中文摘要：

目的建立一种新型的体外三维脑片共培养模型，观察绿色荧光蛋白转染的大鼠C6细胞在脑片模型上的侵袭、迁移形式，模拟胶质瘤细胞在脑内播散的方式。方法脂质体介导pEGFP-N3质粒转染体外常规培养的C6胶质瘤细胞，筛选稳定表达绿色荧光蛋白的细胞，荧光显微镜和流式细胞仪分别观察和计数绿色荧光蛋白的表达；活体取3周龄SD大鼠全脑，制作400μm厚脑片并培养于Millicell插入式细胞培养皿。碘化丙啶（PI）染色鉴定脑片神经元活性；将转染后的绿色荧光细胞C6细胞球接种于脑片表面，形成胶质瘤细胞一脑片共培养模型。荧光显微镜及激光共聚焦显微镜观察C6细胞在脑片上的侵袭和迁移。结果荧光显微镜及流式细胞仪检测转染后C6细胞几乎全部表达绿色荧光蛋白：脑片培养7d后仍然保持完整的皮质、尾状核、胼胝体等组织结构。PI染色证实脑片中神经元保持活性：荧光显微镜下观察显示C6细胞接种脑片表面1、3、5、7d后．迁移范围随时间的增加而逐渐增加；激光共聚焦逐层扫描可见C6细胞在脑片中的侵袭及三维分布，侵袭过程中C6细胞形态保持良好。结论大鼠脑片培养及肿瘤细胞一脑片共培养模型是研究胶质瘤细胞在体内的侵袭、迁移形式以及抗肿瘤治疗的潜在模型。

英文摘要：

Objective To establish an improved experimental 3-D brain slice co-culture model system, which allows us to observe the invasion and migration styles of C6 glioma cells transfected with enhanced green fluorescent protein （EGFP） and to simulate glioma cells disseminating in the brain. Methods C6 glioma cells routinely cultured in vitro were transfected with liposome-mediated pEGFP-N3 plasmid; cells stably expressed GFP were selected; then, fluorescence microscopy and flow cytometry were employed to observe and count the expression of GFP. Three-week-old SD rats were selected; rat whole cerebrum was cut into 400 ~m thick slices and cultured on a Millicell-CM membrane insert; the viability of neurons in the cultured brain slices was evaluated by propidium iodide （PI） staining. We implanted EGFP-expressing glioma cells on the surface of brain slices to form glioma cell-brain slice co-culture models; confocal laser scanning microscope and fluorescence microscope were used to observe the C6 cells migration and invasion styles within brain slice. Results C6 cells transfected with plasmid pEGFP-N3 expressed GFP were verified by fluorescence microscope and flow cytometry. The organotypic organizations, including the laminar structure of the cortex, caudate nucleus and corpus callosum in the brain slices, remained well preserved after 7 days of culture. PI staining conformed that the brain slices kept activity. Glioma cell migration area was gradually expanded 1, 3, 5 and 7 days after co-culture on brain slices-C6 cells co-culture models. Three-D distribution and invasion of glioma cells were observed by laser confocal microscopy serial sections. Cell morphology kept well in brain slice during invasion. Conclusion Rat brain slice culture and glioma cell- brain slice co-culture models are potential models to study the migration, invasion of glioma ceils and anti-invasive treatment analogous to normal brain conditions in vivo.