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目的:构建大鼠胶质细胞源性神经营养因子基因的重组腺病毒并观察其在神经干细胞上的表达,探索胶质细胞源性神经营养因子作为基因治疗神经病变的价值。方法:实验于2004-8/12在四川大学华西医院眼疾病分子遗传实验室进行。以新生SD大鼠大脑皮层细胞RNA为模板,反转录聚合酶链反应扩增出全长的胶质细胞源性神经营养因子基因,经测序验证后,亚克隆至穿梭质粒pAdTrackCMV中,与骨架质粒pAdEasy1同源重组为腺病毒载体。线形化后转染293细胞进行包装扩增,利用AdEasy1系统上的绿色荧光蛋白鉴定病毒表达,并用病毒上清转化大鼠神经干细胞,检测胶质细胞源性神经营养因子基因的表达。结果:①胶质细胞源性神经营养因子基因扩增后进行测序显示扩增片段为653bp个核苷酸,同源性比较该序列正确。②重组腺病毒载体经酶切验证为阳性克隆,转染293细胞包装后的病毒滴度为1x109PFU/mL。③转入胶质细胞源性神经营养因子的重组腺病毒的神经干细胞扩增出653bp的带。结论:成功构建了胶质细胞源性神经营养因子重组腺病毒并见其在大鼠神经干细胞中进行了表达。
OBJECTIVE: To construct a recombinant adenovirus expressing rat glial cell line-derived neurotrophic factor gene and observe its expression on neural stem cells and explore the value of glial cell line-derived neurotrophic factor as a gene therapy for neuropathy. Methods: The experiment was performed at the Molecular Diseases Laboratory of West China Hospital, Sichuan University from August to August 2004. Full-length glial cell line-derived neurotrophic factor gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using RNA from cerebral cortex of neonatal SD rats. After sequencing, the gene was subcloned into the shuttle plasmid pAdTrackCMV, Plasmid pAdEasy1 is homologously recombined into adenoviral vector. After linearization, the 293 cells were transfected into 293 cells for amplification. The green fluorescent protein (AdEasy1) was used to identify the expression of the virus. The neural stem cells were transfected with the virus supernatant to detect the expression of glial derived neurotrophic factor gene. Results: ① The glial cell line-derived neurotrophic factor gene was amplified and sequenced. The result showed that the amplified fragment was 653 bp in length. The sequence was correct by homology analysis. ② The recombinant adenovirus vector was confirmed by enzyme digestion as a positive clone. The virus titer after transfection with 293 cells was 1x109 PFU / mL. ③ into the glial cell line derived neurotrophic factor recombinant adenovirus neural stem cells amplified 653bp band. Conclusion: The glial cell line-derived neurotrophic factor recombinant adenovirus was successfully constructed and expressed in rat neural stem cells.