金黄色葡萄球菌诱导牛原代乳腺上皮细胞的凋亡

The Apoptosis of Bovine Primary Mammary Epithelial Cells Induced by Staphylococcus aureus

【目的】观察金黄色葡萄球菌能否诱导牛原代乳腺上皮细胞发生凋亡。【方法】先以新鲜牛奶为材料分离培养牛乳腺上皮细胞并鉴定,然后用金黄色葡萄球菌侵染牛乳腺上皮细胞,采用普通光镜和扫描电子显微镜观察金黄色葡萄球菌诱导牛乳腺上皮细胞凋亡的形态学变化;用流式细胞仪(Annexin V/PI双染法)定量检测金黄色葡萄球菌感染对牛乳腺上皮细胞凋亡的诱导作用;RT-PCR检测细胞凋亡通路中关键蛋白caspase 3和caspase 8的变化情况。【结果】从乳汁中分离的细胞经原代培养后,细胞呈典型的铺路石状,细胞表层可产生大小不等的乳滴,RT-PCR法扩增出乳腺上皮细胞的骨架蛋白8特异性条带;免疫荧光细胞染色法结果显示分离培养的牛乳腺上皮细胞表达角蛋白8;金黄色葡萄球菌感染牛乳腺上皮细胞3 h后,可诱导牛乳腺上皮细胞发生凋亡,表现为细胞核皱缩,染色质边缘化和细胞浆内空泡增多等典型凋亡特征;Annexin V/PI双染法检测后发现,与对照组相比,感染组细胞凋亡率明显升高,差异极显著(P﹤0.01);与对照组相比,感染组细胞caspase3及caspase 8表达量明显增高。【结论】金黄色葡萄球菌可以诱导牛原代乳腺上皮细胞凋亡,具有细胞凋亡的典型形态特征和生化特性,凋亡通路可能涉及caspase 3和caspase 8参与的外源性凋亡途径。

【Objective】 The objective of this experiment is to observe if apoptosis of the primary bovine mammary epithelial cells could be induced by Staphylococcus aureus in vitro.【Method】Bovine Mammary Epithelial Cells were separated from fresh milk and evaluated by RT-PCR and protein assaies of keratin8.After infection,light microscopy,scanning electron microscopy and Annexin V/PI staining were used to evaluate the apoptosis of bovine mammary epithelial cells,and the apoptosis ratios were calculated.The expression of caspase 3 and caspase 8 were detected by RT-PCR.【Result】 The cells’ shape looked like typical paving stones and keratin 8 was also detected by immunohistochemistry and PCR.Bovine mammary epithelial cells were round shrink aged with the apoptosis morphologies,and the expression of caspase 3 and caspase 8 was up-regulated after infection.【Conclusion】S.aureus could induce apoptosis of bovine mammary epithelial cells,and the apoptosis pathway could be associated with caspase 3 and caspase 8.