化学学报 ›› 2011, Vol. 69 ›› Issue (21): 2609-2617.DOI: 10.6023/A1012161J 上一篇    下一篇

研究论文

应用毒理蛋白质组学技术研究多环芳烃类物质诱导气道上皮细胞毒理作用的机制

闵凌峰1,何淑雅2,陈琼*,1,谢明萱1,彭红兵1,曹兰玉1,王怀石1,朱小燕1   

  1. (1中南大学湘雅医院老年医学科、呼吸内科 长沙 410008)
    (2南华大学生物化学与分子生物学系 衡阳 421001)
  • 收稿日期:2010-12-16 修回日期:2011-06-18 出版日期:2011-11-14 发布日期:2011-07-02
  • 通讯作者: 陈琼 E-mail:qiongch@yahoo.com.cn
  • 基金资助:

    耐辐射奇球菌基因文库构建及其修改蛋白质相互作用网络研究;蛋白质组学技术筛选验证吸烟相关性肺癌预警标志物

Using Proteomic Analysis to Screen for Differentially Expressed Proteins in Airway Epithelial Cells Toxicity Mediated by Polycyclic Aromatic Hydrocarbons

Min Lingfeng1 He Shuya2 Chen Qiong*,1 Xie Mingxuan1 Peng Hongbing1 Cao Lanyu1 Wang Huaishi1 Zhu Xiaoyan1   

  1. (1 Department of Respiratory, Department of Geriatric Medicine, Xiangya Hospital of Central South University, Changsha 410008)
    (2 Department of Biochemistry and Molecular Biology, University of South China, Hengyang 421001)
  • Received:2010-12-16 Revised:2011-06-18 Online:2011-11-14 Published:2011-07-02

为了更好地理解苯并芘(Benzo(a)pyrene, B(a)P)在气道细胞诱导的毒理作用机制. 应用二维凝胶电泳(two-dimensional electrophoresis, 2-DE)分离B(a)P处理组(B(a)P-A549)、对照组(DMSO-A549)的总蛋白质, 图像分析识别差异表达的蛋白质点, 基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异蛋白质点, Western blot验证差异蛋白热激蛋白27 (heat shock protein 27, HSP 27)以及锰超氧化物歧化酶(Mn superoxide dismutase, Mn SOD)的表达, 使用0.1, 1, 10 μmol/L B(a)P处理A549细胞, 应用Western blot及RT-PCR检测各组细胞中Mn SOD的表达, 检测各组细胞中总抗氧化活性(total antioxidant capacity)、总SOD活性(total activity of SOD)、过氧化氢酶活性(catalase activity, CAT)、谷胱甘肽还原酶活性(glutathione reductase, Gr). 本研究首先建立了B(a)P处理组、对照组的2-DE 图谱, 质谱鉴定了23个差异蛋白质, Western blot 证实HSP 27及Mn SOD的表达水平在B(a)P处理组中较对照组明显下调|使用0.1, 1, 10 μmol/L B(a)P处理A549细胞后, 发现随着B(a)P的浓度升高, 虽然Mn SOD的表达及SOD活性出现先诱导后抑制的现象, 但CAT及Gr活性的增高依然提高了机体的总抗氧化活性. 研究结果提示: HSP 27及Mn SOD 与B(a)P在气道细胞诱导的毒理作用相关, CAT及Gr在对抗B(a)P带来的氧化损伤方面可能具有十分重要的作用.

关键词: 苯并芘, 气道上皮细胞, 蛋白质组学, HSP 27, Mn SOD

In order to better understand the molecular mechanisms of Benzo(a)pyrene [B(a)P] mediated toxicity on airway epithelial cells, comparative proteomics involving two-dimensional gel electrophoresis (2-DE) and MALDI-TOF Mass Spectrometry (MS) was performed on total proteins extracts from B(a)P-A549 and its control cell line DMSO-A549. Then the expression of heat shock protein 27 (HSP 27) and Mn superoxide dismutase (Mn SOD), two of the identified differential expressed protein, was analyzed using western blot. After 0.1, 1, 10 μmol/L B(a)P treatment, the Mn SOD expression levels were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, the total antioxidant capacity, total activity of SOD, catalase activity (CAT) and glutathione reductase activity (Gr) were determined also. In this study, 23 differentially expressed proteins was identified. Western blot confirm that B(a)P decreased heat shock protein 27 and Mn SOD expression levels in A549 cells. and the expression levels of this two genes were increased to the control level after detoxification. After 0.1, 1, 10 μmol/L B(a)P treatment on A549 cells, the expression level of Mn SOD and SOD activity were significantly induced after 0.1 μmol/L B(a)P exposure and then reduced after 1 μmol/L and 10 μmol/L B(a)P exposure. With the increased concentration of B(a)P, the catalase activity, the total antioxidant capacity and the glutathione reductase activity increased. Our results indicate that HSP 27 and Mn SOD may be related to the B(a)P mediated toxicity in airway epithelial cells. CAT and Gr may play an important role to antioxidant injury induced by B(a)P.

Key words: PAHs, airway epithelial cells, proteomics, HSP 27, Mn SOD

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