TH和GDNF双转基因工程细胞对多巴胺能神经元的保护作用

目的将人的外源基因TH和GDNF共同转染SH-SY5Y细胞,建立一种可同时高效稳定表达TH和GDNF的工程细胞,探讨其在帕金森病(PD)基因治疗中的作用。方法将人GDNF和TH的cDNA构建于表达载体PcDNA3.0和PcDNA3.1,形成重组真核表达载体PcDNA3.1/hGDNF、和PcDNA3.0/hTH,同时转染至SH-SY5Y细胞系,利用RT-PCR鉴定筛选出高效稳定表达阳性克隆,将此工程细胞与大鼠原代多巴胺(DA)能神经元共培养,免疫细胞化学检测观察DA能神经元的数目和生长状态。结果成功建立了可同时高效稳定表达人TH和GDNF双基因的工程细胞,并且该细胞可防止DA能神经元退变死亡,有助于DA能神经元抵抗MPP+的毒性损伤。结论人TH和GDNF双转基因工程细胞对DA能神经原有防治兼顾的保护作用。、

Protective effect of human TH and GDNF cDNA-engineered SH-SY5Y cells on primary dopaminergic neurons

Objective To explore the possibility of stable co-transfection of PcDNA 3.0/h TH and PcDNA 3.1/h GDNF in SH-SY5Y cells and set up a kind of engineered cells that can secrete human TH and GDNF at the same time. Methods PcDNA 3.0/h Th and PcDNA 3.1/h GDNF were constructed using gene clone technique and recombined UNA technique. With the help of lipofectamine,SH-SY5Y cells were transfected with PcDNA 3.0/h TH and PcDNA 3.1/h GDNF and human TH and GDNF cDNA engineered cells could be obtained by RT-PCR and Immunocytochemisty. Rusults The engineered cells showed high effect and stable expression of both TH and GDNF at the same time.They can prevent DA neuron from undergoing degeneration and death induced by MPP+. Conclusion The present study first set up the engineered SH-SY5Y cells based on the strategy of combining prevention with therapy. They maybe play an important role in gene therapy of PD.