脑恶性胶质瘤微血管内皮和血管拟态形成机制及靶向治疗研究

Recently, more and more attentions were focused on the research of endothelial differention of human stem/progenitor cells. We obtained malignant glioma specimens which were screened by flow cytometry with the endothelial marker CD144 and stem cells marker CD133. In vitro, the screened stem cells were cultured to observe endothelial differention of human stem/progenitor in two- and three-dimensions under conditions of stem cells and endothelial cells medium and the hypoxia circumstance. In order to study the differential genes and its expression, We conducted the experiments by immunohistochemistry, flow cytometry, RT-PCR, Western Blot and gene chip, and demonstrated the relationship between human glioma stem/progenitor cells and microvascular endothelium, vasculogenic mimicry, and molecular mechanisms of endothelial differention of human glioma stem/progenitor cells. In order to study its targeted therapy, in vitro,we observed the formation of endothelial differention of human stem/progenitor cells which were transfected by oncolytic herpes simplex virus(o-HSV) carrying andostain-angiostain fusion gene and deleted ICP47 gene or differential expression of distinct gene/silencing gene---endothelial cells, vasculogenic mimicry and related gene expressions in two- and three-dimensions under conditions of stem cells and endothelial cells medium and the hypoxia circumstance. And in vivo, the glioma stem cells xenografts, which were established in the nude mice by glioma stem /progenitor cells, were transfected by oncolytic herpes simplex virus(o-HSV) carrying andostain-angiostain fusion gene and deleted ICP47 gene or differential expression of distinct gene/silencing gene, we observed endothelial cells, vasculogenic mimicry, related genes and their expressions by molecule technique, and the growth of tumor by MRI , to demonstrate the feature of the glioblastoma size and blood supply, and to observe whether in vivo the balance between the growth of the tumor and the blood supply of the tumor under the actions of the andostain-angiostain fusion gene and distinct differential gene/silencing gene. Consequently we provide a new idea for the engineering tumors stem cells in the treatment of the glioblastoma.

利用人脑恶性胶质瘤组织标本制备单细胞悬液，行胶质瘤干细胞原代培养, 采用肿瘤干细胞及血管相关标记物CD133、CD144行流式细胞筛选，体外诱导条件下进行二维及三维培养，免疫组化、RT-PCR、Western blot及基因芯片技术研究血管相关基因的表达及诱导分化前后基因表达谱的改变，阐明脑胶质瘤干细胞与微血管内皮细胞、血管生成拟态之间的关系及其发生的分子机制；利用删除ICP47片段的基因工程化溶瘤病毒（使差异表达基因过表达/表达沉默及人Endo-Angio融合基因修饰的），观察体内、外靶向治疗对胶质瘤干细胞的内皮细胞转化及血管和血管生成拟态形成的影响和对肿瘤生长的影响，揭示脑恶性胶质瘤的血液供应特征，并探讨基因修饰的溶瘤病毒工程化肿瘤干细胞模型中肿瘤血液供应与肿瘤生长能否达到平衡，从而其生长达到静息稳定状态，为病毒-基因工程化肿瘤干细胞靶向治疗胶质瘤提供新的思路。

胶质瘤干细胞（GSCs）向血管内皮细胞（ECs）跨分化过程是近年发现的一种胶质瘤血管形成机制，该机制中涉及的关键基因和信号通路的表达少有研究，本研究旨在探索胶质瘤血管跨分化形成过程中关键基因在其中的作用。本研究利用基因芯片筛选GSCs向ECs跨分化过程中差异基因的表达，通过基因信息学分析，确定脯氨酰羟化酶 4-alpha-1亚基基因（P4HA1）和脂肪酸合成酶（FASN）为关键候选基因，随后进行了二者在GSCs向ECs跨分化过程中的作用及机制以及靶向治疗研究。. 结果显示，P4HA1在人脑胶质瘤组织标本中存在过表达现象，其表达水平与肿瘤恶性级别、Ki67 表达水平和微血管密度（MVD）呈正相关；敲低 P4HA1基因表达可抑制GSCs细胞增殖活性、迁移能力和管腔形成能力；下调P4HA1表达能抑制颅内肿瘤生长速度、延长荷瘤鼠总生存时间，同时在颅内肿瘤跨分化形成的内皮细胞明显减少；敲低P4HA1表达能抑制胶原IV合成，从而破坏胶质瘤中血管基底膜结构完整性。此外,针对另一关键基因FASN的研究发现，在人脑胶质瘤标本中，FASN在各级别胶质瘤中亦存在过表达现象，并且其表达水平随肿瘤级别升高而增多，并与Ki67、MVD均成正相关； FASN水平下调可导致GSCs增殖活性、迁移能力、管腔形成能力明显下降；体内实验中，FASN表达抑制可致裸鼠颅内肿瘤生长速度明显下降，MVD数目明显减少，总生存时间（OS）明显延长。抑制 FASN 表达及其活性，可降低HIF-1α和VEGF-A的表达，促进血管生成抑制因子 VEGF165b 表达增多。. 本项研究首次阐述了P4HA1基因在胶质瘤跨分化血管形成机制和血管基底膜结构形成中具有重要作用，P4HA1基因可能成为胶质瘤抗血管治疗的潜在靶点；在进行FASN在胶质瘤血管生成过程中的作用研究中，观察到抑制FASN可以阻断GSCs中HIF-1α/VEGF-A信号通路，并上调血管生成抑制因子VEGF165b表达，因此，FASN有可能成为胶质瘤抗血管治疗的又一潜在靶点。通过本项目的研究，丰富了胶质瘤肿瘤血管形成理论及机制，并为临床抗血管治疗胶质瘤提供又一选择。