【摘要】：乳腺炎給奶牛養(yǎng)殖業(yè)造成了巨大經(jīng)濟(jì)損失,金黃色葡萄球菌是引起奶牛乳腺炎主要病原菌之一。血乳屏障是乳腺組織抵御病原微生物入侵的天然屏障,主要由緊密連接結(jié)構(gòu)組成。本試驗通過檢測金黃色葡萄球菌的提取物脂磷壁酸對乳腺上皮細(xì)胞間緊密連接蛋白表達(dá)的影響,為揭示金黃色葡萄菌對奶牛血乳屏障的影響提供試驗依據(jù)。體外試驗通過組織塊培養(yǎng)法和梯度胰酶消化法分離純化奶牛乳腺上皮細(xì)胞,采用Transwell小室建立乳腺上皮屏障模型。試驗分為A、B、C、D、E 5組,A組為對照組,B、C、D、E為LTA刺激組,刺激量分別為0.01μg/m L、0.1μg/m L、1μg/m L、10μg/m L,24 h后檢測乳腺上皮屏障模型通透率的變化,實時熒光定量PCR方法檢測Occludin、Claudin-1和ZO-1 m RNA的表達(dá)水平。體內(nèi)試驗通過乳頭導(dǎo)管注射LTA,建立小鼠乳腺炎模型,通過HE染色觀察組織形態(tài)結(jié)構(gòu),ELISA方法檢測TNF-α表達(dá)水平,Western blot方法檢測Occludin、Claudin-1、ZO-1蛋白定量表達(dá),免疫熒光方法檢測Occludin和Claudin-1蛋白定位表達(dá)。結(jié)果:(1)通過組織塊培養(yǎng)法成功獲得奶牛乳腺上皮細(xì)胞,免疫熒光檢測角蛋白-18表達(dá)為陽性。(2)在Transwell小室PET膜上培養(yǎng)乳腺上皮細(xì)胞,檢測跨膜電阻在96 h后趨于穩(wěn)定,形成單層細(xì)胞屏障。(3)HE染色和ELISA結(jié)果顯示,以1μg和10μg LTA刺激小鼠乳腺組織,可成功構(gòu)建小鼠乳腺炎模型。(4)通透率結(jié)果顯示,與對照組相比,0.1μg/m L LTA使乳腺上皮通透率上升顯著(P0.05),1μg/m L和10μg/m L LTA使乳腺上皮通透率上升極顯著(P0.01)。(5)熒光定量PCR結(jié)果顯示,與對照組相比,Claudin-1m RNA表達(dá)量無變化,Occludin和ZO-1 m RNA表達(dá)量隨著LTA濃度增加呈下降趨勢。(6)Western blot結(jié)果顯示,Occludin和ZO-1蛋白表達(dá)水平與m RNA表達(dá)趨勢一致,與對照組相比,10μg LTA Claudin-1蛋白表達(dá)量下降顯著(P0.05)。(7)免疫熒光結(jié)果顯示,Occluidn和Claudin-1定位于細(xì)胞膜上,10μg LTA引起Occludin熒光強(qiáng)度降低。綜上所述,金黃色葡萄球菌的LTA,可以誘發(fā)乳腺炎癥的產(chǎn)生,增加乳腺上皮的通透率,降低乳腺緊密連接蛋白Occludin和ZO-1的表達(dá)。
[Abstract]:Mastitis has caused huge economic loss to dairy cattle breeding industry. Staphylococcus aureus is one of the main pathogens of dairy cow mastitis. Blood-milk barrier is a natural barrier for breast tissue to resist the invasion of pathogenic microorganisms. In order to reveal the effect of Staphylococcus aureus extract lipoteichoic acid on the expression of tight junction protein in mammary epithelial cells, this study provided experimental basis for revealing the effect of grape aureus on the blood-milk barrier of dairy cattle. Bovine mammary epithelial cells were isolated and purified by tissue mass culture and gradient trypsin digestion in vitro. The mammary epithelial barrier model was established by Transwell chamber. The experiment was divided into two groups: group A: the control group was treated with LTA, and the stimulation amount was 0. 1 渭 g / mL ~ (-1) 渭 g / mL ~ (-1) 10 渭 g / m ~ (-1) L ~ (-1). The permeability of mammary epithelial barrier model was detected by real-time fluorescence quantitative PCR. The expression levels of Occludin-Claudin-1 and ZO-1 m RNA were detected by real-time quantitative PCR. In vivo, the mouse mastitis model was established by injecting LTA-nipple catheter. The expression level of TNF- 偽 was detected by HE staining. The expression of TNF- 偽 was detected by Elisa. The protein expression of Occludin-Claudin-1 ZO-1 protein was detected by Western blot. The localization expression of Occludin and Claudin-1 protein was detected by immunofluorescence method. Results: (1) Dairy mammary epithelial cells were successfully obtained by tissue mass culture, and keratin-18 expression was detected by immunofluorescence. (2) mammary epithelial cells were cultured on PET membrane of Transwell chamber, and the transmembrane resistance tended to stabilize after 96 h. Formation of monolayer cell barrier. (3) the results of HE staining and ELISA showed that the mammary gland tissues of mice were stimulated with 1 渭 g and 10 渭 g LTA, and the mouse mastitis model was successfully constructed. (4) the results of permeability showed that: 1 渭 g and 10 渭 g LTA could induce mastitis in mice. Compared with the control group, 0.1 渭 g / mL LTA significantly increased the permeability of breast epithelium (P0.05) 1 渭 g / mL and 10 渭 g / mL LTA significantly increased the permeability of breast epithelium (P0.01). (5). Compared with the control group, the expression of Claudin-1m RNA showed no change. The expression of Occludin and ZO-1 m RNA decreased with the increase of LTA concentration. (6) the results of) Western blot showed that the expression level of LTA and ZO-1 was the same as that of m RNA. Compared with the control group, the expression of 10 渭 g LTA Claudin-1 protein decreased significantly (P0.05). (7). The results of immunofluorescence showed that Occluidn and Claudin-1 were located on the cell membrane and 10 渭 g LTA induced the decrease of Occludin fluorescence intensity. In conclusion, staphylococcus aureus can induce inflammation of mammary gland, increase the permeability of mammary epithelium and decrease the expression of Occludin and ZO-1.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.23

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 羅金印;李宏勝;李新圃;楊峰;;蘭州地區(qū)奶牛乳房炎流行病學(xué)及病原菌調(diào)查[J];黑龍江畜牧獸醫(yī);2015年21期

2 葛創(chuàng);蔡元坤;;脂磷壁酸相關(guān)最新研究進(jìn)展[J];上海醫(yī)學(xué);2012年07期

3 閆靜靜;張曉燕;;樹突細(xì)胞的特征及其生物學(xué)功能的研究進(jìn)展[J];微生物與感染;2012年02期

4 郭慶;朱偉;易瓊;何炳坤;徐賓朋;王魯;;8味中藥對奶牛乳房炎病原菌的體外抑菌試驗[J];中國獸醫(yī)雜志;2012年05期

5 高嫻;李素云;王海峰;;Toll樣受體與慢性阻塞性肺疾病發(fā)病機(jī)制的研究進(jìn)展[J];中國老年學(xué)雜志;2012年09期

6 莊曉明;曹永暉;樊祥山;吳鴻雁;;緊密連接蛋白claudin-1與乳腺腫瘤的相關(guān)性研究[J];中國普外基礎(chǔ)與臨床雜志;2012年02期

7 樊利軍;張夢澤;韋藝媛;盧勝明;王小珂;王瓏;姚毅;王雅春;俞英;;奶牛金黃色葡萄球菌乳腺炎小鼠模型的建立[J];實驗動物科學(xué);2011年06期

8 儲以微;;2011諾貝爾獎:免疫學(xué)研究的突破及意義[J];復(fù)旦學(xué)報(醫(yī)學(xué)版);2011年06期

9 朱海燕;遲蕾;徐冰;高永紅;婁晉寧;;清開靈注射液對血腦屏障模型通透性的影響[J];中國中醫(yī)藥信息雜志;2011年09期

10 李玉文;秦建華;;唐山市奶牛乳房炎的流行病學(xué)調(diào)查[J];中國奶牛;2011年17期

，

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