| The malignant tumor of hematological system–leukemia is one kind of fatal disease which threat people's lives; scientists have not found the pathogeny and pathogenesis about leukemia. T-Lymphocytes, as the most important functional cell of immune system, mediate cellular immunity. Studying this series cells can lead us to further understand the necrosis and apoptosis of cells, or help us to learn the changes of Subcellular structure induced by injuries outsides.And first and most important thing is that studying these cells will provide straightly evidences for pathogenesy of leukemia. Ionizing radiation is one of pratical therapies to curing the malignant tumor because of the irreversible damages of cells by inradiation. It is the method that can improve and enhance the therapy of cellular level to cure the malignant tumor through studying proteomics of nuclear damages induced by inradiation.Proteomics is one kind of investigative technique in laboratory which study the changes of physiology and pathology undet the external conditions, the major jobs include extracting, separating, analyzing and indentifing the target proteins, the extraction proteins is the direct influence of effect and precision of protein separation. Nuclear protein is typical low abundance protein, and it is also a main disadvantage to interference the improvement of proteomics.In this study, using 2D-DIGE and MALDI-TOF/MS methods to analyzing and indentifing the nuclear proteins induced by 6Gy inradiation.Grouping the cells under the different collection times, using 2D-DIGE to separating the proteins, basing on the annalistic results, assisting with searching engine check the MSCOT database to indentify the disparate proteins, entirely analyze nuclear protein, provide the evidences for deeper studying the injures of T lymphocytes induced by inradiation.The main work is as follow:1 Cell culture, cytoactive level detection and extraction and quantitation of proteins Culture Jurkat the T-Lymphocytes, cellect the cells as the cells density as the experiment acquired and detect the cells cytoactive. Use the different methods to extract the nuclear protein campare and analyze indentify extraction method wether it is suit for the experiment. Use the Bradford method to detect the density of cells.2 Creating the technique to analyze and indentify the nuclea protein with the method of 2D-DIGE and MALDI-TOF/MSSet the proteins in different time as experimental group which have been proved the extraction proteins are enough to take the 2D-DIGE. Set the plasmosin and cell protein as control group. Separate the proteins with 2D-DIGE Based on optimized methods of the experiment manuals. 1987±97 protein spots have been collected, with the software analyzed 76 disaparte proteins have been found, and searching for the MSCOT database the 14 proteins have been indentified. These proteins will be decribed as this:①10 proteins of heritage material in nuclear.②1 proteins involve in metabolism and materials transportation in cells.③2 membrane skeleton proteins.④1stress protein and influcing mitotic cycle.3 The research of differential nuclear proteins of T-Lymphocytes induced by inradiationUse the technique that have been proved to group the 4 time extracted proteins, label fluorescent, separate the proteins with 2D-DIGE, analyze the data by MALDI-TOF/MS and searching the MSCOT database. 10 differential nuclear proteins have been found, classify these 10 proteins, the result is as follow:①Tumor associated antigen of lymphocytes: TGF-βreceptor interacting protein shows down regulation. Growth factor bound protein show down regulation. Tumor rejection antigen shows down regulation of statistical significance.②Metabolism relative nuclear proteins of lymphocytes:Ubiquitin carboxy-terminal hydrolase shows down regulation. Disulfide-isomerase protein shows down regulation. Glutathione S-transferase shows down regulation.③heritage material of nuclear Lymphocytes cells: Heterogeneous nuclear ribonucleoprotein K shows down regulation. Nuclear RNA helicase shows up regulation. G1/S transition control protein shows down regulation.④The proteins relative nuclear stability of lymphocytes: Proteasome activator 28 shows down regulation between 0 to 8 hours, shows up regulation between 8 to 16 hours, shows down regulation between 16 to 24 hours.
|
PDF Full Text Request