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

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 differential expressing proteins, we could access the regulatory pathway involved in lead caused mitochondria damage. After that, we could selecet two key proteins, involved in oxidative stress and cell apoptosis and also showing obviously diversity in expression level, for functional study by RNAi. RNAi of the selected proteins will intefere their expression, and then the result will provide clues for their physiological function and further clarify the mechanism invovle in lead stress. Thus, our research will provide the theoretical fundation for repairment and treatment after lead caused damage..

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

自项目启动以来，在前期的工作基础上，铅胁迫下细胞线粒体损伤关键蛋白的研究主要包括以下方面：①线粒体差异蛋白质组分析：采用Nycodenz不连续密度梯度离心法提纯了日本三角涡虫组织线粒体，以正常对照组与铅处理组的线粒体蛋白行双向电泳，质谱分析后获得差异表达蛋白谱。成功鉴定19个蛋白点，发现其中8个蛋白在铅处理后表达下降，分别为：电压依赖性阴离子通道相关蛋白、琥珀酸脱氢酶黄素亚基、细胞色素C还原酶、ATP合酶α亚基、Prohibitin、溶菌酶、P32蛋白、β-肌动蛋白、α-微管蛋白。表达上调的蛋白有11个，分别为：细胞色素C氧化酶Va型、染色体隔离蛋白SMC、再生相关蛋白、锌指蛋白213、β-微管蛋白、角蛋白等。②线粒体转录水平表达差异分析：日本三角涡虫经醋酸铅(80 mg•L-1)处理48h，试剂盒提取总RNA，通过高通量测序，比较两组数据从而筛选出差异表达的基因总共926条。相对于对照组，铅胁迫组中有451条基因表达量上调，475条基因表达量下调；其中代谢途径最为丰富的是“Metabolic pathways”代谢路径，共有135条差异基因。其次是“Ribosome”代谢路径，共有53条差异基因，“Oxidative phosphorylation”代谢路径，共有38条差异基因；铅胁迫组的核糖体大亚基中有18个下调基因，11个上调基因，核糖体小亚基中上调基因有16个，下调基因有8个；内质网中发现上调基因15个，下调基因18个；在氧化磷酸化通路上，发现了与5个电子传递链复合体: NADH脱氢(复合体Ⅰ)、琥珀酸脱氢酶（复合体Ⅱ）、细胞色素C还原酶（复合体Ⅲ）、细胞色素C氧化酶（复合体Ⅳ）和ATP合酶（复合体Ⅴ）相关的关键性差异表达基因。在铅胁迫条件下，这些差异基因表达量出现下调，表明线粒体电子传递链受损。③线粒体铅损伤关键蛋白及调控：对铅胁迫下涡虫咽部细胞内ROS水平的荧光检测，发现铅处理72小时，涡虫细胞内的ROS水平大幅上升；Western-Blot检测结果显示两个凋亡标志蛋白Bcl-xL，AKT表达上调；激光共聚焦检测涡虫细胞内的P32的分布及其在铅胁迫下变化，铅溶液处理72小时，P32线粒体定位减少；铅胁迫后P32 RNAi涡虫，ND1、ND2、ND6、16SrRNA基因表达趋于正常水平，。此外，研究过程中6名研究生先后参加了本项目的研究工作，并毕业2名。