胶质瘤干细胞对其微环境中的小胶质/巨噬细胞的改造作用及机制研究

It's known that transformation of tumor microenvironment by tumor cells is key to tumor prognosis. But the forms and mechanisms of transformation are unclear. In our bi-color fluorescence protein tracing glioma stem cells (GSCs) transplantation nude mouse model, we discovered that brain oligodendrocytes, abdominal macrophages and subcutaneous fibroblast underwent malignant transformation induced by GSCs. But the mechanism of host cells canceration remained to be answered。Our application designs that GSCs expressed RFP are transplanted into bone marrow-derived cells (BMDCs) expressed RFP nude mice to identify that the forms and mechanisms of transformation of microglia/macrophages in glioma microenvironment induced by GSCs。Base on that BMSC-GFP nude mouse model, in which only bone marrow-derived microglia/macrophages were GFP+, has been established in our preliminary experiments (see research foundation). And we had identified that abdominal macrophages were malignant transformed by fused with GSCs. We are sure of completing the study on transformation of microglia/macrophages in glioma microenvironment induced by glioma stem cells.

大多数学者都认为肿瘤细胞对其微生态环境的改造能力是决定肿瘤转归的关键所在,但这种能力的具体表现和如何发挥不很清楚。我们已利用自建的转基因RFP/GFP胶质瘤原位移植模型证实了胶质瘤干细胞(GSCs)能把微环境中的脑少突胶质细胞,腹腔巨噬细胞和皮下成纤维细胞改造成癌细胞,但具体机制不清,本申请继续用这个模型,探索GSCs改造位于脑组织中的骨髓源性小胶质巨噬细胞成癌细胞的过程及分子调控机制。基于在预实验中建立的BMDCs-GFP裸小鼠模型(见研究基础)已经解决了只示踪微环境中表达GFP的骨髓源性小胶质/巨噬细胞的特殊关键技术和GSCs通过细胞融合机制把巨噬细胞改造癌细胞的融合细胞已锁定,因此有把握通过本申请能完成胶质瘤干细胞对其微环境中的小胶质巨噬细胞的改造作用及机制研究。

背景.胶质瘤干细胞对骨髓间充质干细胞的改造作用尚不明确。本研究旨在明确胶质瘤干细胞诱导胶质瘤微环境中骨髓间充质干细胞发生恶性转化，通过基因芯片获得其恶性转化的差异基因表达谱，并明确其恶性转化的部分分子机制。.研究内容.1，将绿色荧光裸小鼠的骨髓移植到放射性骨髓毁损的非绿色荧光裸小鼠，并将标记红色荧光的胶质瘤干细胞原位移植到上述骨髓重建裸小鼠，建立RFP和GFP分别示踪胶质瘤干细胞和骨髓来源细胞的双色荧光模型。.2，从上述模型的原位移植瘤中单克隆恶性转化的骨髓间充质干细胞（imBMSCs）。.3，建立裸小鼠骨髓间充质干细胞、皮下荷瘤裸小鼠骨髓间充质干细胞以及imBMSCs的mRNA，miRNA和lncRNA表达谱，通过生物信息学分析imBMSCs的关键基因和通路。.4，敲低c-Met验证其是BMSCs恶性转化的关键基因，揭示imBMSCs恶性转化中c-Met调控的新机制。.结果及数据.1，裸小鼠经6Gy辐射达到骨髓清髓性毁损，骨髓移植后完全由GFP+骨髓细胞重建骨髓功能。通过荧光示踪发现骨髓来源细胞被胶质瘤干细胞招募到胶质瘤中参与肿瘤微环境构成。.2，从双色荧光模型中单克隆到imBMSCs表达BMSCs标记Sca-1和CD44，裸小鼠致瘤率100%，增殖活性、克隆形成能力以及侵袭性均高于胶质瘤干细胞。.3，imBMSCs差异表达基因主要与细胞粘附与迁移，免疫应答，血管生成和细胞增殖相关。Met、Csf1r、Pik3cg、Pik3r5、Cxcr4为关键基因。 MAPK、TGF-β、Jak-STAT通路在通路相互作用网络中处于中心位置。.4，敲低c-Met显著抑制imBMSCs恶性表型。实验结果确定LncRNA Ftx通过与 miR-342结合解除其对c-Met的抑制从而促进imBMSCs恶性表型的新机制。.科学意义.1，建立胶质瘤干细胞／骨髓源细胞双色荧光裸小鼠模型，证实胶质瘤干细胞招募骨髓源细胞参与肿瘤微环境构成。.2，证实胶质瘤干细胞诱导微环境中骨髓间充质干细胞恶性转化，且恶性程度高于胶质瘤干细胞。.3，筛选到imBMSCs的关键基因Met、Csf1r、Pik3cg、Pik3r5、Cxcr4和关键通路MAPK、TGF-beta、Jak-STAT。.4，证实LncRNA Ftx通过与 miR-342结合上调c-Met促进imBMSCs恶性表型的分子机制。