胶质母细胞瘤在脑白质内迁移形式及抗肿瘤细胞迁移靶向治疗研究

The characteristics of invasion and migration are the major cause for the poor prognosis of glioblastoma. Based upon our previous studies, we hypothesize that the chemotactic guidance would be required during the tumor cells migration along white matter fibers and their congregation to the microvascular niche in the white matter. The tumor cells in the routes to and aligning around microvascular niche would convert their phenotypes between proliferation and motility, thereafter migrate further in the white matter. In this study，we intend to study the invasion and migration patterns of malignant glioma cells in the white matter by serial collection of tissue samples along tumor migration routes and mirrow sections preparation as well. hMena, CD133,CD34,LFB and GM-130 will be labled to indicate tumor cells in motility, tumor stem cells, microvessels, white matter fibers and Golgi bodies respectively. Immnohistologic staining, western blot, and RT-PCR are employed to analyze the CXCL12/CXCR4 expression along the migration routes and arround the microvascular niche, while the phenotype conversion between proliferation and motility as well as the switch function of miR451 in the conversion are defined. The key experiments above will be repeated in the invasion model of rat brain slices in vitro to decompose the invasion mechanism hypothesized in this project. The effect of anti-migration targeted therapy for glioblastoma will be observed after administration of Rac1or (and) RhoA inhibition agents in the invasion models in vitro and orthotopic implanted model in rats. This research is expected to help us to understand invasion mechanisms of malignant glioma better and supply new clues for anti-invastion therapy for the tumor.

胶质母细胞瘤侵袭迁移的特性是其不良预后的主要原因，在前期研究基础上，我们提出：胶质瘤细胞沿着脑白质纤维束进行迁移及向微血管旁niche聚集需趋向信号引导，在微血管旁niche完成增殖-运动之间的表型转换和迁移接力。本项目拟对人及鼠脑胶质瘤沿迁移路径行系列取材及镜像切片，hMena、CD133、CD34、LFB、GM-130分别标记运动的肿瘤细胞、肿瘤干细胞、微血管、白质纤维、高尔基体，研究肿瘤细胞在白质中的迁移模式，并应用免疫组化、蛋白印迹杂交、RT-PCR技术，分析CXCL12/CXCR4在迁移路径中及微血管旁niche中的表达特点，判定迁移中增殖和运动细胞表型转换及miR451的开关作用；在鼠脑片模型中进行关键环节的体外分解研究。给予Rac1或(和)RhoA抑制剂，在体外及鼠脑胶质母细胞瘤模型中，观察抗细胞运动的靶向治疗效果。项目将为阐明恶性胶质瘤侵袭机理和抗侵袭靶向治疗提供新线索。

恶性胶质瘤侵袭迁移的生物学行为是造成其不良预后的主要原因。在本项目实施期限内，通过胶质母细胞瘤不同部位组织学标本、鼠脑原位肿瘤种植模型、脑片半器官培养、水凝胶及干细胞球三维模型、体外二维侵袭迁移模型等技术平台，开展胶质母细胞瘤在白质纤维束内侵袭迁移的规律、调控的关键信号分子、细胞侵袭运动能力的靶向抑制策略研究，完成了全部课题任务，并拓展了研究内容。研究成果主要为：（1）胶质母细胞瘤中侵袭迁移的细胞主要是胶质瘤干细胞，Rac1阳性的胶质瘤干细胞运动能力强，主要聚集在肿瘤坏死区及微血管周围，并沿着白质纤维束延伸方向分布；在体外细胞侵袭和迁移实验也证实，Rac1阳性的胶质瘤干细胞侵袭能力强。（2）Rac1 通过调节片状伪足的形成、黏着斑的周转和MMP-2、MMP-9表达参与调控细胞的迁移和侵袭。（3）标记染色高尔基体可以在体内及体外指示胶质瘤细胞的运动方向。（4）胶质母细胞瘤在白质纤维束内的迁移和侵袭是受CXCL12/CXCR4信号轴的指引，从血管旁的niche沿着白质纤维束向以远的血管旁的niche接替迁移。（5）miR-451通过结合不同的下游信号分子，在胶质母细胞瘤细胞增殖和运动表型转换中起到分子开关的作用。（6）Arg-cortatin-Arp2/3信号轴的表达受CXCL12/CXCR4的调控，Arg、cortatin、Arp2/3在细胞片状伪足的形成及侵袭运动中起重要作用，发现了他们潜在的关系及调节细胞运动的机制；（7）抑制Rac1或Arp2/3的表达能够显著抑制胶质母细胞瘤的侵袭和迁移。（8）本项目针对SOCE对胶质母细胞瘤侵袭迁移的调控机制进行了拓展研究。本项目培养14名研究生，其中9名硕士及1名博士已经毕业；发表论文19篇，其中SCI论文5篇，另2篇英文论文投稿中；获得专利1项；在国际及国内学术会议上宣讲及报告10余次。本研究进一步阐明了胶质母细胞瘤在白质纤维束中侵袭迁移的规律和调节机制以靶向抑制方案，为改善临床治疗提供了理论依据。