蛋白质的磷酸化修饰在侵袭相关垂体GH型腺瘤发生发展中的作用及机制

Protein phosphorylation is one of the most important post-translational modifications. Phosphorylation and dephosphotylation processes are the most general and important regulations of intracellular signal transduction, gene expression, and many other life processes, and many other life processes, which draw the widespread attention in the biological function research, but its role in aggressive pituitary somatotrophinomas remain unclear. We will investigate mechanism of aggressive pituitary somatotrophinomas which are directly or indirectly regulated by the reversible phosphorylation of proteins using iTRAQ labeling and LC-MS / MS. This work is on the basis of the models which are frozen specimens of aggressive pituitary somatotrophinomas , primary cultured human growth hormone pituitary adenoma cells and xenografts mouse models. We will screen key proteins and key signaling pathways with the occurrence and development of aggressive pituitary somatotrophinomas, several key proteins will be optimized and chosen through bioinformatic analysis, in addition, functional analysis of proteins will be conducted by immunohistochemistry on the basis of suitable antibody, at the same time, critical regulatory junction and its biological significance will be further analyzed and identified. The results of this research will be the foundation for early diagnosis and targeted therapy of aggressive pituitary somatotrophinomas.

蛋白质的磷酸化和去磷酸化过程调节着细胞信号转导、分化、生长、凋亡等几乎所有的生命活动。但其在侵袭相关垂体GH腺瘤发生发展中的作用尚不清楚。本课题拟在前期工作基础上，以人侵袭性垂体GH腺瘤冻存标本与原代培养的侵袭性GH腺瘤细胞及其动物异种移植瘤为主要模型，采用LC-MS/MS技术平台研究蛋白质磷酸化修饰对于侵袭性GH型腺瘤发生发展的影响，包括细胞信号转导、增殖、凋亡等；筛选与侵袭性GH腺瘤发生、 发展密切相关的关键蛋白质分子和关键信号通路,通过生物信息学分析精简出关键磷酸化蛋白质分子。优化关键磷酸化蛋白质分子检测试剂,筛选适用于免疫组化分析的抗体并开展功能分析，确定关键调控节点及其生物学意义 , 为侵袭相关GH腺瘤发生机制的研究及药物作用靶点的筛选提供新的思路，并为侵袭相关GH腺瘤临床诊断、治疗和预后判断提供有意义的可能线索。

基于磷酸化蛋白组学研究，探讨代谢信号通路异常与垂体生长激素（GH）腺瘤的相关性。垂体GH腺瘤标本取材于2010年8月至2017年8月于首都医科大学附属北京天坛医院神经外科行手术治疗的12例患者，健康垂体组织取材于6例因交通意外后尸体捐献者。采用超高效液相色谱和蛋白质质谱分析垂体GH腺瘤和健康垂体组织间的差异表达蛋白质，进一步通过生物信息分析方法识别与垂体GH腺瘤代谢相关的信号通路，之后采用免疫蛋白印迹方法验证该信号通路中关键分子在垂体GH腺瘤和健康垂体组织中的表达情况。蛋白质谱分析结果显示，垂体GH腺瘤与健康垂体之间存在1 213个差异磷酸化蛋白位点（P＜0.05, 差异倍数＞1.5或＜0.67。在垂体GH腺瘤中，表达上调的磷酸化蛋白位点有342个，表达下调的磷酸化蛋白位点有871个。这1 213个差异磷酸化蛋白位点分布在667个蛋白中。IPA生物信息学分析显示，667个差异表达的磷酸化蛋白质主要富集在AMPK（腺苷酸活化蛋白激酶）信号通路和糖代谢相关信号通路（P＜0.05。在糖代谢通路富集的10个磷酸化蛋白中，有9个在垂体GH腺瘤中低表达（正文结果中无此表达）。IPA通路活性分析显示，AMPK信号通路活性抑制。免疫蛋白印迹证实，在垂体GH腺瘤和健康垂体之间AMPK蛋白质的表达未见明显差异，而在垂体GH腺瘤中磷酸化AMPK（ser496）蛋白质表达明显低于在健康垂体中的表达。 磷酸化蛋白质组学分析显示，垂体GH腺瘤存在异常代谢。AMPK信号通路的低表达可能与垂体GH腺瘤异常糖代谢相关。