Robo4在胶质瘤血管新生中的作用及机制

Study about the mechanisms in glioma angiogenesis and the inhibition of angiogenesis bring new approach for the therapy of gliomas.Inhibition on it will provide a new approach of therapy for gliomas.Robo4 is a kind of the growth related conservative cross membrane protein, which is expressed on microvessel enthelial cells specifically. Nevertheless,the expression of robo4 in gliomas as well as the relationship between Robo4 and tumor angiogenesis has not been reported yet. Our preliminary studies have shown that robo4 is highly expressed in brain glioma, and the expression level of its ligand Slit2 was positively correlated with the pathological grade. Moreover, we have found that the co-culture with glioma cells promoted the expression of Robo4 on endothelial cells and antibody against Robo4 could inhibit the proliferation of endothelial cells effectively. Hence, based on domestic and international researches and our preliminary studies, we assume that Robo4 promotes the angiogenesis in gliomas by regulating the Src-Rac1 signaling pathway. In this study, we first aim to clarify the roles of Robo4 in the proliferation of endothelial cells and production of microwessels as well as the function and mechanism of downstream signaling pathways. Then we will perform in vivo studies to assess the role of Robo4 in glioma angiogenesis and the inhibitory effects of Robo4 antibody on glioma by reseaches of experimental animals. Finally we aim to explore the correlation between the expression level of Robo4 and the grade of glioma and prognosis of glioma patients. The main aim of this study is to clarify the relationship between Robo4 and gliomas and to provide the theoretical support for the application of anti-Robo4 in the genetic therapy for gliomas.

胶质瘤血管新生的机制研究以及抑制其血管生成是胶质瘤治疗的一个新途径。Robo4是一类与发育有关的保守跨膜蛋白，特异表达于微血管内皮细胞。Robo4在胶质瘤中的表达及其与血管新生的作用尚无报道。我们前期研究表明Robo4在脑胶质瘤中高表达，其配体Slit2表达水平与肿瘤的病理级别呈正相关。同时我们发现与胶质瘤细胞的共培养可促进内皮细胞表达Robo4，其抗体有效抑制内皮细胞的增殖。结合国内外研究和前期实验结果，我们推测Robo4通过调节Src-Rac1等信号通路，促进胶质瘤的血管新生。本研究拟首先在细胞水平明确Robo4在内皮细胞增殖及血管新生中的作用以及下游信号通路的作用机制；然后通过实验动物研究体内Robo4对胶质瘤血管新生的作用以及其抗体对胶质瘤的抑制效果；最后明确临床上Robo4与胶质瘤级别、预后的相关性，进一步明确其与胶质瘤的关系，为抗Robo4应用于胶质瘤的基因治疗提供理论依据。

血管新生在胶质瘤的发展中具有重要作用，抑制血管新生是其治疗的新途径。Robo4是一类与发育有关的保守跨膜蛋白，特异表达于微血管内皮细胞。在此之前Robo4在胶质瘤中的表达及其与血管新生的作用尚无报道。胶质瘤血管新生的机制研究以及抑制血管生成是胶质瘤治疗中的一个重要课题，可作为胶质瘤治疗的新途径。本研究拟明确Robo4基因其与胶质瘤的血管新生直接关系，为抗Robo4应用于胶质瘤的基因治疗提供实验理论依据。首先我们调查了Robo4在微血管内皮细胞表面的表达情况以及Robo4与胶质瘤血肿瘤屏障的关系。结果发现Robo4在正常脑组织中呈低水平表达，而在胶质瘤组织中高表达，随后发现Robo4表达于胶质瘤组织的微血管内皮细胞的细胞膜及胞浆中。同时与正常血脑屏障相比，在血肿瘤模型中内皮细胞的Robo4表达较高。我们发现抑制Robo4的表达能够显著抑制内皮细胞的紧密连接相关蛋白的表达，并且在这一过程中Src及Erk1/2通路发挥作用，证实Robo4在胶质瘤的发生发展中发挥作用。敲除Robo4表达后，人脑恶性胶质瘤血肿瘤屏障的通透性增加，而过表达Robo4后其通透性显著下降。证实该基因与胶质瘤血肿瘤屏障通透性相关。随后发现在胶质瘤细胞培养基的作用下，微血管内皮细胞Robo4的表达受到显著抑制。而Robo4的过表达能够明显抑制胶质瘤细胞诱导的微血管内皮细胞的体外增殖、迁移及微管形成。当与其配体Slit2结合后，Robo4能够对胶质瘤诱导的微血管生成产生影响。敲除Robo4的表达能够上调胶质瘤微血管内皮细胞的 VEGFR2、PI3K、AKT及FAK的磷酸化表达水平。