【摘要】：奶牛乳腺炎過程中伴隨著免疫細(xì)胞在乳腺內(nèi)的聚集、炎癥因子釋放等病理學(xué)特征,同時(shí)造成產(chǎn)奶量下降與淘汰率升高,給奶牛養(yǎng)殖業(yè)帶來不可估量的損失。在動(dòng)物體內(nèi),硒主要以硒蛋白的形式存在,除作為一種重要的飼源性微量元素外,硒在機(jī)體防御體系中也發(fā)揮著重要作用。臨床研究表明飼料中添加適量的硒,能顯著降低奶牛乳腺炎的發(fā)病率或減輕乳腺的病變程度。因此,本實(shí)驗(yàn)體外研究了硒的抗炎作用以及硒對炎癥相關(guān)信號通路的影響,以豐富奶牛乳腺炎的發(fā)病機(jī)理并為乳腺炎的防治提供理論基礎(chǔ)。金黃色葡萄球菌是乳腺炎的主要致病菌,揭示其致病機(jī)理以及機(jī)體對其感染的防御機(jī)制,具有重要的現(xiàn)實(shí)意義。實(shí)驗(yàn)采用經(jīng)乳導(dǎo)管灌注金黃色葡萄球菌的方法構(gòu)建干奶期奶牛乳腺炎急性感染模型,通過血液常規(guī)檢查、生化相關(guān)指標(biāo)和相關(guān)炎癥細(xì)胞因子基因的檢測,觀察金黃色葡萄球菌侵襲奶牛乳腺組織后奶牛血液和乳腺組織局部的變化。結(jié)果顯示,金黃色葡萄球菌侵入奶牛乳腺組織24 h后血液白細(xì)胞數(shù)顯著增加；血清總蛋白、球蛋白的含量以及乳酸脫氫酶的活性升高；乳腺組織中TNF-α、IL-1β、IL-6和IL-8的mRNA表達(dá)均出現(xiàn)顯著或極顯著的升高,這些結(jié)果表明金黃色葡萄球菌誘導(dǎo)奶牛乳腺組織發(fā)生炎性損傷,并誘發(fā)全身性先天免疫應(yīng)答反應(yīng)。采集處于泌乳期的健康荷斯坦奶牛乳腺組織培養(yǎng)奶牛原代乳腺上皮細(xì)胞,構(gòu)建金黃色葡萄球菌感染奶牛原代乳腺上皮細(xì)胞(bMEC)模型。檢測在金黃色葡萄球菌感染后bMEC細(xì)胞因子表達(dá)的變化。結(jié)果表明,金黃色葡萄球菌感染乳腺上皮細(xì)胞后,IL-1β、TNF-α、IL-6和IL-8的mRNA表達(dá)于4～6 h內(nèi)均出現(xiàn)顯著或極顯著升高(p0.01)且所有基因的表達(dá)均于8h達(dá)到峰值,說明金黃色葡萄球菌誘導(dǎo)奶牛乳腺上皮細(xì)胞炎癥反應(yīng)存在時(shí)間依賴性。為研究硒對奶牛乳腺上皮細(xì)胞活性的影響,采用不同濃度(0、1、2、4、8、16、32和64 μmol/L)的硒處理奶牛乳腺上皮細(xì)胞12 h, MTT法檢測細(xì)胞活性。結(jié)果表明,0-16μM的硒不影響乳腺上皮細(xì)胞活性,當(dāng)硒濃度在32 μmol/L和64 μmol/L時(shí)細(xì)胞活性極顯著低于對照組(p0.001),表明高濃度的硒對乳腺上皮細(xì)胞有毒性作用。為研究硒在細(xì)胞炎癥反應(yīng)過程中對TLR2和Nod2信號通路的調(diào)控作用,在培養(yǎng)液中添加不同濃度的硒(2、4和8 μmol/L檢測硒對TLR2和Nod2信號通路的影響。實(shí)驗(yàn)結(jié)果表明,硒對TLR2信號通路的傳導(dǎo)具有抑制作用。金黃色葡萄球菌感染乳腺上皮細(xì)胞后,TLR2基因表達(dá)上升,在感染后8h,4μmol/L和8 μmol/L的硒能極顯著抑制這種反應(yīng)(p0.01)；在感染后8 h,金黃色葡萄球菌極顯著提高M(jìn)yd88基因的表達(dá)量(p0.001)；2、4和8 μmol/L的硒均能顯著抑制金黃色葡萄球菌誘導(dǎo)的奶牛乳腺上皮細(xì)胞Myd88的表達(dá)；金黃色葡萄球菌極顯著(p0.01或p0.001)提高乳腺上皮細(xì)胞Irak4和Irak1基因的表達(dá)量,但是硒對這一反應(yīng)抑制作用微弱；金黃色葡萄球菌顯著或極顯著提高Traf6基因的表達(dá)(p0.05或p0.001)；硒能顯著或極顯著抑制這一反應(yīng)(p0.01或p0.001)。硒可減弱Nod2信號通路的傳導(dǎo)。在金黃色葡萄球菌感染后,乳腺上皮細(xì)胞Nod2基因表達(dá)升高或極顯著升高,但是硒對這一效果的抑制作用微弱；金黃色葡萄球菌感染乳腺上皮細(xì)胞后RIP2基因的表達(dá)升高或極顯著提高,硒能顯著或極顯著的抑制這一效果(p0.05～p0.001)。硒對乳腺上皮細(xì)胞炎癥細(xì)胞因子基因的表達(dá)具有調(diào)控作用。金黃色葡萄球菌能顯著或極顯著的提高乳腺上皮細(xì)胞炎癥細(xì)胞因子TNF-a、IL-1β和IL-6基因的表達(dá),硒能顯著或極顯著抑制這種表達(dá)(p0.05～p0.001)。同時(shí),金黃色葡萄球菌能極顯著提高激活蛋白AP-1/c-jun和c-fos的表達(dá),硒通過降低c-jun和c-fos異源二聚體的形成而減弱AP-1的表達(dá)。為從蛋白水平揭示硒對炎癥信號通路的影響,采用Western Blot技術(shù)對NF-κB和MAPK信號通路中關(guān)鍵蛋白的表達(dá)進(jìn)行檢測。實(shí)驗(yàn)分5組,以不添加亞硒酸鈉和金黃色葡萄球菌的細(xì)胞作為空白對照組,以金黃色葡萄球菌刺激0.5 h的細(xì)胞作為陽性對照組,以不同濃度的亞硒酸鈉(2、4和8 μmol/L)預(yù)孵育12h并用金黃色葡萄球菌刺激0.5 h的細(xì)胞作為實(shí)驗(yàn)組,分別檢測IκBα、p65、p38和Erk蛋白的磷酸化表達(dá)。結(jié)果顯示,金黃色葡萄球菌刺激乳腺上皮細(xì)胞0.5 h后,IκBα、p65、p38和Erk蛋白的磷酸化水平與空白對照組相比極顯著上升(p0.001)；與陽性對照組相比,4pM和8 μM的硒能極顯著抑制金黃色葡萄球誘導(dǎo)的NF-κB IκBα和p65的磷酸化(p0.001),同時(shí)極顯著降低金黃色葡萄球菌誘導(dǎo)的MAPK p38和Erk的磷酸化(p0.001),這表明4gM和8 μM的硒通過對TLR2以及Nod2信號通路的調(diào)控進(jìn)而達(dá)到對NF-κB以及MAPK的抑制效果,最終下調(diào)了金黃色葡萄球菌誘導(dǎo)奶牛乳腺上皮細(xì)胞炎癥細(xì)胞因子基因的表達(dá),減弱了金黃色葡萄球菌誘導(dǎo)的奶牛乳腺上皮細(xì)胞的炎癥損傷。最后,本實(shí)驗(yàn)構(gòu)建了金黃色葡萄球菌感染巨噬細(xì)胞模型,通過向培養(yǎng)基內(nèi)添加不同濃度的硒(1、1.5和2μmol/L),并采用熒光定量PCR和ELISA的方法檢測硒對感染金黃色葡萄球菌感染巨噬細(xì)胞而引起的細(xì)胞因子表達(dá)與釋放的影響。結(jié)果顯示,與空白對照組相比,陽性對照組TNF-α、IL-1β和IL-6的表達(dá)與釋放在10h內(nèi)均極顯著上升(p0.001)；與陽性對照組相比,不同濃度的硒下調(diào)或顯著下調(diào)了巨噬細(xì)胞炎癥細(xì)胞因子的表達(dá)與釋放。為檢測NF-μB以及MAPK信號通路是否參與巨噬細(xì)胞炎癥反應(yīng)過程中,采用Western Blot技術(shù)在蛋白水平上對IκBα、p65、p38、Erk和Jnk蛋白的磷酸化進(jìn)行檢測。結(jié)果顯示,金黃色葡萄球菌刺激巨噬細(xì)胞0.5 h后,與空白對照組相比IκBα、p65、p38、Erk和Jnk蛋白的磷酸化水平極顯著升高,說明NF-κB和MAPK信號通路被激活,與陽性對照組相比,硒能極顯著抑制NF-κB和MAPK信號通路的傳導(dǎo),這種影響是通過抑制IκB、p65、p38和Jnk以及Erk的磷酸化完成的。
[Abstract]:With the accumulation of immune cells in the mammary gland and the release of inflammatory factors in the process of mastitis in dairy cows, the milk yield decreased and the elimination rate increased, which brought inestimable losses to the dairy cattle breeding industry. Selenium also plays an important role in the body defense system. Clinical studies have shown that dietary selenium supplementation can significantly reduce the incidence of mastitis or reduce the degree of breast lesion in dairy cows. Staphylococcus aureus is the main pathogen of mastitis, revealing its pathogenic mechanism and the body's defense mechanism against its infection has important practical significance. The changes of blood and mammary gland tissue in dairy cows were observed by routine blood test, biochemical indexes and related inflammatory cytokine gene detection. The expression of TNF-a, IL-1beta, IL-6 and IL-8 mRNA in mammary gland tissues were significantly or extremely significantly increased. These results indicated that Staphylococcus aureus could induce inflammation injury in mammary gland tissues of dairy cows and induce systemic innate immune response. The expression of bMEC cytokines in primary mammary epithelial cells of cows infected with Staphylococcus aureus (S. aureus) was detected after S. aureus infection. The expression of - 8 mRNA increased significantly or extremely significantly within 4 to 6 hours (p0.01) and all the genes reached the peak at 8 hours, indicating that the inflammation of dairy cow mammary epithelial cells induced by Staphylococcus aureus was time-dependent. To study the effect of selenium on the activity of dairy cow mammary epithelial cells, different concentrations (0, 1, 2, 4, 8, 16, 32 and 64) were used. The results showed that selenium of 0-16 Mu did not affect the activity of mammary epithelial cells. When selenium concentration was 32 and 64 mu mol/L, the cell activity was significantly lower than that of the control group (p0.001), indicating that high concentration of selenium was toxic to mammary epithelial cells. TLR2 and Nod2 signaling pathways were regulated by selenium (2,4 and 8 micromol/L). The results showed that selenium inhibited the transmission of TLR2 signaling pathways. TLR2 gene expression was observed in breast epithelial cells infected with Staphylococcus aureus. Selenium at 4, 4 and 8 micromol/L significantly inhibited this reaction (p0.01), Staphylococcus aureus significantly increased Myd88 gene expression (p0.001), 2, 4 and 8 micromol/L selenium significantly inhibited Myd88 expression in dairy cow mammary epithelial cells induced by Staphylococcus aureus. Staphylococcus aureus significantly or extremely significantly increased the expression of Irak4 and Irak1 genes in mammary epithelial cells (p0.01 or p0.001), but selenium had a weak inhibitory effect on this reaction; Staphylococcus aureus significantly or extremely significantly increased the expression of Traf6 gene (p0.05 or p0.001); selenium could significantly or extremely significantly inhibit this reaction (p0.01 or p0.001). Selenium could weaken the Nod2 message. The expression of Nod2 gene in mammary epithelial cells was elevated or significantly elevated after S. aureus infection, but the inhibition effect of selenium on this effect was weak. The expression of RIP2 gene in mammary epithelial cells infected by S. aureus was elevated or significantly elevated after S. aureus infection, and selenium could significantly or significantly inhibit this effect (p Selenium can regulate the expression of inflammatory cytokines in mammary epithelial cells. Staphylococcus aureus can significantly or extremely significantly increase the expression of inflammatory cytokines TNF-a, IL-1beta and IL-6 in mammary epithelial cells. Selenium can significantly or extremely significantly inhibit the expression of inflammatory cytokines TNF-a, IL-1beta and IL-6 in mammary epithelial cells (p0.05-p0.001). In order to reveal the effect of selenium on inflammatory signaling pathway at protein level, Western Blot technique was used to detect the expression of key proteins in NF-kappa B and MAPK signaling pathway. The cells without sodium selenite and Staphylococcus aureus were used as blank control group, the cells stimulated by Staphylococcus aureus for 0.5 h were used as positive control group, and the cells pre-incubated with different concentrations of sodium selenite (2,4 and 8 micromol/L) for 12 h and stimulated by Staphylococcus aureus for 0.5 h were used as experimental group. I kappa B alpha, p65, p38 and Erk were detected respectively. The results showed that the phosphorylation levels of I-kappa B-alpha, p65, p38 and Erk proteins in breast epithelial cells stimulated by Staphylococcus aureus were significantly higher than those in the blank control group (p0.001); compared with the positive control group, selenium at 4pM and 8mu M significantly inhibited the phosphorus levels of NF-kappa B I-kappa B-alpha and p65 induced by Staphylococcus aureus. Acidification (p0.001) and significantly decreased the phosphorylation of MAPK p38 and Erk induced by Staphylococcus aureus (p0.001) suggested that 4gM and 8mu M of selenium could inhibit NF-kappa B and MAPK by regulating TLR2 and Nod2 signaling pathways, and ultimately down-regulated the inflammatory cytokines induced by Staphylococcus aureus in dairy cow mammary epithelial cells. The expression of subgene weakened the inflammation damage of dairy cow mammary epithelial cells induced by Staphylococcus aureus. Finally, the macrophage model of Staphylococcus aureus infection was established. Different concentrations of selenium (1,1.5 and 2 micromol/L) were added to the culture medium, and the effect of selenium on the infection of dairy cow mammary epithelial cells was detected by fluorescence quantitative PCR and ELISA. The results showed that the expression and release of TNF-a, IL-1beta and IL-6 in the positive control group were significantly increased within 10 hours (p0.001) compared with the blank control group. Compared with the positive control group, different concentrations of selenium decreased or significantly decreased the inflammation of macrophages. In order to detect the expression and release of cytokines, the phosphorylation of I-kappa B-alpha, p65, p38, Erk and Jnk proteins at protein level was detected by Western Blot technique. The results showed that Staphylococcus aureus stimulated macrophages for 0.5 hours and compared with the blank control group, I-kappa was detected. The phosphorylation levels of Ba, p65, p38, Erk and Jnk proteins were significantly elevated, suggesting that NF-kappa B and MAPK signaling pathways were activated. Compared with the positive control group, selenium could significantly inhibit the transmission of NF-kappa B and MAPK signaling pathways. This effect was achieved by inhibiting the phosphorylation of I-kappa B, p65, p38 and Jnk and Erk.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S858.23

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王月影;朱河水;王艷玲;;乳腺上皮細(xì)胞的鈣轉(zhuǎn)運(yùn)研究進(jìn)展[J];江西農(nóng)業(yè)學(xué)報(bào);2008年06期

2 楊櫟;李鍵;吳媛媛;田甜;畢琳;梁天宇;;綠色熒光蛋白基因在牦牛乳腺上皮細(xì)胞中表達(dá)的研究[J];西南民族大學(xué)學(xué)報(bào)(自然科學(xué)版);2009年03期

3 吳媛媛;李鍵;楊櫟;畢琳;田甜;;牦牛乳腺上皮細(xì)胞的體外培養(yǎng)的研究[J];西南民族大學(xué)學(xué)報(bào)(自然科學(xué)版);2009年04期

4 弓超;王凌云;周歡敏;;牛乳腺上皮細(xì)胞體外分離和培養(yǎng)[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2011年07期

5 李吉霞;葛秀國;王文秀;張涌;;牛乳腺上皮細(xì)胞的分離培養(yǎng)[J];黑龍江畜牧獸醫(yī);2012年07期

6 符梅;熊顯榮;高川;李宇;王樹茂;李鍵;;麥洼牦牛乳腺上皮細(xì)胞系的建立及其生物學(xué)特性[J];西北農(nóng)業(yè)學(xué)報(bào);2012年11期

7 郝明超;劉r，

本文編號：2192285