| 李慧瑾,李正堃,连媛媛,贺春霞,秦 魏,孙丽君,孙晶莹,赵 栋,曹慧玲.H1N1流感病毒血凝素对人肺细胞的损伤作用及机制研究[J].,2018,(6):1029-1033 |
| H1N1流感病毒血凝素对人肺细胞的损伤作用及机制研究 |
| Effects and Mechanisms of Hemagglutinin of H1N1 Influenza Virus on the Human Lung Cells |
| 投稿时间：2017-10-18 修订日期：2017-11-13 |
| DOI：10.13241/j.cnki.pmb.2018.06.006 |
| 中文关键词: H1N1流感病毒 血凝素 人胚肺成纤维细胞 线粒体 |
| 英文关键词: H1N1 influenza virus Hemagglutinin Human Embryonic Lung Fibroblasts Mitochondria |
| 基金项目:国家自然科学基金青年基金项目（81202373）；陕西省高校科协青年人才托举计划项目（20170408）；西安医学院2016年博士科研启动基金项目(2016DOC13) |
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| 中文摘要: |
| 摘要 目的：研究H1N1流感病毒血凝素HA对人胚肺成纤维细胞的损伤作用并初步探讨其机理。方法：合成2009 H1N1的HA基因全长，分别将HA的PCR产物和pEGFP-N1质粒经Hind III和EcoRI酶切电泳、纯化、连接并转化大肠杆菌DH5α，构建真核表达质粒pEGFP-N1/HA。质粒转染293细胞，检测其转染效率，并收获细胞总蛋白进行Western blotting检测。将阳性质粒转染MRC-5细胞，CCK-8检测HA的表达对细胞增殖活性，线粒体膜电位检测试剂盒检测其对细胞的早期凋亡的影响，ATP检测试剂盒检测HA的表达对ATP水平的改变。结果：PCR电泳检测获得分子量为1700 bp左右的目的条带，菌落PCR鉴定HA片段成功插入pEGFP-N1载体。荧光显微镜下检测pEGFP-N1/HA转染293细胞有明显荧光，Western blotting结果显示pEGFP-N1/HA转染细胞有单一的目的蛋白表达。HA的表达可明显抑制MRC-5细胞活力，HA的高表达降低MRC-5细胞的线粒体膜电位及ATP水平。结论：H1N1流感病毒HA对人肺细胞的损伤作用可能与影响肺细胞的线粒体功能有关。 |
| 英文摘要: |
| ABSTRACT Objective: To study the effects of hemagglutinin HA of H1N1 influenza virus on human embryonic lung fibroblasts and explore its mechanisms. Methods: HA gene of H1N1 was synthesized, PCR production of HA and pEGFP-N1 were digested by Hind III and EcoR I respectively, the recombinant plasmid pEGFP-N1/HA was constructed after ligation and transformation into Escherichia coli DH5α. The transfection efficiency was tested after the positive plasmid was transfected into 293 cells and the HA expression level was analyzed by Western blotting. The cell proliferation was detected by CCK-8 assay, the early apoptosis was determined by mitochondrial membrane potential, ATP level was detected by ATP assay kit after the plasmid pEGFP-N1/HA was transfected into MRC-5 cells. Results: The target band with molecular weight of about 1700 bp was obtained by PCR. The plasmid pEGFP-N1/HA was successfully identified by PCR. The strong fluorescence signals and a single band were detected in 293 cells after being transfected by pEGFP-N1/HA. CCK-8 test results showed that overexpression of HA significantly inhibited the viability of MRC-5 cells. MitoTracker Red CMXRos staining indicated that overexpression of HA reduced the mitochondrial membrane potential of MRC-5 cells. The results of ATP assay showed that overexpression of HA decreased the ATP level in MRC-5 cells. Conclusion: HA of H1N1 influenza virus-induced damage of human lung MRC-5 cells may be due to impaired mitochondrial function. |
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