Objective: To investigate the tumor microenvironment of immune tolerance induced by glioma stem cells (GSC). Methods: Human GSC SU3 cells transfected with red fluorescent protein (SU3-RFP) gene were implanted into the brain, subcutis (armpit and foot), liver and abdominal cavity of transgenic green fluorescence protein (GFP) nude mice to establish RFP(+) /GFP(+) dual fluorescence solid tumor model. The re-cultured cells derived from implanted tumor tissues, SU3-RFP cells co-cultured with peritoneal fluid of transgenic GFP nude mice and malignant ascites of tumor-bearing mice were observed by fluorescence microscopy and real-time video image tracing to analyze the microenvironment of immune tolerance mediated by RFP(+) /GFP(+) implanted tumor. Results: Dual fluorescence labeled frozen section showed that all of cells in the tumor microenvironment were GFP(+) , while the pressed tissue-patch showed that the tumor blood vessels exhibited a RFP(+) /GFP(+) double-positioning yellow. In the GFP single fluorescence labeled tumor tissue, all of cells in the microenvironment were green, including tumor edge, necrotic foci and blood vessel. Among them, CD68(+) , F4/80(+) , CD11c(+) , CD11b(+) and CD80(+) cells were observed. In the dual fluorescence labeled co-cultured cells, the phagocytosis and fusion between green host cells and red tumor cells were also observed, and these fusion cells might transfer to the malignant dendritic cells and macrophages. Conclusions: The tumor microenvironment of immune tolerance induced by GSC is not affected by the tissue types of tumor-inoculated sites, and the immune tolerance mediated by inflammatory cells is associated with the inducible malignant transformation, which may be driven by cell fusion.
目的： 探讨胶质瘤干细胞诱发肿瘤免疫耐受微环境的细胞情况。 方法： 将转染红色荧光蛋白(RFP)基因的SU3细胞(SU3－RFP)接种于转染绿色荧光蛋白(GFP)的裸小鼠脑内、皮下(腋下和足掌)、肝脏和腹腔等部位，建立RFP(＋)/GFP(＋)实体瘤模型。对移植瘤组织再培养细胞、SU3－RFP细胞与表达GFP的裸小鼠腹腔冲洗液共培养细胞、荷瘤鼠恶性腹水等进行荧光显微镜和实时摄影跟踪观察，分析RFP(＋)/GFP(＋)移植瘤免疫耐受微环境情况。 结果： 双色荧光示踪的冰冻切片显示，肿瘤微环境中的细胞均为GFP(＋)细胞，而在组织压片上，肿瘤血管显示RFP(＋)/GFP(＋)的黄色共定位细胞。在GFP单色荧光示踪移植瘤组织中，肿瘤微环境中的细胞为绿色，可见于肿瘤组织边缘、中间和肿瘤血管中，并有CD68(＋)、F4/80(＋)、CD11c(＋)、CD11b(＋)和CD80(＋)细胞存在。在双色荧光示踪的共培养细胞中，观察到绿色宿主细胞和红色肿瘤细胞之间发生的吞噬和融合，且融合细胞可转化为恶性的树突状细胞和巨噬细胞。 结论： 胶质瘤干细胞诱导的肿瘤免疫耐受微环境不受肿瘤寄生地固有组织细胞类型的限制，免疫炎性细胞的免疫耐受与其被诱导恶性转化有关，而这种恶性转化可能受到细胞融合的驱动。.
Keywords: Glioma; Green fluorescent protein; Immune tolerance; Red fluorescent protein; Stem cell; Tumor microenvironment.