人酪氨酸羟化酶和胶质细胞源性神经营养因子双转基因工程细胞对MN9D细胞内多巴胺含量的影响

Effect of human TH and GDNF cDNA-engineered SH-SY5Y cells on dopamine levels in MN9D cells

目的建立一种可同时高效稳定表达酪氨酸羟化酶(TH)和胶质细胞源性神经营养因子(GDNF)的双基因工程细胞,探讨其在帕金森病(PD)基因治疗中的作用。方法应用分子克隆技术构建真核表达载体PcDNA3.1/hGDNF和PcDNA3.0/hTH,并同时转染至SHSY5Y细胞系,利用半定量逆转录聚合酶链反应(RTPCR)鉴定筛选出的阳性细胞克隆TH和GDNF的表达水平,将双转工程细胞与MN9D细胞共培养,高效液相色谱分析工程细胞对MN9D细胞生长状态及细胞内单胺类递质含量的影响。结果双转基因工程细胞可防止MN9D细胞退变死亡,与对照组相比较,DA、HVA和DOPAC提高了1.9、2.1和1.7倍(P<0.01)。双转基因工程细胞可对抗MPP+对MN9D的毒性损伤作用,与对照组相比较,DA、HVA和DOPAC提高了3.3、1.7和2.0倍(P<0.01),而且其效果明显优于TH单基因工程细胞。结论成功构建可分泌人TH和GDNF的双转基因工程细胞,其对多巴胺能神经元具有明显的保护作用。

Objective To construct a kind of engineered cells that can secrete human TH and GDNF at the same time,and study its possible effect on gene therapy of Parkinson's disease.Methods PcDNA^3.0/ hTH and PcDNA^3.1/ hGDNF were constructed using gene clone technique and recombined DNA technique.With the help of lipofectamine,SH-SY5Y cells were transfected with PcDNA^3.0/ hTH and PcDNA^3.1/ hGDNF and positive clones of human TH and GDNF cDNA engineered cells could be determined by RT-PCR.The levels of dopamine in MN9D cells co-cultured with engineered cells with or without MPP+ were determined by HPLC.Results The levels of DA,HVA and DOPAC in MN9D cells protected by double gene-engineered cells were increased 1.9,2.1 and 1.7 times respectively in comparision with the control cells (P<0.01).The levels of DA,HVA and DOPAC in MN9D cells protected by double gene-engineered cells against MPP+ were increased 3.3,1.7 and 12.0 times respectively ^(P< ^0.01). The effects were much better than TH engineered cells.Conclusion The present study first set up the engineered SH-SY5Y cells secreting human TH and GDNF based on the strategy of combining prevention with therapy.They obviously protect dopaminergic neurons against degeneration and maybe play an important role in gene therapy of PD.