利用慢病毒载体构建GDNF基因修饰神经干细胞的实验研究

Experimental study on construction of GDNF gene modified neural stem cells using lentiviral vectors

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:GDNF primer 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 and the recombinant plasmid,GDNF gene was 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 293 T and 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 293 T and HEK293 cells,a large number of positive protein(green fluorescent protein)were observed.(3)Neural stem cells infected by packaged lentivirus could express GDFN gene in large quantities.Conclusion:The lentivirus containing GDNF gene 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.