神经胶质瘤血管新生的动态蛋白质组学研究

Gliomas are highly angiogenic. The new-formed vascular system has a distinct abnormal histological structure that includes a significantly increased vessel density, a more tortuous shape, an increased arterio-venous shunting, and the lack of a blood-brain barrier; which creates conditions for glioma growth, proliferation, invasion, and metastasis. Tumor vascular system is a promising target for therapy and progression monitoring of a glioma. However, no effective molecular markers have been proven to be useful in a clinic setting. This proposal will use high-throughput proteomic strategies to identify glioma angiogenesis-specific biomarkers for a better understanding of glioma pathogenesis, and the discovery of new therapeutic targets and progression-monitoring indices. We will focus on the protein expression, tyrosine nitration, and tyrosine phosphorylation in human glioma blood-vessels. Tyrosine nitration and phosphorylation are extensively associated with tumor angiogenesis and can occur at the same tyrosine site. Interference with those modifications could have a therapeutic impact on a glioma. .This present application is a ready-to-go research program that combines basic scientists and clinical scientists. They will define the unrecognized glioma angiogenesis biomarkers with analyses of the proteome, nitrotyrosine proteome, and phosphotyrosine proteome of glioma blood vessels. We will preferentially isolate the blood vessels from different grade glioma and control tissues with laser-capture microdissection (LCM), followed by high-throughput proteomic strategies to identify differentially expressed proteins (DEPs), differential nitrotyrosine proteins (DNTPs), and differential phosphotyrosine proteins (DPTPs) among different grade glioma and control LCM-vessels. The significant pathways that involve DEPs, DNTPs, and DPTPs will be identified with pathway analysis. The nitrotyrosine sites and phosphotyrosine sites will be determined with tandem mass spectrometry. The protein-domains that contain those modified sites will be determined with domain analysis. Also, we will determine the relative degree of nitration and phosphorylation in glioma relative to controls. Those data will be validated in a set of new glioma samples and controls. .Those quantitative data will be used to discriminate different grade gliomas from controls for discovery of tumor progression-monitoring indices. We will comprehensively analyze all data and rationalize those data in the glioma biology system to discover novel therapeutic targets for anti-angiogenic therapy.

高度异常的血管新生是神经胶质瘤的重要特征，与其增殖、侵袭密切相关。目前以肿瘤血管新生为靶点的治疗已成为肿瘤治疗领域的主要方向之一。肿瘤血管新生受到多种因素调节，且构成这些新生血管的蛋白质常常有复杂的修饰如酪氨酸硝基化和酪氨酸磷酸化。血管生成因子和抗血管生成因子间的失衡被认为是肿瘤血管新生的机制，然而，还没有一个因子被证实在临床上真正有用。因此，非常有必要从蛋白质整体角度出发，采用系统综合策略对其进行动态、深入研究来更全面、真实地揭示肿瘤血管新生的过程和本质。该项目拟采用高通量蛋白质组学和系统生物学策略集中研究不同分级神经胶质瘤新生血管的蛋白质组、酪氨酸硝基化蛋白质组、酪氨酸磷酸化蛋白质组、及其蛋白质网络的改变, 以鉴定、识别神经胶质瘤血管新生的生物标志物来更好地阐明神经胶质瘤发生发展的病理机制、发现新的治疗靶和进展监测指标。

高度异常的血管新生是神经胶质瘤的重要病理生理特征，与其增殖、侵袭密切相关。肿瘤血管新生受到多种因素调节，血管生成因子和抗血管生成因子间的失衡被认为是肿瘤血管新生的机制；然而，还没有一个因子被证实在临床上真正有用。本项目旨在从蛋白质组和系统生物学角度揭示肿瘤血管新生的过程和本质，以鉴别神经胶质瘤血管新生的生物标志物和有效抗血管生成治疗靶标。激光捕获显微切割（LCM）技术被用来分离不同级别神经胶质瘤新生血管，采用定量蛋白质组学和系统生物学方法研究不同级别神经胶质瘤新生血管的蛋白质组、硝基化蛋白质组、磷酸化蛋白质组，鉴定差异表达蛋白质（DEP）、差异硝基化蛋白质（DNTP）、差异磷酸化蛋白质（DPTP）及其蛋白质分子网络的改变。结果：（1）LCM成功分离了不同级别（II，III，IV）神经胶质瘤新生血管，但I级神经胶质瘤新生血管由于太少而未成功分离。（2）iTRAQ定量蛋白质组学方法分析不同级别神经胶质瘤新生血管与对照血管，共鉴定了837个差异表达蛋白质（DEPs），其中一些DEPs如成纤维细胞IV生长因子、转化生长因子、血管生成素、基质金属蛋白酶、基质金属蛋白酶抑制剂、整合素和凝血酶敏感蛋白等是血管生成调节因子或肿瘤调节因子，参与肿瘤血管生成的调节。（3）基于iTRAQ-TiO2富集的定量磷酸化蛋白质组学分析神经胶质瘤新生血管与对照血管，共鉴定了1828个磷酸化肽，708个磷酸化蛋白质。（4）基于双向凝胶电泳（2DGE）的硝基化酪氨酸免疫亲和偶联串联质谱在星形胶质细胞瘤鉴定了57个硝基化酪氨酸免疫阳性的2D胶点，18个硝基化蛋白质和20个硝基化位点。这些数据构成了目前最大的神经胶质瘤血管新生的蛋白质组数据库，为深入研究神经胶质瘤血管生成的分子机制、鉴定肿瘤血管生成的生物标志物打下了基础，为寻找有效治疗靶标以预防和治疗神经胶质瘤提供了科学依据。

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