人类肝脏氧化应激反应和氧化还原调控网络的初步研究

发布时间：2018-04-28 13:22

本文选题：肝脏 + 氧化应激　；参考：《中国人民解放军军事医学科学院》2006年博士论文

【摘要】：肝脏作为人体的代谢中心，其旺盛的代谢活动必然会伴随ROS(reactive oxygen species)的大量生成，ROS的大量产生和／或抗氧化防御机制的不足将引起氧化应激，另外，酒精、药物、肥胖引起的过量自由脂肪酸代谢等都极易使肝细胞产生氧化应激，最终引发肝损伤及肝病。因此，各种氧化应激反应信号通路及氧化还原调控蛋白在保护肝脏免受氧化损伤中发挥重要的作用，但这些蛋白在肝脏中的作用及其作用机制尚无系统性研究。规模化蛋白质相互作用研究是揭示蛋白质的作用机制，发现新的蛋白质功能、新的调控途径以及不同调控途径间内在联系等的重要手段。本文以肝脏氧化应激和氧化还原调控关键蛋白为诱饵，采用高通量酵母双杂交技术筛选人肝脏cDNA文库，从蛋白质相互作用角度出发，对肝脏氧化应激反应和氧化还原调控网络进行初步研究。本文成功进行了27个诱饵的文库筛选，获得528个候选阳性克隆，除去非编码序列和不符合读框的序列，得到223条读框正确的猎物序列，相对于97对、涉及20个诱饵的相互作用。除11对已知相互作用外，其余为新发现的相互作用，提示在肝脏氧化应激反应和氧化还原调控网络中可能还有很多的成员未被发现。为了进一步考察得到的相互作用的可靠性，，我们首先对其技术假阳性和生物学假阳性进行评价。采用酵母回转验证实验对技术假阳性进行了初步评价，在随机挑选的61对相互作用中，回转阳性率为52.5％，而已知相互作用的回转阳性率也仅为53.3％。表明我们的数据中存在一定的假阳性，同时在回转验证中存在明显的假阴性，提示我们不能轻易地去除回转呈阴性的相互作用。对技术假阳性的有效评价还需要借助其它实验手段。在生物学假阳性评价方面，我们采用多种生物信息分析方法，包括PubMed和HPRD数据库搜索、整合转录组和基因敲除表型数据、GO注释、相互作用结构域分析、模式生物同源性比较等。发现我们得到的相互作用中，有22对相互作用(23％)的诱饵和猎物基因名共同出现在相应文献中；26对相互作用(27％)的诱饵和猎物基因表达相关；9对相互作用(9％)的诱饵和猎物有相同或相
[Abstract]:As the metabolic center of human body, the vigorous metabolic activity of liver is bound to be accompanied by ROS(reactive oxygen species-based production of Ros and / or deficiency of antioxidant defense mechanism will lead to oxidative stress, in addition, alcohol, drugs, Excessive free fatty acid metabolism caused by obesity can easily lead to oxidative stress in hepatocytes and eventually lead to liver injury and liver disease. Therefore, various oxidative stress signaling pathways and redox regulatory proteins play an important role in protecting the liver from oxidative damage. However, the role of these proteins in the liver and its mechanism have not been systematically studied. The study of large-scale protein interaction is an important means to reveal the mechanism of protein action, to discover new protein functions, new regulatory pathways and internal relations between different regulatory pathways. In this paper, the cDNA library of human liver was screened by high-throughput yeast two-hybrid technique with the key proteins of liver oxidative stress and redox regulation as bait. The oxidative stress response and redox regulatory network of liver were studied. In this paper, 27 decoys were successfully screened, and 528 candidate positive clones were obtained. After removing the non-coding sequence and the unmatched reading frame sequence, the correct prey sequences of 223 reading frames were obtained. Compared with 97 pairs, the interaction of 20 decoys was involved. In addition to 11 pairs of known interactions, the others are newly discovered, suggesting that many members may not be found in the liver oxidative stress response and redox regulatory network. In order to further investigate the reliability of the interaction, we first evaluate the technical false positive and biological false positive. The technical false positive rate was preliminarily evaluated by yeast gyration verification experiment. Among 61 pairs of interactions randomly selected, the positive rate of gyration was 52.5 and the positive rate of known interaction was only 53.3%. It indicates that there are some false positives in our data and obvious false negative in gyration verification, which indicates that we can not easily remove the interaction between gyratory negative and negative. The effective evaluation of false positive technique also needs other experimental means. In the aspect of biological false positive evaluation, we used a variety of biological information analysis methods, including PubMed and HPRD database search, integration of transcription and gene knockout phenotypic data go annotation, interaction domain analysis, model biological homology comparison, and so on. It is found that there are 22 pairs of interaction bait and prey gene name in the corresponding literature.) the bait and prey gene expression are related to the gene expression of prey.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R341

【引证文献】