 21 d and 30 d collected serum, were measured by enzyme activity and ion concentration in serum by automatic biochemical analyzer; changes of inflammatory cytokines and acute phase use the ELISA method to detect the protein in serum. The primary bovine endometrial epithelial cells (BEECs), mechanism of assessment of BEECs damage induced by anti E.coli L.rhamnosus GR-1. The experiment was divided into four groups: control group (CONT); Escherichia coli group (ECOL); early 3H added rhamnose lactobacillus bacteria GR-1 group (LRGR); 3 h ahead with Lactobacillus rhamnosus GR-1 after adding Escherichia coli group (LRGR+ECOL).E concentration of coli was 1 * 105 CFU/mL, L.rhamnosus concentration of GR-1 was 1 * 107 CFU/mL. by inverted microscope, scanning electron microscopy, transmission electron microscopy observation of BEECs morphology and ultrastructure changes by flow cytometry; Apoptosis was detected by BEECs; the Western Blot method to detect cell tight junction proteins and signal pathway protein expression; the expression of Real-time PCR method to detect cell related receptors and inflammatory factors. Results: after 7 d, CE group of serum AST level was significantly higher than that of HC group; postpartum 14 d, 21 d and 30 d, CE group the serum level of CK was significantly higher than that of HC group and SE group, postpartum 21d, serum CK level was significantly higher than that of HC group. After 21d and 30d, TNF- alpha CE group in serum was significantly higher than that of HE group and SE group; 14d, postpartum 7d, 21d and 30d, CE in serum Hp level significantly in group HC, and 14d and 21d in postpartum 7d, SE group, serum Hp level was significantly higher than that of group HC.L.rhamnosus GR-1 decreased the damage of cell structure induced by E.coli, significantly decreased the apoptosis rate of.L.thamnosusGR-1 decreased significantly in E.coli infected cells leads to pattern recognition receptors (TLR2, TLR4, the NOD1 and NOD2), The inflammasome (NLRP3, ASC and Caspase-1), TLR4 (MyD88 and TICAM2 downstream adaptor molecule), a negative regulator of TOLLIP transcription factor (NF- kappa B and IRF3), inflammatory cytokines (TNF- alpha, IL-1 beta, IL-6, IL-8, IL-10, IL-18 and IFN beta) the immune responses of mRNA molecules the expression levels of.L.rhanmsosus increased GR-1 significantly increased the tight junction protein claudin-1/2 induced by E.coli, Occludin and ZO-1 expression decreased significantly, decreased the expression of NF- kappa B2 and p65. Conclusion: TNF- and Hp levels in serum can be used as an auxiliary diagnosis of clinical endometritis. Compared with other fault diagnosis index, the sensitivity of dairy cows the serum Hp of higher inflammation can be used as auxiliary diagnostic index.L.rhamnosus GR-1 subclinical endometritis by attenuating MyD88 dependent and non dependent signal transduction pathway, and strengthen the interaction between NLRs and TLRs With the inhibition of E.colE induced bovine endometrial epithelial cells NF- B signaling pathway activation, thereby reducing the release of inflammatory cytokines, and alleviate the E.coli is closely connected to the cell destruction, reduce the rate of apoptosis, thereby reducing the inflammatory injury of E induced by coli in cow endometrial epithelial cells.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.23

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