苦参碱干预金黄色葡萄球菌对奶牛乳腺上皮细胞粘附作用的研究

发布时间：2018-02-20 16:30

  本文关键词： 乳腺上皮细胞 粘附作用 苦参碱 抗氧化能力　出处：《宁夏大学》2017年硕士论文　论文类型：学位论文

【摘要】：奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)作为病原菌穿越乳导管进入奶牛乳腺后首先接触的细胞,其与病原微生物的相互作用对于疾病的发展进程有非常重要的意义。粘附作为病原菌入侵宿主细胞的先决条件之一,病原菌首先粘附到宿主细胞,然后定植在宿主细胞从而发挥致病作用。在奶牛乳房炎发生的过程中,对于病原菌感染乳房,粘附更是关键的一步。本课题研究苦参碱干预金黄色葡萄球菌对BMECs粘附作用的影响,为解释病原菌对BMECs粘附作用的特点及病原菌侵袭BMECs所引起的细胞凋亡机制提供理论基础。首先试验采用组织块培养法和乳汁分离法分离培养BMECs,后对细胞进行纯化和鉴定。其次,探究不同时间、pH和温度对金黄色葡萄球菌粘附BMECs的影响;将不同浓度的苦参碱加入到细胞培养液中进行BMECs的培养,检测苦参碱对BMECs增殖和凋亡的各项指标,综合评价苦参碱对BMECs增殖、凋亡及抗氧化能力的作用特点,从而选出既能促进BMECs的增殖又能提高细胞抗氧化能力的苦参碱浓度。用白藜芦醇、地塞米松、紫花苜蓿黄酮作为对照,以苦参碱为主研究药物对粘附作用的影响。研究发现,组织块培养法和乳汁分离法均可培养出BMECs;此细胞核型正常(2n=60,XX),角蛋白表达呈阳性,说明在体外成功建立了 BMECs模型;显微镜下观察,纯化的P4～P10细胞呈多角型或乳滴型,P12～P14奶牛乳腺上皮细胞形态发生变化,形成明显的“网状”结构;P4～P14细胞生长曲线呈“S”型,符合生物学生长的一般规律;但P12与P14细胞生长相对缓慢;通过瑞氏-姬姆萨染色后,发现P12和P14细胞之间出现空泡结构。通过研究时间、pH和温度对金黄色葡萄球菌粘附BMECs的影响,确定了粘附的最佳时间为2.5 h,最适pH值7.0～7.5之间,最适温度为37℃;通过检测苦参碱对BMECs增殖和抗氧化能力的影响,发现苦参碱既可促进细胞增殖又可提高细胞抗氧化能力的最适浓度为50 μg/mL;将此浓度的药物进行粘附实验,发现与白藜芦醇、地塞米松、紫花苜蓿黄酮相比,苦参碱对粘附作用具有显著的抑制作用。以上研究结果揭示,纯化后的细胞为BMECs,其中P4～P10细胞性状更为稳定,可用于后续实验;金黄色葡萄球菌粘附BMECs的最佳时间为2.5 h,最适pH值7.0～7.5之间,最适温度为37℃;苦参碱(适宜浓度50μg/mL)对金黄色葡萄球菌粘附BMECs具有显著的抑制作用。
[Abstract]:Bovine mammary epithelial cells (BMECs) are the first cells that the pathogen contacts after passing through the milk catheter into the mammary gland of the cow. The interaction with pathogenic microorganisms is very important for the development of disease. As one of the prerequisites for pathogen invading host cells, pathogen first adheres to host cells. They are then colonized in the host cells to play a pathogenic role. In the course of mastitis in cows, when the pathogen infects the breast, The effect of matrine on the adhesion of Staphylococcus aureus to BMECs was studied. In order to explain the characteristics of the adhesion of pathogenic bacteria to BMECs and the mechanism of apoptosis induced by pathogen invading BMECs, BMECs were isolated and cultured by tissue mass culture method and milk separation method, and the cells were purified and identified. To explore the effect of pH and temperature on the adhesion of Staphylococcus aureus to BMECs at different time, to culture BMECs with different concentrations of matrine in cell culture medium, and to detect the indexes of BMECs proliferation and apoptosis. The effects of matrine on the proliferation, apoptosis and antioxidant ability of BMECs were comprehensively evaluated, and the concentration of matrine, which could not only promote the proliferation of BMECs but also increase the antioxidant ability of cells, was selected, and resveratrol and dexamethasone were used. The effect of matrine on the adhesion of alfalfa flavonoids was studied. It was found that BMECs could be obtained by tissue culture and milk separation, and the keratin expression was positive in this nuclear type. The results showed that the BMECs model was successfully established in vitro, and the purified P4P10 cells were observed to be polygonal or milk-droplet type, and the morphology of mammary epithelial cells in P12 / P14 dairy cows was changed, and the growth curve of P4 / P14 cells was "S". The growth of P12 and P14 cells was relatively slow. The effect of pH and temperature on the adhesion of Staphylococcus aureus to BMECs was studied. The optimum time of adhesion was 2.5 h, the optimum pH value was 7.0 鈩，

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