【摘要】 目的研究重组腺相关病毒载体介导的增强型绿色荧光蛋白基因在永生化大鼠神经前体细胞的转染效率及其表达情况.方法携带增强型绿色荧光蛋白基因的重组腺相关病毒转染永生化大鼠神经前体细胞,持续观察转基因神经前体细胞绿色荧光蛋白的表达情况,并计算转染效率.应用巢蛋白抗体和抗猿肾病毒40大T抗原抗体进行细胞鉴定,50mL/L胎牛血清诱导细胞分化后,应用神经元和星形胶质细胞特异性标志物抗体检测其分化能力.结果荧光显微镜观察显示转染后24h神经前体细胞即有绿色荧光蛋白表达,转染后72h,大部分细胞表达绿色荧光蛋白,转染效率可达90%.经传代培养8wk后,表达绿色荧光蛋白的阳性细胞仍可高达70%~75%.免疫细胞化学结果显示转绿色荧光蛋白神经前体细胞巢蛋白和抗猿肾病毒40大T抗原抗体染色均为阳性,50mL/L胎牛血清可诱导其分化为神经元和星形胶质细胞.结论携带增强型绿色荧光蛋白基因的重组腺相关病毒可高效转染永生化大鼠神经前体细胞,并可在细胞中长期稳定地表达,绿色荧光蛋白作为良好的示踪剂可应用于神经前体细胞的体内移植.更多还原

【Abstract】 AIM: To study the transfection efficiency and the expression of enhanced green fluorescent protein(eGFP) in immortalized neural progenitor cells(INPCs) mediated by recombinant adeno-associated virus(rAAV). METHODS: The viral vector of rAAV containing eGFP was transfected into INPC. The expression of green fluorescent protein was observed since 24 h after transfection and the transfection efficiency was measured. Anti-nestin antibodies and simian virus 40 large T antigen gene (SV40Tag) antibodies were used to identify the transfected cells. The specific molecular markers of neurons and astrocytes were detected using immunocytochemistry method to investigate the capability of differentiation of the transfected cells after being induced by 50 mL/L fetal bovine serum. RESULTS: EGFP expression was detected as early as 24 h after transfection. The number of eGFP positive cells reached about 90% after 72 h. After being cultured for 8 weeks, 70%-75% INPCs were still eGFP positive. The transfected cells were confirmed as nestin positive and SV40Tag positive cells with immunocytochemistry and could be differentiated into neurons and astrocytes by the induction of fetal bovine serum. CONCLUSION: The viral vector of rAAV containing eGFP can be highly transfected into INPCs and the transfected cells show a stable and long-term eGFP expression in vitro. EGFP can be a valuable tracking tool for the study of neural progenitor cell transplantation.更多还原