Notch/RBP-J靶向调节HSPG2在肿瘤血管正常化中的作用和机制研究

Tumor vascular targeting therapy mainly depends on the combination of tumor vessel normalization and chemotherapeutic drugs. However, drug resistance and other problems restrict the clinical application of vascular targeting drugs represented by bevacizumab. So it is urgent to explore the mechanism of tumor vascular structure formation and look for new target. It has been shown that Notch/RBP-J signaling plays an important role in vascular development, but the role and mechanism of Notch/RBP-J signaling in tumor vascular structure formation is not completely clear. We designed and synthesized vascular targeted Notch signal activator D1R independently in our previous research. Results showed that D1R activated Notch signaling of tumor vascular endothelial cells specificly and inhibited tumor growth by inhibiting tumor angiogenesis. D1R also promoted the normalization of tumor vessels. The differential expressed gene HSPG2 was selected by mRNA chip and 3 binding sites of transcription factors RBP-J was found in HSPG2 promoter region. This study proposed on the basis of previous work and we will use tumor-bearing mouse model and in vitro tube formation model to investigate the role of Notch signaling and HSPG2 in the formation of tumor vascular structure. Our research will clarity that Notch/RBP-J can directly regulate the expression of HSPG2 to influence the basement membrane formation, pericytes recruitment and cell junction formation, and promote the normalization of tumor vasculature. The research of this study has important theoretical and practical significance for understanding the mechanism of tumor vascular structure formation and finding new targets for tumor vascular targeting.

肿瘤血管靶向治疗主要依赖血管正常化联合化疗药物策略，但耐药等问题限制了以贝伐单抗为代表的血管靶向药物的临床应用，因此急需深入探索肿瘤血管结构形成机制及寻找新靶点。研究表明Notch/RBP-J信号在血管发育中发挥重要作用，但在血管结构形成中的作用和机制还不完全清楚。前期我们设计合成了血管靶向Notch信号激活剂D1R，其激活内皮细胞Notch信号后通过抑制血管生成抑制了肿瘤生长，且促进肿瘤血管正常化。芯片筛选出差异基因HSPG2，其启动子区有3个转录因子RBP-J结合位点。本课题拟利用荷瘤小鼠和体外管腔形成模型，研究Notch信号和HSPG2在肿瘤血管结构形成中的作用，阐明Notch/RBP-J通过直接调节HSPG2的表达实现对肿瘤血管基底膜、细胞连接形成和周细胞募集的调控以促进肿瘤血管正常化。本课题的研究对于深入了解肿瘤血管结构形成机制及寻找血管靶向治疗新靶点具有重要的理论和实际意义。

肿瘤血管结构的异常导致药物不能很好的到达肿瘤内部是肿瘤药物治疗效果不佳的主要原因之一。Notch信号是进化上高度保守的一条信号通路，在生理性血管发育及肿瘤血管生成中都起着非常重要的作用，然而其在血管结构形成和功能中的作用及机制目前还不是很清楚。本课题中我们利用遗传修饰小鼠、荷瘤小鼠等动物模型，综合应用病毒感染、免疫荧光染色、蛋白免疫印迹和基因表达谱芯片等技术，系统研究了Notch信号和HSPG2在肿瘤血管结构形成中的作用。我们的研究发现：Notch信号核心转录因子RBP-J的剔除减缓了LLC肿瘤的生长，促进了肿瘤血管生成，使肿瘤血管结构和功能恶化；同时RBP-J的剔除破坏了血脑屏障和血视网膜屏障，造成小鼠大脑和视网膜组织血液的渗漏。内皮细胞过表达Notch信号会导致肿瘤组织血管生成减少，缺氧加剧，继而导致肿瘤生成减缓，但是促进了肿瘤血管“正常化”；同时内皮细胞过表达Notch信号会导致新生小鼠视网膜血管对周细胞的募集，促进新生血管成熟。蛋白免疫印迹和免疫荧光染色结果显示：Notch信号上调可以通过促进黏附分子VE-Cadherin、beta-Catenin的表达促进内皮细胞黏附链接的形成；Notch信号的抑制可以通过抑制黏附分子VE-Cadherin、beta-Catenin的表达阻碍内皮细胞黏附链接的形成。表达谱芯片分析筛选出下游基因Hspg2，Notch信号的激活可上调内皮细胞中Hspg2的表达，Notch信号的抑制可下调内皮细胞中Hspg2的表达。Hspg2的干扰可以抑制hD1R蛋白激活Notch信号所导致黏附分子VE-Cadherin、beta-Catenin表达的上调，表明Notch信号调节血管结构和功能很可能通过调节Hspg2的表达实现的。本研究为深入理解Notch信号在血管结构形成中的作用和机制，以及在此基础上以Notch信号为靶点的血管靶向药物的研发与应用奠定理论基础。