胶质瘤干细胞与微环境中骨髓间充质干细胞或巨噬细胞融合的鉴定及生物学特性分析  

Identification of fusion cells between glioma stem cells and bone marrow mesenchymal stem cells or macrophages in tumor microenvironment and their characteristics analysis

作　　者：王海洋[1] 陈俊杰[1] 王麒龙 王德林[1] 陈金生[1] 代兴亮[1] 王爱东[1] 黄强[1] 董军[1] 

机构地区：[1]苏州大学附属第二医院神经外科,苏州215004

出　　处：《中华神经医学杂志》2016年第3期217-223,共7页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81472739、81172400）;江苏省自然科学基金（BK20151214）

摘　　要：目的观察胶质瘤干细胞与其微环境中骨髓间充质干细胞（BMSCs）或巨噬细胞（Mφ）能否发生融合，并鉴定融合细胞的相关表型。方法将红色荧光蛋白（RFP）基因稳定转染的人胶质瘤干细胞株SU4-RFP和源于表达绿色荧光蛋白（EGFP）的Balb/c裸小鼠BMSCs和Mφ共培养，激光共聚焦显微镜下观察SU4-RFP与BMSCs、Mφ间的融合，分别命名为F-BMSCs、F-Mφ，单克隆高增殖力的RFP/EGFP双阳性细胞，用荧光标记的原位杂交技术（FISH）、Western blotting分别检测SU4-RFP、F-BMSCs、F-Mφ和BMSCs中RFP、EGFP基因和蛋白的表达；免疫细胞化学染色检测SU4-RFP、BMSCs、Mφ和F-BMSCs、F-Mφ分子标记物的表达；染色体核型分析检测SU4-RFP、BMSCs、F-BMSCs有丝分裂相的染色体形态；克隆形成实验、CCK-8法和Transwell实验分别检测F-BMSCs、F-Mφ、SU4-RFP的增殖能力和侵袭能力。结果激光扫描共聚焦显微镜下观察显示融合细胞表达红、绿2种荧光。FISH、Western blotting检测显示F-BMSCs、F-Mφ共表达RFP、EGFP 2种基因和蛋白。免疫细胞化学染色显示SU4-RFP细胞仅巢蛋白（Nestin）表达阳性。BMSCs仅CD105和CD44表达强阳性，Mφ细胞仅CD68表达阳性；F-BMSCs共表达Nestin和CD105、CD44，F-Mφ共表达Nestin和CD68。染色体核型分析显示F-BMSCs的染色体核型中小鼠的端着丝粒染色体占大多数，同时可见少量人的中着丝粒染色体。F-BMSCs、F-Mφ的克隆形成率高于SU4-RFP，差异有统计学意义（P〈0.05）。CCK-8实验显示F-BMSCs、F-Mφ的生长速度高于SU4-RFP；Transwell侵袭实验显示F-BMSCs、F-Mφ的穿膜细胞数（429.4±17.9、421.6±14.6）高于SU4-RFP（216.2±22.6），差异有统计学意义（P〈0.05）。 结论肿瘤干细胞可与宿主BMSCs或Mφ自发融合，且融合后细胞恶性程度更高，提示其可能为肿瘤恶性进展的机制之一。Objective To identify and analyze the fusion cells between glioma stem cells with bone marrow mesenchymal stem cells （BMSCs） or macrophages （Mφ） in vitro, and research the biological phenotypes of the fusion cells. Methods Red fluorescent protein （RFP） gene was stably transfected into human glioma stem cell line SU4. BMSCs and Mφ were harvested from Balb/c nude mice with enhanced green fluorescent protein （EGFP） expression. SU4 cells were co-cultured with BMSCs and Mφ, respectively, in the dual-color tracing platform. RFP/EGFP double positive cells with high proliferation ability, named F-BMSCs or F-Mφ, were monocloned. The gene and protein expressions of RFP and EGFP were detected by Western blotting and fluorescently-labeled in situ hybridization （FISH） in SU4-RFP, F-BMSCs, F-Mφ and BMSCs; immunocytochemistry was employed to detect the molecular markers. Chromosome karyotype was used to analyze the mitotic cell division phase; the biological characteristics of SU4-RFP, F-BMSCs, F-Mφ and BMSCs were further analyzed by colony formation, CCK8 and Transwel invasion assay. Results Two kinds of fluorescences expressed in the fusion cells observed by confocal microscopy. FISH and Western blotting showed that F-BMSCs, F-Mφ had gene and protein co-expressions of RFP and EGFP. Immunocytochemistry displayed nestin positive expression only in the SU4-RFP cells, CD105 and CD44 strongly positive expression only in the BMSCs, and CD68 positive expression only in the Mφ cells; co-expressions ofnestin, CD105 and CD44 were noted in the F-BMSCs; co-expressions ofnestin and CD68 were noted in the F-Mφ. Karyotype analysis showed that mice terminal centromere chromosomes were mainly the F-BMSCs karyotype, while a small number of people metacentric chromosomes could be noted. The clone formation rate of F-BMSCs and F-Mq0 was significantly higher than that of SU4-RFP （P〈0.05）. CCK8 assays showed that the growth rate of F-BMSCs and F-Mφ was significantly higher than that of SU4-RFP （P〈0.05）; Tr

关 键 词：胶质瘤干细胞 骨髓间充质干细胞 巨噬细胞 细胞融合 双色荧光示踪 

分 类 号：R739.4[医药卫生—肿瘤]