本文關(guān)鍵詞：奶牛子宮內(nèi)膜炎早期診斷及益生菌抗大腸桿菌誘導(dǎo)的子宮內(nèi)膜上皮細(xì)胞炎性損傷作用機(jī)理　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 奶牛子宮內(nèi)膜炎 早期診斷 大腸桿菌 鼠李糖乳桿菌 炎癥

【摘要】：背景與目的:大腸桿菌(Escherichia coli,E.coli)是引起奶牛子宮內(nèi)膜炎的主要致病菌之一,該病給奶牛養(yǎng)殖業(yè)帶來了嚴(yán)重的經(jīng)濟(jì)損失,探究早期奶牛子宮內(nèi)膜炎的診斷指標(biāo),做到早發(fā)現(xiàn)、早治療顯得尤為重要。益生菌作為潛在的抗生素替代品,治療和預(yù)防奶牛子宮內(nèi)膜炎的機(jī)制還不甚明確。因此,本試驗(yàn)通過研究鼠李糖乳桿菌GR-1(Lactobacill rhamnosus GR-1,L.rhamnosus GR-1)抗E.coli致奶牛子宮內(nèi)膜上皮細(xì)胞炎性損傷的作用機(jī)制,為其臨床應(yīng)用提供理論依據(jù)。方法:根據(jù)對(duì)產(chǎn)后200頭荷斯坦奶牛第21 d陰道內(nèi)分泌物性狀和子宮內(nèi)容物細(xì)胞學(xué)檢查,選取21頭奶牛分為三組:臨床型子宮內(nèi)膜炎組(CE,n = 7);亞臨床型子宮內(nèi)膜炎組(SE,n = 7)和健康對(duì)照組(HC,n = 7)。分別于產(chǎn)后1d、7 d、14 d、21 d和30 d采集血清,使用全自動(dòng)生化分析儀測(cè)定血清中酶活性及離子濃度變化;使用ELISA方法檢測(cè)血清中炎性細(xì)胞因子和急性期蛋白的變化。利用原代奶牛子宮內(nèi)膜上皮細(xì)胞(BEECs),評(píng)估L.rhamnosus GR-1抗E.coli致BEECs損傷的作用機(jī)制。試驗(yàn)分為四個(gè)組:對(duì)照組(CONT);大腸桿菌組(ECOL);提前3h加入鼠李糖乳桿菌GR-1組(LRGR);提前3 h加入鼠李糖乳桿菌GR-1后再加入大腸桿菌組(LRGR+ECOL)。E coli添加濃度為 1×105 CFU/mL,L.rhamnosus GR-1 添加濃度為 1×107 CFU/mL。通過倒置顯微鏡、掃描電鏡、透射電鏡觀察BEECs形態(tài)學(xué)和超微結(jié)構(gòu)變化;利用流式細(xì)胞術(shù)檢測(cè)BEECs的凋亡情況;Western Blot方法檢測(cè)細(xì)胞緊密連接蛋白及信號(hào)通路蛋白表達(dá);Real-time PCR方法檢測(cè)細(xì)胞相關(guān)受體及炎性因子表達(dá)。結(jié)果:產(chǎn)后7 d,CE組血清中AST的水平顯著高于HC組;產(chǎn)后14 d、21 d和30 d,CE組血清中CK的水平顯著高于HC組,且產(chǎn)后21d,SE組血清中CK的水平顯著高于HC組。產(chǎn)后21d和30d,CE組血清中TNF-α的水平顯著高于HE組和SE組;產(chǎn)后7d、14d、21d和30d,CE組血清中Hp的水平顯著高于HC組,且在產(chǎn)后7d、14d和21d,SE組血清中Hp的水平顯著高于HC組。L.rhamnosus GR-1減弱了E.coli誘導(dǎo)的細(xì)胞結(jié)構(gòu)損傷,顯著降低了細(xì)胞凋亡率。L.thamnosusGR-1顯著降低了E.coli感染細(xì)胞導(dǎo)致的模式識(shí)別受體(TLR2,TLR4,NOD1和NOD2),炎性體(NLRP3,ASC 和 Caspase-1),TLR4 下游銜接分子(MyD88 和 TICAM2),負(fù)調(diào)控因子TOLLIP,轉(zhuǎn)錄調(diào)控因子(NF-κB和IRF3),炎性細(xì)胞因子(TNF-α,IL-1β,IL-6,IL-8,IL-10,IL-18和IFNβ)等免疫反應(yīng)分子mRNA表達(dá)水平的增加。L.rhanmsosus GR-1顯著提高了E.coli導(dǎo)致的緊密連接蛋白claudin-1/2、Occludin及ZO-1表達(dá)減少,顯著降低了 NF-κB2和p65的表達(dá)。結(jié)論:奶牛血清中TNF-α和Hp水平可以作為臨床型子宮內(nèi)膜炎的輔助診斷指標(biāo)。與其他診斷指標(biāo)相比,奶牛血清中Hp對(duì)炎癥的敏感性更高可以作為亞臨床型子宮內(nèi)膜炎的輔助診斷指標(biāo)。L.rhamnosus GR-1通過衰減MyD88依賴性和非依賴性信號(hào)傳遞途徑的信號(hào)轉(zhuǎn)導(dǎo),以及加強(qiáng)NLRs和TLRs之間的相互作用,抑制E.colE誘導(dǎo)的奶牛子宮內(nèi)膜上皮細(xì)胞NF-κB信號(hào)通路激活,進(jìn)而減少了炎性細(xì)胞因子的釋放,同時(shí)緩解了E.coli對(duì)細(xì)胞緊密連接的破壞,減少了細(xì)胞的凋亡率,從而減輕了E coli誘導(dǎo)的奶牛子宮內(nèi)膜上皮細(xì)胞的炎性損傷作用。
[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.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.23

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