本文關鍵詞：奶牛子宮內膜炎早期診斷及益生菌抗大腸桿菌誘導的子宮內膜上皮細胞炎性損傷作用機理　出處：《中國農業(yè)大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 奶牛子宮內膜炎 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)是引起奶牛子宮內膜炎的主要致病菌之一,該病給奶牛養(yǎng)殖業(yè)帶來了嚴重的經濟損失,探究早期奶牛子宮內膜炎的診斷指標,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,治療和預防奶牛子宮內膜炎的機制還不甚明確。因此,本試驗通過研究鼠李糖乳桿菌GR-1(Lactobacill rhamnosus GR-1,L.rhamnosus GR-1)抗E.coli致奶牛子宮內膜上皮細胞炎性損傷的作用機制,為其臨床應用提供理論依據。方法:根據對產后200頭荷斯坦奶牛第21 d陰道內分泌物性狀和子宮內容物細胞學檢查,選取21頭奶牛分為三組:臨床型子宮內膜炎組(CE,n = 7);亞臨床型子宮內膜炎組(SE,n = 7)和健康對照組(HC,n = 7)。分別于產后1d、7 d、14 d、21 d和30 d采集血清,使用全自動生化分析儀測定血清中酶活性及離子濃度變化;使用ELISA方法檢測血清中炎性細胞因子和急性期蛋白的變化。利用原代奶牛子宮內膜上皮細胞(BEECs),評估L.rhamnosus GR-1抗E.coli致BEECs損傷的作用機制。試驗分為四個組:對照組(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)學和超微結構變化;利用流式細胞術檢測BEECs的凋亡情況;Western Blot方法檢測細胞緊密連接蛋白及信號通路蛋白表達;Real-time PCR方法檢測細胞相關受體及炎性因子表達。結果:產后7 d,CE組血清中AST的水平顯著高于HC組;產后14 d、21 d和30 d,CE組血清中CK的水平顯著高于HC組,且產后21d,SE組血清中CK的水平顯著高于HC組。產后21d和30d,CE組血清中TNF-α的水平顯著高于HE組和SE組;產后7d、14d、21d和30d,CE組血清中Hp的水平顯著高于HC組,且在產后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誘導的細胞結構損傷,顯著降低了細胞凋亡率。L.thamnosusGR-1顯著降低了E.coli感染細胞導致的模式識別受體(TLR2,TLR4,NOD1和NOD2),炎性體(NLRP3,ASC 和 Caspase-1),TLR4 下游銜接分子(MyD88 和 TICAM2),負調控因子TOLLIP,轉錄調控因子(NF-κB和IRF3),炎性細胞因子(TNF-α,IL-1β,IL-6,IL-8,IL-10,IL-18和IFNβ)等免疫反應分子mRNA表達水平的增加。L.rhanmsosus GR-1顯著提高了E.coli導致的緊密連接蛋白claudin-1/2、Occludin及ZO-1表達減少,顯著降低了 NF-κB2和p65的表達。結論:奶牛血清中TNF-α和Hp水平可以作為臨床型子宮內膜炎的輔助診斷指標。與其他診斷指標相比,奶牛血清中Hp對炎癥的敏感性更高可以作為亞臨床型子宮內膜炎的輔助診斷指標。L.rhamnosus GR-1通過衰減MyD88依賴性和非依賴性信號傳遞途徑的信號轉導,以及加強NLRs和TLRs之間的相互作用,抑制E.colE誘導的奶牛子宮內膜上皮細胞NF-κB信號通路激活,進而減少了炎性細胞因子的釋放,同時緩解了E.coli對細胞緊密連接的破壞,減少了細胞的凋亡率,從而減輕了E coli誘導的奶牛子宮內膜上皮細胞的炎性損傷作用。
[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.

【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S858.23

【參考文獻】

相關期刊論文 前10條

1 那立冬;王東升;董書偉;張世棟;閆寶琪;楊洪早;桑夢琪;嚴作廷;;奶牛子宮內膜炎病因學研究進展[J];動物醫(yī)學進展;2016年09期

2 都日塔哈拉;曹金山;高龍;付長其;郭羽麗;;奶牛子宮內膜上皮細胞體外培養(yǎng)及鑒定[J];中國畜牧獸醫(yī);2016年06期

3 鄺曉嬌;張世棟;董書偉;王東升;魏立琴;嚴作廷;;奶牛子宮內膜細胞體外培養(yǎng)及應用研究概況[J];動物醫(yī)學進展;2014年12期

4 陳松;孫苗;;唐山地區(qū)奶牛子宮內膜炎發(fā)病情況調查[J];中國奶牛;2014年14期

5 張世棟;王東升;王旭榮;潘虎;李宏勝;嚴作廷;;荷斯坦奶牛子宮內膜上皮細胞的體外培養(yǎng)[J];中國畜牧獸醫(yī);2013年04期

6 杜金梁;秦建華;褚景生;徐麗娜;馬玉忠;;復方益母生化散對奶牛子宮內膜細胞CYP450表達和免疫功能的影響[J];中國農業(yè)科學;2010年06期

7 張雷;張振新;王亨;韓超;齊長明;;奶牛子宮內膜上皮細胞和間質細胞的組織塊培養(yǎng)及鑒定[J];中國獸醫(yī)雜志;2008年04期

8 杜光波;;淮安地區(qū)奶牛子宮內膜炎的發(fā)病規(guī)律和相關原因的調查分析[J];中國奶牛;2007年10期

9 孔祥峰;孔祥國;胡元亮;;奶牛子宮內膜炎病因學研究進展[J];畜牧與獸醫(yī);2005年11期

10 吳金節(jié),章孝榮,劉亞,郭勇,楊克禮;奶牛子宮內膜炎防治前后血液流變學參數(shù)的變化[J];南京農業(yè)大學學報;2000年02期

相關博士學位論文 前3條

1 朱琪;魚腥草素鈉抗LPS誘導的奶牛子宮內膜上皮細胞炎癥的分子機制[D];中國農業(yè)大學;2015年

2 趙樹臣;中藥組方治療奶牛子宮內膜炎的研究[D];內蒙古農業(yè)大學;2007年

3 郭世寧;防治奶牛子宮內膜炎新型復方藥物制劑宮炎清的研究[D];東北農業(yè)大學;2002年

相關碩士學位論文 前3條

1 吳岳;IFN-τ調控奶牛子宮上皮細胞表達MICB及BoLA-A的研究[D];華中農業(yè)大學;2012年

2 王爽;奶牛子宮內膜炎主要病原菌分離鑒定及多重PCR診斷方法的建立[D];黑龍江八一農墾大學;2008年

3 陳現(xiàn)偉;復方中草藥制劑對奶牛子宮內膜炎的防治效果及免疫增強功能研究[D];山東農業(yè)大學;2007年

，

本文編號：1389996