| 摘要: |
| 【目的】研究产PV-杀白细胞素（Panton Valentine leukocidin,PVL）金黄色葡萄球菌ATCC49775（产PVL标准菌株）、ΔPVL 49775（PVL基因缺失株）以及体外重组PVL（rPVL）对奶牛乳腺上皮细胞（bovine mammary epithelial cells,BMECs）内质网应激和自噬的影响，为系统阐述金黄色葡萄球菌及PVL对BMECs损伤的分子机制奠定基础。【方法】用感染复数（MOI）为30的金黄色葡萄球菌菌株ATCC49775和ΔPVL 49775感染BMECs，于感染后3和6 h取样；用100 ng/mL rPVL处理BMECs，于处理后1，3和6 h取样；以未处理的BMECs为对照（CK）。用免疫荧光法检测样品BMECs细胞的自噬体荧光强度；提取样品细胞的总蛋白，以GADPH为内参蛋白，采用Western blot法检测各处理组内质网应激相关蛋白（CHOP和GRP78）和自噬相关蛋白（LC3 Ⅱ、ATG5、Beclin1和p62）的相对表达量。【结果】ATCC49775、ΔPVL 49775感染后不同时间,BMECs自噬体荧光强度均极显著（P＜0.01）高于CK,内质网应激相关蛋白（CHOP和GRP78）和自噬相关蛋白（LC3-Ⅱ、ATG5、Beclin1和p62）的表达水平也大多显著(P<0.05)或极显著（P＜0.01）高于CK;同一处理时间下， ATCC49775感染组自噬体荧光强度和相关蛋白表达水平均显著(P<0.05)或极显著（P＜0.01）高于ΔPVL49775感染组。 rPVL处理后不同时间,BMECs的自噬体荧光强度均极显著（P＜0.01）高于CK，内质网应激和自噬相关蛋白的表达水平大多极显著（P＜0.01）高于CK。【结论】产PVL金黄色葡萄球菌ATCC49775、ΔPVL 49775以及rPVL均加剧了BMECs的内质网应激和自噬，说明PVL在金黄色葡萄球菌感染BMECs的过程中有重要作用。 |
| 关键词: 金黄色葡萄球菌 PV-杀白细胞素 奶牛乳腺上皮细胞 内质网应激 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金项目（32060816） |
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| Effects of PVL-producing Staphylococcus aureus on endoplasmic reticulum stress and autophagy in BMECs |
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HAI Zhenzhen,FAN Yongxin,MA Xiaoxia,et al
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| Abstract: |
| 【Objective】This study investigated the effects of Panton Valentine leukocidin (PVL) producing Staphylococcus aureus ATCC49775 (standard strain）,ΔPVL 49775 （PVL gene deletion strain） and recombinant PVL (rPVL) on endoplasmic reticulum stress and autophagy in bovine mammary epithelial cells (BMECs) to provide basis for explaining the mechanism of BMECs injury caused by S. aureus and PVL.【Method】Samples were taken at 3 and 6 h after infected with MOI of 30 S. aureus ATCC49775 and ΔPVL 49775 in BMECs and at 1,3 and 6 h after treated with 100 ng/mL rPVL in BMECs,and untreated BMECs were used as control (CK).The fluorescence intensity of autophagy was detected by immunofluorescence method.Total protein of sample cells was extracted using GAPDH as the reference protein.The expression levels of endoplasmic reticulum stress related proteins（CHOP and GRP78） and autophagy-rela-ted proteins （LC3-Ⅱ,ATG5,Beclin1 and p62） in each treatment group were detected by Western blot.【Result】Fluorescence intensity of autophagosome of BMECs infected by ATCC49775 and ΔPVL 49775 were extremely significantly (P<0.01) higher than that of the control and expressions of most endoplasmic reticulum stress related proteins（CHOP and GRP78） and autophagy related proteins（LC3-Ⅱ,ATG5,Beclin1 and p62） were up regulated significantly (P<0.05) or very significantly (P<0.01).Under same treatment time,the fluorescence intensity and protein expression levels in S. aureus ATCC49775 infection group were significantly (P<0.05) or extremely significantly (P<0.01) higher than those in ΔPVL 49775 infection group.The fluorescence intensity of autophagosome was extremely significantly (P<0.01) increased by rPVL and expressions of most endoplasmic reticulum stress and autophagy-related proteins were also significantly up-regulated (P<0.01).【Conclusion】PVL-producing S. aureus ATCC49775,ΔPVL 49775 and rPVL aggravated endoplasmic reticulum stress and autophagy in BMECs,suggesting that PVL played an important role in S. aureus infection of BMECs. |
| Key words: Staphylococcus aureus Panton-Valentine leukocidin bovine mammary epithelial cells endoplasmic reticulum stress |
