中文摘要：

目的构建携带增强绿色荧光蛋白（ enhanced green fluorescent protein, EGFP）和胶质细胞源性神经营养因子（glial cell line-derived neurotrophic factor, GDNF）基因的慢病毒载体，转染原代培养的神经干细胞（neural stem cell，NSC），观察能否获得持续的GDNF表达。方法采用聚合酶链反应扩增，限制性内切酶酶切，T4DNA连接酶连接，将GDNF插入慢病毒主体载体pGC-FU，构建重组慢病毒载体（1enti-CMV-GDNF-EGFP）。将构建成功的慢病毒三质粒系统通过Lipofectamine 2000转染于人胚肾细胞系（293T），测定病毒滴度。感染NSC，荧光显微镜下观察EGFP的表达。流式细胞仪检测转染效率，Western blot检测GDNF的表达，酶联免疫吸附测定法检测NSC转染后释放GDNF的量。结果构建的慢病毒载体能高效感染NSC，转染效率达77．7％，3个月后仍有73．7％，Western blot的结果表明GDNF成功在细胞内表达，而酶联免疫吸附测定法表明转染后细胞外分泌GDNF的量随着时间的推移增多，与对照组比较差异有统计学意义（P〈0.05）。结论构建携带EGFP和GDNF基因的慢病毒载体，并感染NSC，能使之持续表达GDNF。（中国眼耳鼻喉科杂志，2008，8：283-285）

英文摘要：

Objective To construct the lentiviral vector containing enhanced green fluorescent protein （EGFP） and glial cell line-derived neurotrophic factor （GDNF） and to transfect the rat neural stem cells （NSC） for a sustained expression of GDNF. Methods Polymerase chain reaction amplification, restricted endonuelease digestion and TdDNA ligase connections were used to construct recombinant lentiviral vector （lenti-CMV-GDNF-EGFP） by inserting GDNF into the main virus vector pGC-FU. Lentivirus three plasmid system was transfeeted into human embryonic kidney cell line-293 T through Lipofectamine 2000, then the virus titer was examined. The NSC was transfected with viral production and the expression of EGFP was observed under fluorescent microscope. The transfection efficiency was examined by flow eytometry. The expression of GDNF was examined by Western blot. The assay of GDNF after transfection was examined by enzyme linked immunosorbent assay （ELISA） method. Results The constructed lentiviral vector （lenti-CMV-GDNF-EGFP） could infect NSC and the transfection efficiency amounted to 77.7% , with the efficiency being 73.7% three months later. Western blot showed the GDNF expression in the cells. ELISA test showed that the extracellular secretion of GDNF was significantly higher than the control group （ P 〈 0.05）. Conclusions Construction of EGFP and GDNF gene lentiviral vector can transfect NSC leading to a sustained expression of GDNF. （Chin J Ophthalmol and Otorhinolaryngol, 2008,8：283-285 ）