电离辐射诱导T淋巴细胞损伤的核内差异蛋白质组学研究

发布时间：2018-02-27 00:06

本文关键词： 核蛋白荧光标记双向差异凝胶电泳基质辅助激光解吸电离飞行时间质谱 Jurkat T淋巴细胞　出处：《吉林大学》2010年博士论文　论文类型：学位论文

【摘要】： JurkatT细胞株为白血病T淋巴细胞的一种,利用该类细胞作为研究对象可进行外界损伤性因素导致细胞坏死和凋亡的研究。电离辐射能够导致细胞不可逆性损伤,是恶性肿瘤的疾病的重要治疗方法。蛋白组学是研究细胞内蛋白构成以及在外界条件作用下发生生理或病理变化的实验室研究技术。将电离辐射与蛋白组学实验技术相结合,研究核内蛋白差异蛋白质变化规律,对寻求对白血病治疗有益的关键性核内调控蛋白因子十分重要。本研究验证了目前应用普遍的蛋白提取方法,建立了荧光免疫双向差异电泳结合质谱学分离和鉴定核内蛋白的实验技术平台,并对分离的差异蛋白质进行鉴定,共得到14个差异蛋白质点,为进一步全面了解核内蛋白质表达差异奠定了基础。 T淋巴细胞在辐射强度为6Gy条件下进行辐射诱发细胞损伤,在不同的时间点收集细胞,提取核内蛋白,应用2D-DIGE电泳进行分离,并利用质谱进行分析,根据质谱分析结果和MSCOT数据库检索,对细胞核蛋白做整体、全面的分析,鉴定了表达差异蛋白,共得到5组10个与T淋巴细胞细胞核内物质表达以及核外转运、参与整个细胞生长、增殖和转归的关键性蛋白。其分类如下:①淋巴细胞核内肿瘤相关抗原包括肿瘤生长因子-β受体相关蛋白呈现上调表达、细胞生长因子结合蛋白和核糖核酸酶—血管生长因子抑制剂呈现下调表达,而肿瘤排斥抗原表现出统计学意义的下调表达,在整个细胞试验流程中表达比较恒定。②淋巴细胞代谢相关核内相关蛋白包括泛素羧基末端水解酶,二硫化物异构酶蛋白和谷胱甘肽S-转移酶均呈现下调表达。③淋巴细胞核内遗传物质相关蛋白包括不均-核糖核蛋白K和G1期/S期转换控制蛋白呈现下调表达,核RNA解旋酶呈上调表达。④淋巴细胞细胞核稳定性相关蛋白包括蛋白酶体激活蛋白PA28,在0~8小时时程范围内呈下调表达,在8~16小时范围内呈现明显上调表达,随后的16~24小时范围内蛋白表达呈现下降趋势。本研究为进一步研究电离辐射损伤淋巴细胞打下了理论基础。
[Abstract]:JurkatT cell line is one of the leukemic T lymphocytes, which can be used as a research object to study the apoptosis and necrosis caused by external injury. Ionizing radiation can lead to irreversibly damaged cells. Proteomics is a laboratory technique for the study of intracellular protein composition and physiological or pathological changes under external conditions. It combines ionizing radiation with proteomics. It is very important to study the variation of differential protein in nuclear proteins in order to seek the key factors which are beneficial to the treatment of leukemia. In this study, we established an experimental platform for the separation and identification of nuclear proteins by fluorescence immunotwo-dimensional differential electrophoresis combined with mass spectrometry, and identified the isolated differential proteins. A total of 14 differentially expressed protein spots were obtained, which laid a foundation for further understanding the differences in protein expression in the nucleus. T lymphocytes were induced by radiation at a radiation intensity of 6 Gy. The cells were collected at different time points, the nuclear proteins were extracted, separated by 2D-DIGE electrophoresis, and analyzed by mass spectrometry. According to the results of mass spectrometry analysis and MSCOT database retrieval, the nuclear proteins were analyzed, and the differentially expressed proteins were identified. A total of 10 groups and 10 groups were obtained, which expressed substance in the nucleus of T lymphocytes and transported them outside the nucleus. The key proteins involved in cell growth, proliferation and outcome are classified as follows: 1. Tumor associated antigens, including tumor growth factor- 尾 receptor associated proteins, are up-regulated, The expression of cell growth factor binding protein and ribonuclease-vascular growth factor inhibitor was down-regulated, while tumor rejection antigen showed a statistically significant down-regulation. In the whole cell test process, the expression of relatively constant .2 lymphocyte metabolism-related nuclear related proteins, including ubiquitin carboxyl terminal hydrolase, Both disulfide isomerase protein and glutathione S-transferase showed down-regulated expression, including heterogeneous ribonucleoprotein K and G1 phase r-S phase transition control protein. The expression of nuclear RNA helicase was up-regulated, including proteasome activator PA28, which was down-regulated in 0h and 816h, respectively. The protein expression showed a downward trend in the 24-hour range. This study provides a theoretical basis for the further study of lymphocytes damaged by ionizing radiation.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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