【作者】 柳亦松；

【导师】 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 分泌蛋白(secretory protein)参与细胞信号的传导、细胞增殖、分化和凋亡的调控、生物体的发育等重要生命过程。了解不同发育、生长时期和不同生理、病理条件下及不同类型细胞分泌蛋白表达的特点对认识生命过程具有重要意义。分泌蛋白质组的研究有助于我们找到直接与特定生理或病理状态相关的生物分子,并且为从分子水平上认识疾病的发生和发展过程提供理论依据,也为设计作用于特定靶分子(target molecule)的药物筛选奠定基础。本研究选择了几种不同的原代细胞和细胞系为研究的模式材料,利用蛋白质组学技术对分泌蛋白进行研究,进行了分泌蛋白组的方法学,表达谱,以及特异性的生物标志物的相关研究。首先,开展大规模的蛋白质组研究的必备条件是高分辨蛋白质分离技术和高通量、高灵敏度的鉴定技术。因此在本研究的第二章我们首先优化了窄范围pH梯度胶的二维电泳(2-DE)的分离条件,并利用优化后的参数,建立了鼻咽癌分泌蛋白质2-DE表达谱。随后探索了胶内蛋白质鉴定的策略,建立了基于2-DE,自动切胶和自动酶解基础上的高通量胶上蛋白质点鉴定技术。其次,利用在第二章鼻咽癌分泌蛋白质组研究中所摸索出来的分泌蛋白质纯化与富集策略,我们开展了嗅鞘细胞分泌蛋白质组表达谱的研究。所不同的是我们采用了优化的一维凝胶电泳和液相色谱串联质谱联合的复杂蛋白质混合物鉴定技术。嗅鞘细胞是一种特殊类型的星型胶质细胞,它兼具了胶质细胞和雪旺式细胞的一些特点。据报道将嗅鞘细胞移植到损伤的中枢神经系统部位可以刺激其不同程度的再生,髓鞘再生和功能恢复。在本论文第三章的研究中,我们应用了SDS-PAGE胶分离与高通量液相串联质谱结合的蛋白质组学技术鉴定嗅鞘细胞条件培养基中的分泌蛋白,同时辅以在线的基因功能分类体系(Gene ontology)来对这些蛋白进行生物学功能预测和研究。我们在嗅鞘细胞条件培养基中鉴定到了168个非冗余蛋白并根据它们的生物学功能来进行分类,其中的大部分蛋白与神经分化和神经再生相关的。非标定量蛋白质组学分析被应用在嗅鞘细胞分泌蛋白质组与胶质细胞分泌蛋白质组的活性组分差异比较研究中,通过蛋白质鉴定的总次数比较(Spectral counting,半定量),我们找到了23个蛋白质在嗅鞘细胞分泌蛋白中上调,14个蛋白质在胶质细胞分泌蛋白中上调。蛋白质印迹法验证了载脂蛋白-E和胰岛素样生长因子结合蛋白质-2等5个在嗅鞘细胞分泌中上调的蛋白。因此,我们认为本实验不但首次大量的鉴定了嗅鞘细胞分泌蛋白质组中的与神经生长和再生相关的蛋白,而且为嗅鞘细胞条件培养基对于中枢神经系统修复的多方面调控作用提供了一些实验证据。总而言之,通过本研究论文,我们建立了大规模的人鼻咽癌细胞系分泌蛋白质组表达谱以及嗅鞘细胞和星形胶质细胞的分泌蛋白质组表达谱。据我们目前所知,我们所鉴定的嗅鞘细胞分泌蛋白质表达谱的蛋白质数据是目前国际上最大的一个溴鞘细胞蛋白质数据库。其次,本研究从蛋白质组学的不同策略出发,建立起了一套行之有效的基于2-DE和1-DE凝胶电泳技术与质谱连用的分泌蛋白质组研究策略,可以有效的对于不同类型的细胞分泌蛋白质组进行富集和简化分析体系,并且实现潜在的疾病标志物的发掘。该体系将同样的适用于其他的肿瘤或疾病相关的蛋白质标志物的研究。更多还原

【Abstract】 Secretory protein is involved in cell signal transduction, cell proliferation, differentiation/apoptosis, the organism’s development and other important life processes. The understanding of the secretory protein expression characteristics of different growth period and different physiological or pathological conditions is of great significance. Secreted proteome research will not only help us to find the specific physiological /pathological condition related to biological molecules directly, and understanding of disease occurrence of molecular level or provide a theoretical basis for the development process, but also for the design of the role of specific target molecules and lay the foundation for drug discovery. This study selected several different types of primary cells and cell lines to study the model material, proteomics technology was used to study the secreted protein, it also carried out a secretory protein group methodology, expression profiling, as well as specific biomarkers of related research.First of all, we carry out a large-scale proteome research as a prerequisite for high-resolution protein separation techniques and high-throughput, high sensitivity identification technology. Therefore, in the second chapter of this study, we optimized the narrow range pH gradient two-dimensional gel electrophoresis (2-DE) separation conditions, and the use of the parameters being optimized to establish a nasopharyngeal secretion of 2-DE protein expression profiling. We then explored the in-gel protein identification strategy, which established based on high-throughput 2-DE, automatic cutting and automatic in-gel digestion of gel protein spots identification technology.Secondly, we take the advantage of the analysis of nasopharyngeal secreted proteins purification and enrichment strategy in the second chapter, and launched the olfactory ensheathing cells secreted protein expression profile studies. The difference of this chapter is that we use an optimized one-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry combined protein mixture identification technology. Olfactory ensheathing cells are a special type of glial cell which have characteristics of both astrocytes and Schwann cells. They express a high level of growth factors which play a very important role as neuronal support. In this study, ID-shotgun proteomics followed with gene ontology analysis was used to screen proteins present in the conditioned medium from primary culture rat olfactory ensheathing cells. One hundred and sixty eight non-redundant proteins were identified, the majority of which were never before reported to be produced by olfactory ensheathing cells. Furthermore, extracellular proteins were classified based on their biological and molecular functions, most of which were involved in neuronal differentiation and development. Moreover, we carried out semi-quantitative proteomic analysis to identify the active components of the olfactory ensheathing cells secretome that different from astrocytes. Spectral counting analysis revealed that twenty-three proteins were up-regulated in olfactory ensheathing cells whereas fourteen were up-regulated in astrocytes. Changes of the expression level of secreted proteins were further confirmed by western blot. In conclusion, this study provided a large number of proteins involved in neuronal development and regeneration in the olfactory ensheathing cell secretome and it may shed light on the role of extracellular matrix proteins from conditioned medium in the repairing process of central nervous system.In total, through the research papers, we have established a large-scale human nasopharyngeal carcinoma cell line secreted proteome expression profile, as well as olfactory ensheathing cells and astrocytes secreted proteome expression profile. As far as we know, our olfactory ensheathing cells secreted protein expression profile data is now the largest international database of olfactory ensheathing cells secreted protein. Moreover, this study of different strategies starting from the proteomics research has established a set of effective technology based on 2-DE and 1-DE gel electrophoresis and mass spectrometry used in conjunction with the secretion proteome research strategy can be effective for the different types of cells to secrete the protein group enrichment and simplify the analysis system, and realize potential markers of disease discovered. This secreted proteins research system will be equally applicable to other tumors or disease-related protein markers.更多还原