【摘要】 目的建立稳定表达绿色荧光蛋白(EGFP)的C6胶质瘤细胞株。方法用含有CMV和EF1α两个启动子的慢病毒转染C6细胞,经流式细胞仪进行克隆筛选,荧光显微镜下观察EGFP阳性细胞,PCR及DNA测序鉴定基因组中EGFP的整合,并通过形态学检查、细胞增殖能力测定及致瘤性评价等进行了转染C6细胞的生物学特性分析。结果慢病毒转染率高达40%;通过流式细胞仪筛选,建立了一种稳定、长期表达EGFP的C6/EGFP细胞株;PCR及DNA测序证实EGFP基因已成功整合入C6细胞;生物学特性分析表明转染前后的C6细胞没有明显改变。结论以EGFP为生物标记物的C6/EGFP细胞株成功建立,可为将来研究脑胶质瘤提供理想的材料。更多还原

【Abstract】 Objective To establish a stable C6/EGFP glioma cell line. Methods The C6 glioma cell line was transfected with the lentivirus vector containing two enhancer-promoter CMV and EF1α. Enhanced green fluorescent protein-positive C6 cells were sorted out by fluorescence-activated cell sort. Expression of EGFP was observed by fluorescent microscopy. EGFP gene in C6 genome was assessed by PCR and DNA sequencing. Original and transfected cells were compared biologically and cytomorphologically. Results Lentivirus vector transfection produced up to 40% EGFP-positive cells. After fluorescence-activated cell sort selection, a pure cell line C6/EGFP was established. Polymerase chain reaction and DNA sequencing revealed integration of EGFP gene in C6 cell genome. Analysis of cell characteristics revealed no difference between transfected and original cells. Conclusions A C6/EGFP cell line expressing EGFP as a marker is established. This cell line can serve as a promising tool for further basic research and gene therapy studies.更多还原