Wang Shuyan, Ren Ping, Xie Shu, Zhu Wanwan, Wang Yang, Guan Yunqian, Zhang Yu
Cell Therapy Center, Xuanwu Hospital, Capital Medical University, Beijing 100053, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Dec;24(12):2061-7.
We transfected human neural stem cells using lentiviral vectors encoding glial cell line derived neurotrophic factor (GDNF) to study its expression level in vitro and to get a stable cell line expressing GDNF. First, GDNF gene was sub-cloned into the lentiviral transfer vectors. Then, the recombinant lentiviral supernatants were packaged by 293T cells through three plasmids transient co-transfection method using standard lipofectamine reagent. The viral titers were tested by the transfection efficiency of 293T cells. At the same time, human neural stem cells (hNSC) were transfected under different multiplicity of infection. GDNF gene expression level and protein secretion level of hNSC were tested by real-time PCR and ELISA methods after transfection. Lentiviral vectors encoding GDNF were constructed. Using lentiviral vectors encoding GDNF we successfully transfected human neural stem cells, and got a stable neural stem cell lines over-expressing GDNF. Furthermore, the results indicated that GDNF expression was influenced by the multiplicity of infection. Human neural stem cells could over-express GDNF through lentivial vectors tranfection. Its gene expression level and protein expression level correlate with the multiplicity of infection.
我们使用编码胶质细胞源性神经营养因子（GDNF）的慢病毒载体转染人神经干细胞，以研究其在体外的表达水平，并获得稳定表达GDNF的细胞系。首先，将GDNF基因亚克隆到慢病毒转移载体中。然后，通过标准脂质体转染试剂，采用三质粒瞬时共转染方法，由293T细胞包装重组慢病毒上清液。通过293T细胞的转染效率检测病毒滴度。同时，在不同感染复数下转染人神经干细胞（hNSC）。转染后，通过实时PCR和ELISA方法检测hNSC的GDNF基因表达水平和蛋白分泌水平。构建了编码GDNF的慢病毒载体。利用编码GDNF的慢病毒载体，我们成功转染了人神经干细胞，并获得了稳定过表达GDNF的神经干细胞系。此外，结果表明GDNF的表达受感染复数的影响。人神经干细胞可通过慢病毒载体转染而过表达GDNF。其基因表达水平和蛋白表达水平与感染复数相关。
