胶质细胞源性神经营养因子（GDNF）在肝癌新生血管生成中的作用机制研究

Communication between tumor cells and vascular endothelial cells is known to be important in tumor-induced angiogenesis. Glial cell line-derived neurotrophic factor (GDNF) is an important neurotrophic polypeptide. In previous studies, we found that GDNF was highly expressed in the tumor tissues of hepatocellular carcinoma (HCC), and GDNF could stimulate or enhance the angiogenesis in vivo in a VEGF-independent manner, which could be blocked by GDNF monoclonal antibody. According to the related literatures and our prestent results, we supposed that, GDNF over-expressed by tumor cells could be secreted into the microenvironment of tumors, which would promote the tumor-induced angiogenesis. In this study, we will further explore the relationship between the expression of GDNF in HCC tissues and the tumorous microvessel dinsity (MVD), determine the dominant GDNF receptors on the surface of vascular endothelial cells that mediate GDNF's effect on angiogenesis and their densities, investigate the relationship between this kind of receptor pathway and the other angiogenesis-promoting pathway such as VEGF/VEGFR, Slit2/Robo1, and evaluate the abilities of GDNF to increase the proliferation and migration of vascular endothelial cells, so as to elucidate the mechanisms by which GDNF induce angiogenesis. Besides, the HCC xenograft mice models will be used to explore whether GDNF could enhance the tumorous angiogenesis and accelerate the proliferation of tumors, and whether these effects could be blocked by GDNF monoclonal antibody, and/or its receptor-inhibitors. Finally，we would like to clarify that GDNF and its receptor pathway is a brand new approach to facilitate the tumorous angiogenesis, and therefore to supply the therapeutic targets for the development of novel tumor angiogenesis inhibitors.

肿瘤细胞和血管内皮细胞的交流在肿瘤诱导的血管生成中起重要作用。胶质细胞源性神经营养因子（GDNF）是重要的促神经生长多肽，前期已发现，它在肝癌中高表达，可促进体外新生血管生成，此作用不依赖于VEGF,可被GDNF单克隆抗体抑制。结合相关文献，我们提出假说：肿瘤过表达的GDNF分泌到微环境中，可对新生血管生成起促进作用。本项目拟检测肝癌表达GDNF与肿瘤微血管密度的相关性，研究血管内皮细胞上介导GDNF促血管生成主要受体及密度、此受体途径与VEGF/VEGFR、Slit2／Robo1通路的关联，以及GDNF促进血管内皮细胞增殖、迁移的能力，以阐明GDNF促血管生成机制。在肝癌移植瘤模型中，验证GDNF是否可促进新生血管生成及肿瘤增殖，使用其单抗及受体抑制剂，是否可阻断此作用。通过该研究，拟最终明确GDNF及其受体通路是促进肿瘤新生血管生成的全新途径，为研制新的抗肿瘤新生血管生成药物提供靶标。

胶质细胞系源性神经营养因子(glial cell line-derived neurotrophic factor，GDNF)是转化生长因子β超家族的一员，具有多种重要的生物学功能。随着对GDNF研究的不断进展，发现其不仅在神经元的存活、生长、分化、神经再生、突触形成与突触可塑性等发面发挥重要作用，而且其与神经退行性疾病的发生、药物成瘾以及肿瘤的生长、迁移和化疗耐药也密切相关，GDNF逐渐显示出其功能多样性及治疗肿瘤的一定潜力。.本项目通过培养脂肪干细胞（ASC）并收集其培养上清（ASC-CM）作用于人脐静脉内皮细胞（HUVEC），观察ASC-CM对HUVEC生物学行为的影响，发现ASC-CM可以显著地刺激HUVEC成管，并且这种作用不依赖于已知的血管内皮生长因子（VEGF）。通过ELISA方法检测ASC-CM中多种细胞因子的浓度，发现ASC-CM中有相当数量的GDNF存在，在ASC-CM中加入足量的VEGF中和抗体和GDNF的中和抗体后发现ASC-CM促进HUVEC成管的现象显著下降，从而发现GDNF可以促进HUVEC的成管并且这种作用不依赖于血管内皮生长因子（VEGF）。同时发现外源性给予HUVEC人重组GDNF可以刺激HUVEC成管、促进HUVEC增殖、迁移，并且这种作用可以被RET的特异性抑制剂RPI-1阻断，且GDNF刺激HUVEC后，细胞内RET/p-RET、AKT/p-AKT以及STAT3/p-STAT3的表达升高，转录因子E2F1的表达明显升高，GDNF显示出营养HUVEC细胞的作用。在此基础上，通过构建稳定敲低GDNF的HUVEC细胞系，发现与对照组相比，HUVEC的增殖、迁移及成管能力显著降低，外源性给予人重组GDNF后可以逆转此现象。此外，本项目通过免疫组化发现肝癌组织相较癌旁组织，GDNF表达量明显升高，表明GDNF在肝癌的发生发展和新生血管生成中发挥了重要作用。.综上，本项目现已按照计划完成研究内容并取得重要进展，发表SCI论文4篇。该项目所提供材料详实，数据真实可靠，研究成果有助于解析肝癌发生发展和靶向治疗耐药的分子机制，有望为抑制肝癌血管生成和分子治疗提供新的靶点和理论依据。