本文選題：奶牛子宮內(nèi)膜炎　切入點：脂多糖　出處：《中國農(nóng)業(yè)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：奶牛產(chǎn)后子宮內(nèi)膜炎是由致病微生物上行感染母牛生殖道而引起的炎癥。大腸桿菌是主要的致病菌之一,但其發(fā)病機(jī)制并不十分清楚。魚腥草素鈉(Sodium houttuyfonate, SH)是魚腥草有效成分魚腥草素的亞硫酸氫鈉加成化合物,穩(wěn)定性強(qiáng),臨床上用于治療炎癥疾病。目前魚腥草素鈉對抗炎癥疾病的分子機(jī)制還不完全清楚,我們假設(shè)NF-κB充當(dāng)關(guān)鍵調(diào)解物,從體外細(xì)胞水平探明其抗炎分子機(jī)制。并在體內(nèi)對小鼠子宮內(nèi)膜炎模型和奶牛子宮內(nèi)膜炎的療效進(jìn)行觀察。從而深入研究SH抗炎活性和其藥理機(jī)制。 本研究運用體外原代培養(yǎng)細(xì)胞的方法,分離鑒定了奶牛子宮內(nèi)膜上皮細(xì)胞和基質(zhì)細(xì)胞。并對培養(yǎng)獲得的原代細(xì)胞進(jìn)行了不同染色方法的形態(tài)學(xué)觀察和透射電鏡觀察。在此基礎(chǔ)上,建立LPS誘導(dǎo)的奶牛子宮內(nèi)膜上皮細(xì)胞(bEECs)炎癥模型。為了評估SH的體內(nèi)外抗炎作用,首先用MTT法和LDH分析檢測梯度濃度的SH對bEECs的毒性；其次用ELISA和qRT-PCR方法分別從蛋白分泌和nRNA表達(dá)兩個水平檢測細(xì)胞炎性因子TNF-α、IL-1β、IL-6和IL-8,同時用western blotting的方法檢測細(xì)胞內(nèi)炎癥信號轉(zhuǎn)導(dǎo)通路MAPKs/NF-κB的關(guān)鍵蛋白ERK、JNK、p38、IκBα和NF-κBp65;最后用臨床診斷的方法評估SH治療小鼠和奶牛子宮內(nèi)膜炎的療效。 研究表明,運用組織培養(yǎng)和復(fù)合酶消化結(jié)合差時技術(shù)可以分離出純度很高的bEECs,為后續(xù)的實驗提供了足夠的細(xì)胞。不同染色方法對子宮內(nèi)膜細(xì)胞觀察可知其細(xì)胞結(jié)構(gòu)清晰,胞漿胞膜分明；超微結(jié)構(gòu)觀察結(jié)果顯示,體外培養(yǎng)的子宮內(nèi)膜上皮細(xì)胞可清晰看到微胞凸,胞內(nèi)各種細(xì)胞器清晰可見。細(xì)胞毒性實驗顯示,SH (120μg/mL)濃度范圍內(nèi)對bEECs無毒副作用；15,30and60μg/mL高中低濃度的SH可以抑制LPS誘導(dǎo)bEECs的TNF-α、IL-1β、IL-6和IL-8的蛋白分泌水平,并呈濃度依賴關(guān)系；對炎性細(xì)胞因子的mRNA有同樣的調(diào)控作用。而且,在LPS激活的bEECs中,SH抑制了IκBα的降解和NF-κB p65的磷酸化；壓制了ERK、JNK、p38磷酸化水平。體內(nèi)療效實驗證實,SH能夠顯著減弱模型組小鼠子宮內(nèi)膜的炎性反應(yīng),子宮指數(shù)與正常對照組差異不顯著,鏡檢子宮組織顯示顯微結(jié)構(gòu)基本正常,表明SH對實驗性子宮內(nèi)膜炎模型小鼠有很好的治療效果。用SH治療奶牛臨床型子宮內(nèi)膜炎受孕率達(dá)到86%,發(fā)情頭數(shù)占總頭數(shù)的91.7%。而用土霉素治療奶牛受孕率為50%,發(fā)情頭數(shù)占總頭數(shù)的67%。在發(fā)情頭數(shù)和受孕率上SH的治療效果優(yōu)于土霉素。
[Abstract]:Postpartum endometritis in dairy cows is caused by the upward infection of pathogenic microorganisms in the reproductive tract of cows. Escherichia coli is one of the major pathogens. But its pathogenesis is not very clear. Sodium houttuyfonate (SH) is a sodium bisulfite addition compound of houttuynin, which has strong stability. Clinical use in the treatment of inflammatory diseases. The molecular mechanism of houttuynin sodium against inflammatory diseases is not fully understood, and we assume that NF- 魏 B acts as a key mediator. The anti-inflammatory molecular mechanism of SH and its pharmacological mechanism were investigated in vitro, and in vivo, the model of endometritis in mice and the therapeutic effect of endometritis in dairy cows were observed in order to study the anti-inflammatory activity of SH and its pharmacological mechanism. In this study, we used the method of primary culture of cells in vitro, The endometrial epithelial cells and stromal cells of dairy cattle were isolated and identified. The primary cells were observed by different staining methods and transmission electron microscopy. The inflammatory model of bovine endometrial epithelial cells induced by LPS was established. In order to evaluate the anti-inflammatory effect of SH in vitro and in vivo, the toxicity of gradient concentration of SH to bEECs was detected by MTT assay and LDH analysis. Secondly, ELISA and qRT-PCR were used to detect the cytokines TNF- 偽, IL-1 尾, IL-6 and IL-8, respectively, from the two levels of protein secretion and nRNA expression. Meanwhile, western blotting was used to detect MAPKs / NF- 魏 B, the key proteins of MAPKs / NF- 魏 B and NF- 魏 Bp65. The therapeutic effect of SH on endometritis in mice and cows was evaluated. The results showed that high purity bEECscould be isolated by tissue culture and enzyme digestion, which provided enough cells for subsequent experiments. Different staining methods showed that the cell structure of endometrial cells was clear. The ultrastructural observation showed that the endometrial epithelial cells cultured in vitro could clearly see the microcellular convexity. The cytotoxicity test showed that SH in the range of 120 渭 g / mL had no toxic side effect on bEECs. SH at high or low concentrations of 1530 渭 g / mL and 60 渭 g / mL could inhibit the protein secretion of TNF- 偽, IL-1 尾, IL-6 and IL-8 induced by LPS in a concentration-dependent manner. Moreover, in bEECs activated by LPS, SH inhibited the degradation of I 魏 B 偽 and phosphorylation of NF- 魏 B p65. The therapeutic effect experiment in vivo showed that SH-H could attenuate the inflammatory response of endometrium in the model group, and the uterine index was not significantly different from that of the normal control group. The microscopic examination showed that the microstructure of the uterus was basically normal, but no significant difference was found between the model group and the normal control group. The results showed that SH had a good therapeutic effect on experimental endometritis model mice. The pregnancy rate of clinical endometritis in dairy cows treated with SH was 86%, and the number of estrous heads accounted for 91.7%. The pregnant rate of cows treated with oxytetracycline was 50%, and the pregnancy rate was 50%. The therapeutic effect of SH was better than that of oxytetracycline in estrus number and pregnancy rate.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S858.23

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