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利用慢病毒载体构建GDNF基因修饰神经干细胞的实验研究
作者:李江  徐斌  张军  李智斌  谭龙旺 
单位:陕西中医药大学附属医院 脊柱病区, 陕西 咸阳 712000
关键词:基因修饰 病毒载体 慢病毒 神经干细胞 胶质细胞源性神经营养因子基因 脊髓损伤 
分类号:R394
出版年·卷·期(页码):2021·49·第十一期(1255-1261)
摘要:

目的:构建胶质细胞源性神经营养因子(GDNF)慢病毒载体,感染神经干细胞,使其能够过表达目的基因。方法:依据GenBank数据库中基因信息设计GDNF基因引物,聚合酶链反应(PCR)扩增GDNF基因片段。运用基因重组技术、质粒载体双酶切反应及重组产物转化将GDNF基因克隆至pLVX-mCMV-ZsGreen载体,经酶切、测序鉴定重组质粒。将成功构建的质粒转染293T和HEK293细胞,慢病毒包装并测定滴度。最后将包装好的病毒感染神经干细胞,进行Q-PCR检测。结果:(1)经酶切和测序鉴定,成功建立了pLVX-rGDNF-mCMV-ZsGreen的基因重组慢病毒载体;(2)包装的病毒感染293T及HEK293细胞细胞后,可见大量的阳性蛋白表达(绿色荧光蛋白);(3)包装好的慢病毒感染神经干细胞后,能够大量表达GDNF基因。结论:成功构建含有GDNF基因的慢病毒,且具有较强感染能力。神经干细胞经包装慢病毒感染后,能够大量表达GDNF基因。本研究为基因修饰神经干细胞治疗脊髓损伤提供了实验基础。

Objective:To construct lentiviral vector of glial cell-derived neurotrophic factor (GDNF),infect neural stem cells to overexpress the target gene. Methods:GDNFprimer was designed according to the gene information in the GenBank database, the target gene fragments were amplified. Using gene recombination, the plasmid vector double-enzyme digestion andthe recombinant plasmid, GDNF genewas cloned into pLVX-mCMV-ZsGreen vector.The double restriction endonuclease digestion and sequencing analysis confirmed the authenticity of the recombinant plasmids. The recombinant plasmids were transfected into 293Tand HEK293 cells, lentivirus particles were packaged and the titer was measured. Finally, packaged lentivirus particle infected neural stem cells for Q-PCR detection. Results:(1) pLVX-rGDNF-mCMV-ZsGreen recombinant lentiviral vector was successfully established, confirmed by enzyme digestion and sequencing. (2) After infection with 293T and HEK293 cells, a large number of positive protein(green fluorescent protein) were observed. (3)Neural stem cells infected by packaged lentivirus could express GDFNgene in large quantities. Conclusion: The lentivirus containing GDNFgene was successfully constructed with strong infection ability. Nerve stem cells can express GDNF target gene in large quantities after lentiviral vector infection. Our study provides a experimental basis for gene-modified neural stem cells in the treatment of spinal cord injury.

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