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摘要目的：以已构建的人鼻咽癌细胞（Human nasopharyngeal carcinoma cell, CNE-2)为模型，以体内生理情况下存在的NO供体巯基亚硝基-谷胱甘肽(S-nitrosoglutathione,GSNO)为工具药，分析离体鼻咽癌细胞蛋白潜在的S-亚硝基化修饰靶点。方法：采用GSH和NaNO2（1:1）在酸性避光条件下反应的方法合成GSNO，利用分光光度计法定量。培养GNE-2细胞，抽提蛋白质，测量蛋白浓度。以GSNO为工具药进行体外孵育。采用生物素转化技术（biotin switch method）纯化巯基亚硝基化蛋白，以iTRAQ试剂盒标记蛋白肽段结合LC-MS/MS技术寻找和鉴定蛋白。采用GO功能分析、String分析差异蛋白质的功能和细胞中的定位。综合分析离体鼻咽癌细胞蛋白潜在的S-亚硝基化修饰靶点。结果：根据筛选条件，我们共筛选出差异蛋白85个。GO分析发现这些差异蛋白涉及的生物过程有细胞生理过程、生物调控、代谢过程等。具有的分子功能有电子传递体、结合功能、转运活性、核酸结合转运因子活性、结构分子活性、分子转导活性等。主要定位于大分子复合物、膜封闭腔、细胞外基质、细胞器、细胞连接细胞、类核等。构建了差异蛋白相互作用网络图。51946
毕业论文关键词：人鼻咽癌细胞； GSNO； 巯基亚硝基化； 蛋白组学
Objective：The human nasopharyngeal carcinoma cell line(CNE-2) has been constructed as a model, the in vivo physiological conditions of NO donor thiolnitroso - glutathione S-nitrosoglutathione (GSNO) as a tool in medicine, to analysis the potentially S-nitrosylated modification target of in vitro nasopharyngeal carcinoma cell protein. Methods: GSNO was synthesized by the reaction of GSH and NaNO2 (1:1) under the condition of acid light avoidance. Cultured GNE-2 cells, extracted protein, measuring protein concentration. Incubation in vitro with GSNO as a tool. Using biotin switch method purified s-nitrosylation protein. Respectively with iTRAQ reagents box labeled peptide combined with LC-MS / MS technology to search for and identification of protein . GO function analysis, String analysis were used to analyze the differences in protein function and cell location. Comprehensive analysis the potential protein S-nitrosylation targets of nasopharyngeal carcinoma cells in vitro. Results: According to the screening conditions, we screened a total of 85 different proteins. GO analysis revealed that the biological processes involved in are cell physiology process, biological regulation, metabolic process, et al. Having a molecular function has the electronic transmission body, a combination of function and transport activity and nucleic acid binding transport activity factor, structure active molecules, molecular transduction activity. Mainly located in the macromolecular complex, membrane enclosed chamber, extracellular matrix, cell organelles, cell connections, nuclear, et al. The differential protein interaction network diagram was constructed.
Keyword:Human nasopharyngeal carcinoma cell；GSNO；S-nitrosylate；proteomic
目录 3
1 引言 4
2 材料与方法 4
2.1 材料 4
2.1.1 细胞株 4
2.1.2 试剂 5
2.1.3 实验仪器 5
2.2 方法 5
2.2.1 GSNO的合成（避光） 5
2.2.2