【作者】 伦艳妮；

【导师】 朱迅；

【作者基本信息】 吉林大学 ， 免疫学， 2009， 博士

【摘要】 为了发掘出在活化巨噬细胞杀伤肿瘤细胞的过程中,发挥主要介导作用的效应分子,我们利用质谱技术分别对BCG活化和TGC诱导的小鼠巨噬细胞膜蛋白进行了鉴定。通过对两组蛋白的比较和分析,我们得到了421个差异表达在BCG活化巨噬细胞的膜蛋白。然后,我们按照GO分类标准对各个蛋白分子的功能预测进行了注释,其中42个成员被鉴定为亚细胞定位在细胞质膜上的膜蛋白。其余分子功能分布广泛,从细胞组成到代谢组分等等。我们进而对所鉴定出的差异性膜蛋白进行了生物信息学分析。通过聚类分析、表达模式分析及表达模式关系预测等研究手段,我们发现,这些分子中的大部分,在巨噬细胞中的表达水平都随着活化时间的延长表现出上调表达的趋势。并且,这些蛋白相互间的表达模式相关性非常复杂,几乎每一种蛋白的表达变化都与多种其它蛋白的表达变化紧密相关。本研究鉴定出的大量活化相关蛋白分子,为深入研究巨噬细胞活化机制及肿瘤细胞杀伤机制积累了必要的数据资料。作为下一阶段对目标蛋白进行功能研究的初始阶段,我们选取了一个全新蛋白INATase。通过构建其重组表达载体,我们获得了一定量的重组蛋白,并制备了兔血清多克隆抗体。为了检测这一蛋白在活化巨噬细胞中的表达情况,我们进行了活化巨噬细胞的免疫组织化学鉴定,初步证实了INATase在活化巨噬细胞中的存在。为下一步进行活化巨噬细胞功能机制的研究奠定了一定的基础。对于活化巨噬细胞的基因水平变化,已有相关的研究结果。本研究首次从转录水平和蛋白质组水平两个方面对巨噬细胞活化过程中的大分子变化进行了分析和研究,鉴定出相当数量的膜蛋白分子,进一步丰富了巨噬细胞膜蛋白数据库,并成功获得了一个有潜在研究价值的重组蛋白。本研究不但为巨噬细胞接触依赖性杀伤机制及巨噬细胞活化的研究提供了大量非常有价值的资料,而且为后续工作提供了一种可靠的研究思路和可行的研究模式。更多还原

【Abstract】 Activated macrophages possess tumoricidal ability associated with a cell-cell contact mechanism. However, the nature of the cell surface proteins involved in this tumoricidal process is not yet known. In this research, macrophages activated by bacterium Mycobacterium bovis bacillus Calmette-Guerin (BCG) and by thioglycolate (TGC) were chosen as two comparative models for the large-scale detection of cell surface protein alterations involved in this process. A proteomics approach involving SDS-PAGE followed by LC-MS/MS was used to characterize membrane proteins of the two models. A total of 421 uniquely expressed proteins were identified on the BCG-activated macrophage membrane. Functional groups indicating signal transduction, transport, and cell adhesion, among others, were significantly enriched in this group of proteins. In addition, 42 proteins associated with the plasma membrane were detected. These membrane proteins may contact tumor cells directly and play important roles in the tumoricidal process. In general, this study provides an initial database of candidate proteins that can now be screened as potential regulators of the adherence-dependent tumoricidal properties associated with macrophages.1. LC-MS/MS analysis on BCG-activated and TGC-induced macrophages membrane proteinsMembrane proteins from BCG-activated and TGC-induced macrophages were prepared and 150μg protein was applied to a 12% bis-Tris SDS-PAGE gel. After visualization by Coomassie blue staining, entire gel lanes were cut into eight pieces of equal size and subjected to in-gel tryptic digestion. The extracted peptides were analyzed using a LCQ deca plus system equipped with a capillary HPLC system (Thermo Finnigan, San Jose, CA, USA). First, microcore RP column (RP-C18, 0.15mm×120mm, ThermoHypersil, San Jose, CA, USA) was used to separated the protein digests The mobile phases used for reverse phase were 0.1% formic acid in water, pH 3.0 (A), and 0.1% formic acid in 100% v/v ACN (B). Peptides were eluted using a 0 to 80% linear gradient of solvent B over 60 min. The flow rate of the pump was 200μl/min and reduced to about 2μl/min after the split. An ESI IT mass spectrometer (LCQ Deca XP, Thermo Finnigan) was used for peptide detection. The positive ion mode was employed and the spray voltage was set at 3.2 kV. The spray temperature was set at 170℃for peptides. Collision energy was automatically set by the LCQ Deca XP system. After acquisition of a full scan, three MS/MS scans were acquired for the next three most intense ions using dynamic exclusion for 6 hours.2. Unique proteins on BCG-activated macrophages membraneComparing the two lists of identified proteins, we filtered out redundant data and consequently confirmed that there were 421 unique proteins on BCG-activated macrophage membranes.To elucidate potential functions of these proteins, we categorized them by GO analysis. The functional categories included the following groups: transport (15%), protein metabolism (14%), other metabolic processes (13%), RNA metabolism (11%), cell organization and biogenesis (10%), signal transduction (8%), developmental processes (8%), stress responses (7%), cell cycle and proliferation (4%), cell adhesion (3%), death (3%), DNA metabolism (2%) and cell-cell signaling (2%). The unique proteins were also assigned subcellular locations by GO classification. They were found to distribute throughout the cell, in subcellular organs such as Golgi apparatus, ribosomes etc. Of the initial 421 unique proteins, 42 were determined to be plasma membrane proteins.3. Bioinformatics analysis on unique membranous proteins of BCG-activated macrophagesThrough GO analysis, 42 proteins were localized to the plasma membrane proteins, making them likely candidates for cell contact. Since the activated macrophages mediate tumor cell killing in a contact-dependent manner, any of these 42 plasma membrane proteins may be involved in mediating the tumoricidal process. The top three functional categories of these 42 members were signal transduction (31%), transport (24%) and cell adhesion (21%). All of these functions are extensively involved in either the physiological processes or the pathology of cells.An attempt was made to predict the interaction relationships among the proteins, based on the protein interaction information databases such as DIP, BIND and IntAct. Querying the KEGG pathway database (http://www.genome.jp/kegg/), these members appeared to be proteins involved in regulating cell communication, including adherents such as integrin and ezrin (EZR). Both integrin and EZR can interact with more than one protein. Ezrin has been reported as a potential regulator of cell-cell and cell-matrix adhesion and plays an important role in the control of adhesion and invasiveness of cancer cells.4. Primary study on INATaseThe recombinant pET-28 plasmids were transformed into E. coli strain BL21. When the cultures were grown to A600=0.6, 0.7mM isopropyl-1-thio-β- D-galactopyranoside (IPTG) was added to induce expression of recombinant proteins. After centrifugation, the cells were resuspended in lyse buffer. The fusion proteins were purified following the protocol of inclusion body purification in MOLECULES CLONING 3.A female rabbit weighed 3kg was primed by 0.5mg purified proteins by hypodermal injection. Another three injections were carried out every two weeks to increase the immunology response. Fifteen days after the last injection, venous blood was obtained to prepare the polyclonal antibody. The antibody titer was determined by ELISA.Using the polyclonal antibody as first antibody, an immunohistochemistry assay was carried out to explore INATase expressed in BCG-activated macrophages.Changes in gene expression profile of macrophages in response to BCG have been analyzed. However, the membrane protein alternations involved are still undiscovered. In the current study, we have further analyzed activated macrophage membrane proteins. We report on 421 unique proteins seen in response to BCG activation but not to TGC activation. According to their functional features, we have categorized these unique proteins into groups including transport, cell organization and biogenesis, signal transduction, cell adhesion, and apoptosis. By providing an initial database of likely candidates for cytotoxicity responses, we set the stage for future detailed gene expression and functional studies that will clarify the tumoricidal mechanism used by activated macrophages.更多还原