本文關鍵詞： 乳腺上皮細胞 粘附作用 苦參堿 抗氧化能力　出處：《寧夏大學》2017年碩士論文　論文類型：學位論文

【摘要】：奶牛乳腺上皮細胞(bovine mammary epithelial cells,BMECs)作為病原菌穿越乳導管進入奶牛乳腺后首先接觸的細胞,其與病原微生物的相互作用對于疾病的發(fā)展進程有非常重要的意義。粘附作為病原菌入侵宿主細胞的先決條件之一,病原菌首先粘附到宿主細胞,然后定植在宿主細胞從而發(fā)揮致病作用。在奶牛乳房炎發(fā)生的過程中,對于病原菌感染乳房,粘附更是關鍵的一步。本課題研究苦參堿干預金黃色葡萄球菌對BMECs粘附作用的影響,為解釋病原菌對BMECs粘附作用的特點及病原菌侵襲BMECs所引起的細胞凋亡機制提供理論基礎。首先試驗采用組織塊培養(yǎng)法和乳汁分離法分離培養(yǎng)BMECs,后對細胞進行純化和鑒定。其次,探究不同時間、pH和溫度對金黃色葡萄球菌粘附BMECs的影響;將不同濃度的苦參堿加入到細胞培養(yǎng)液中進行BMECs的培養(yǎng),檢測苦參堿對BMECs增殖和凋亡的各項指標,綜合評價苦參堿對BMECs增殖、凋亡及抗氧化能力的作用特點,從而選出既能促進BMECs的增殖又能提高細胞抗氧化能力的苦參堿濃度。用白藜蘆醇、地塞米松、紫花苜蓿黃酮作為對照,以苦參堿為主研究藥物對粘附作用的影響。研究發(fā)現(xiàn),組織塊培養(yǎng)法和乳汁分離法均可培養(yǎng)出BMECs;此細胞核型正常(2n=60,XX),角蛋白表達呈陽性,說明在體外成功建立了 BMECs模型;顯微鏡下觀察,純化的P4～P10細胞呈多角型或乳滴型,P12～P14奶牛乳腺上皮細胞形態(tài)發(fā)生變化,形成明顯的“網(wǎng)狀”結構;P4～P14細胞生長曲線呈“S”型,符合生物學生長的一般規(guī)律;但P12與P14細胞生長相對緩慢;通過瑞氏-姬姆薩染色后,發(fā)現(xiàn)P12和P14細胞之間出現(xiàn)空泡結構。通過研究時間、pH和溫度對金黃色葡萄球菌粘附BMECs的影響,確定了粘附的最佳時間為2.5 h,最適pH值7.0～7.5之間,最適溫度為37℃;通過檢測苦參堿對BMECs增殖和抗氧化能力的影響,發(fā)現(xiàn)苦參堿既可促進細胞增殖又可提高細胞抗氧化能力的最適濃度為50 μg/mL;將此濃度的藥物進行粘附實驗,發(fā)現(xiàn)與白藜蘆醇、地塞米松、紫花苜蓿黃酮相比,苦參堿對粘附作用具有顯著的抑制作用。以上研究結果揭示,純化后的細胞為BMECs,其中P4～P10細胞性狀更為穩(wěn)定,可用于后續(xù)實驗;金黃色葡萄球菌粘附BMECs的最佳時間為2.5 h,最適pH值7.0～7.5之間,最適溫度為37℃;苦參堿(適宜濃度50μg/mL)對金黃色葡萄球菌粘附BMECs具有顯著的抑制作用。
[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，

本文編號：1519440