脂磷壁酸对乳腺上皮紧密连接蛋白表达的影响
发布时间：2018-08-06 13:49
【摘要】：乳腺炎给奶牛养殖业造成了巨大经济损失,金黄色葡萄球菌是引起奶牛乳腺炎主要病原菌之一。血乳屏障是乳腺组织抵御病原微生物入侵的天然屏障,主要由紧密连接结构组成。本试验通过检测金黄色葡萄球菌的提取物脂磷壁酸对乳腺上皮细胞间紧密连接蛋白表达的影响,为揭示金黄色葡萄菌对奶牛血乳屏障的影响提供试验依据。体外试验通过组织块培养法和梯度胰酶消化法分离纯化奶牛乳腺上皮细胞,采用Transwell小室建立乳腺上皮屏障模型。试验分为A、B、C、D、E 5组,A组为对照组,B、C、D、E为LTA刺激组,刺激量分别为0.01μg/m L、0.1μg/m L、1μg/m L、10μg/m L,24 h后检测乳腺上皮屏障模型通透率的变化,实时荧光定量PCR方法检测Occludin、Claudin-1和ZO-1 m RNA的表达水平。体内试验通过乳头导管注射LTA,建立小鼠乳腺炎模型,通过HE染色观察组织形态结构,ELISA方法检测TNF-α表达水平,Western blot方法检测Occludin、Claudin-1、ZO-1蛋白定量表达,免疫荧光方法检测Occludin和Claudin-1蛋白定位表达。结果:(1)通过组织块培养法成功获得奶牛乳腺上皮细胞,免疫荧光检测角蛋白-18表达为阳性。(2)在Transwell小室PET膜上培养乳腺上皮细胞,检测跨膜电阻在96 h后趋于稳定,形成单层细胞屏障。(3)HE染色和ELISA结果显示,以1μg和10μg LTA刺激小鼠乳腺组织,可成功构建小鼠乳腺炎模型。(4)通透率结果显示,与对照组相比,0.1μg/m L LTA使乳腺上皮通透率上升显著(P0.05),1μg/m L和10μg/m L LTA使乳腺上皮通透率上升极显著(P0.01)。(5)荧光定量PCR结果显示,与对照组相比,Claudin-1m RNA表达量无变化,Occludin和ZO-1 m RNA表达量随着LTA浓度增加呈下降趋势。(6)Western blot结果显示,Occludin和ZO-1蛋白表达水平与m RNA表达趋势一致,与对照组相比,10μg LTA Claudin-1蛋白表达量下降显著(P0.05)。(7)免疫荧光结果显示,Occluidn和Claudin-1定位于细胞膜上,10μg LTA引起Occludin荧光强度降低。综上所述,金黄色葡萄球菌的LTA,可以诱发乳腺炎症的产生,增加乳腺上皮的通透率,降低乳腺紧密连接蛋白Occludin和ZO-1的表达。
[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- 伪 was detected by HE staining. The expression of TNF- 伪 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.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23
本文编号：2167923
[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- 伪 was detected by HE staining. The expression of TNF- 伪 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.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23
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