双色荧光蛋白示踪人脑胶质瘤鼠移植模型中肿瘤血管壁细胞的构建及机制研究

Neovascularization was considered to be an important biological phenomenon during tumor initiation and malignant progression. We have demonstrated that human glioma stem/progenitor cells contribute to neovascularization via transdifferentiation of endothelial-like cells in vivo and in vitro，but its mechanism is unclear. In this application, we intend to analysis the dynamic variation of tumor cell and endothelial cell in the transplanted tumor vessels based on human glioma xenografted mouse model that could trace tumor cells (red fluorescent) and host cells (green fluorescent)，to prove the TSPCs give rise to tumour endothelium during the process of constructing TSPCs niche under the regulation of tumor angiogenic molecule when multiplying progeny cells. This process is multielement in the niche, as TSPCs including tumor initiating cell and cancerated host cells，ECs containing differentiation of TSPCs and migration of host endothelial progenitor cells. TSPCs niche also include immue inflammatory cells，fibroblast etc. Cytokine released by those cells promot tumor vasculogenesis under molecular regulation. If the hypothesis could be proved，we will elucidate the heterogeneity of TSPCs niche and related molecular regulation by the tracer technique of fluorescence cell innovatively.

肿瘤血管生成是生物学的重要现象。我们已在体内外证明人脑胶质瘤干祖细胞通过向血管内皮样细胞转分化参与肿瘤血管形成，但机制未明。本申请拟利用我们建立的、具有肿瘤细胞(红色荧光)和宿主细胞(绿色荧光) 双示踪功能的可移植性人脑胶质瘤动物模型，动态分析移植瘤血管壁上肿瘤细胞与内皮细胞的组成变化，以证明肿瘤干祖细胞(TSPCs)繁衍子代细胞的同时，在肿瘤血管生成分子调控下，在构筑TSPCs niche(巢)的过程中生成新血管。该过程呈多元化，niche中的TSPCs包括肿瘤起始细胞和被诱导癌变的宿主细胞；内皮细胞包括由TSPCs转化而来，和由宿主循环内皮祖细胞(EPC)迁移而来；TSPCs niche成员还包括免疫炎性细胞、成纤维细胞等,其分泌的细胞因子接受分子调控，促进肿瘤血管生长。如假设得到证明，将在国际上首次以荧光示踪细胞技术证明TSPCs niche的异质性和相关的分子调控机制。

（1）在新建一批稳定表达红色荧光蛋白的SU3-RFP、C6-RFP、U87-RFP和SHG44-RFP等胶质瘤细胞系，和优化表达绿色荧光蛋白的免疫缺陷裸小鼠基础上,将双色荧光示踪的人脑或鼠脑胶质瘤原位或异位移植瘤模型肿瘤组织中肿瘤源性和宿主源性细胞成分清晰明辨,确切地分析了荧光蛋白示踪胶质瘤移植模型在包括肿瘤血管在内的肿瘤细胞学和分子生物学特征(2篇SCI收录论著已发表)。（2）在移植瘤模型中，首次应用1mm厚切片进行组织压片观察到在完整的肿瘤脉管系统中同时存在肿瘤细胞来源的红色，宿主细胞来源的绿色和两者发生融合成黄色的三类血管,并在体外共培养SU3-RFP和骨髓间充质细胞中发现两者是可以发生融合的，据此提岀了细胞融合是肿瘤血管生成和发生的重要途径，这对进一步研究融合细胞分化和转分化在肿瘤血管形成中的作用机制有重要意义。