铅胁迫引起日本三角涡虫线粒体损伤关键蛋白的发掘与功能研究

项目名称： 铅胁迫引起日本三角涡虫线粒体损伤关键蛋白的发掘与功能研究

项目编号： No.31201762

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 生物科学

项目作者： 叶海燕

作者单位： 陕西师范大学

项目金额： 25万元

中文摘要： 铅对细胞造成的损伤包括亚细胞超微结构的改变和细胞器功能的改变，其中由铅引起的氧化应激压力增加和细胞凋亡都和线粒体有着密切关系。本项研究首次全面发掘由铅胁迫引起线粒体功能损伤涉及的全部蛋白，并选择与氧化应激和细胞凋亡相关的2个重要蛋白研究其在铅损伤中的作用和功能，希望系统地阐明细胞线粒体铅损伤的分子机制。 本项研究拟选择水体污染的指示动物、模式生物- - 日本三角涡虫为实验材料，Nycodenz密度梯度超速离心得到高质纯化的线粒体，采用2D电泳技术，通过比较蛋白质组方法得到差异蛋白谱，对特异表达、表达上调和下调的蛋白点进行质谱分析，获得线粒体铅损伤相关全部蛋白的质谱信息。通过分析蛋白质的种类和表达量的变化，发掘铅胁迫对线粒体损伤涉及蛋白的调控路径，阐明损伤机制。选择差异显著，与氧化应激和细胞凋亡相关的2个蛋白，通过RNAi抑制表达，研究其功能和调控机制，为后续损伤后修复和治疗研究奠定基础

中文关键词： 线粒体损伤；铅胁迫；蛋白质组；转录组；RNA干扰

英文摘要： Cellular damage caused by lead includes pathological changes in subcellular microstructure and organellar function. Among these changes, oxidative stress and cell apoptosis are closely related to mitochondria. Thus, we focus on exploring total proteins invovled in lead caused mitochondria damage, and select two important proteins related to oxidative stress and cell apoptosis respectively, then study their roles in lead caused damage. Thereby we expect to illucidate the molecular mechanism of mitochondria damage caused by lead. We will use Dugesia japonic, the model animal for water pollution study, as experimental materials. We plan to use a comprehensive approach to determine the proteome of Dugesia japonic's mitochondria. Firstly, The highly pure mitochondria were separated by Nycodenz gradient density separation technique from Dugesia japonica body that yields mitochondria largely free of contamination by cytosol, peroxisomes, plastids, and other membranes.Secondly, we will use 2-D PAGE and comparative proteomic analysis to find the specifically expressed as well as upregulated and downregulated expressed proteins. Thirdly, using LC-MS/MS analysis we will identify all related polypeptides and their related proteins in lead stress. By analysing the changes in the species and expression level of the differenti

英文关键词： Mitochondrial damage；Leed stress；Protome；Transcriptome；RNAi