绿色荧光蛋白转染胶质瘤细胞的体内实验研究

In Vivo Experiment on Brain Glioma Cells Transfected with Green Fluorescent Protein

目的 利用体外转染绿色荧光蛋白 (Enhanced Green Fluorescent Protein,EGFP)基因的胶质瘤细胞建立大鼠移植模型 ,并探索利用该模型研究肿瘤体内侵袭的可行性。方法 携有EGFP基因的 p EGFP- N3质粒体外转染 C6胶质瘤细胞 ,筛选稳定表达 GFP的细胞克隆并以立体定向法植入 SD大鼠脑实质内建立肿瘤移植模型。 4周后处死大鼠并制作连续石蜡切片 ,相邻切片分别作 HE、免疫组化染色和荧光显微镜检测。结果 GFP于体内稳定表达 ,在荧光显微镜下可较容易区分肿瘤细胞和非肿瘤细胞 ,并能检测到侵袭至远端的单个肿瘤细胞 ,其检测效率明显高于HE和免疫组化染色。结论 EGFP基因体外转染 C6胶质瘤细胞并建立大鼠脑内肿瘤移植模型 ,是研究胶质瘤体内侵袭的有效模型和方法。

Objective To determine whether fluorescence from C6 glioma cells transfected with the enhanced green fluorescent protein gene in vitro and xenotransplanted into the brain of SD rats would permit the detection of brain tumor invasion in vivo. Methods C6 glioma cells were transfected with a plasmid vector (pEGFP\|N3) containing the EGFP gene. Stable EGFP\|expressing clone were isolated and stereotactically injected into the parenchyma of rats. Four weeks later,rats were killed and continuous brain sections were examined using fluorescence microscopy after adjacent sections were performed by immunohistochemistry or routine HE staining for detection of tumor cell invasion. Results We demonstrate that EGFP transfected C6 glioma cells maintain stable high level EGFP expression in the central nervous system during their growth in vivo. EGFP fluorescence clearly demarcated the primary tumor margins and readily allowed for the visualization of distant micrometastasis and local invasion on the single cell level. Small locally invasive foci, including those immediately adjacent to the leading invasive edge of the tumor, were virtually undetectable by routine HE and immunohistochemistry staining. Conclusion We showed that EGFP\|transfected C6 glioma cells can be visualized by fluorescence microscopy after intracranial implantation. This method is superior to routine HE staining and immunohistochemistry for the detection and study of relevant patterns of brain tumor invasion in vivo.