【摘要】 目的　将人的酪氨酸羟化酶(hTH)和胶质细胞源性神经营养因子(hGDNF)基因共同转染SH SY5Y细胞,筛选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 hTH和 hGDNF cDNA插入表达载体 pcDNA3 .0 和 pcDNA3 .1,构建重组真核表达载体 pcDNA3 1/hGDNF和 pcDNA3 0/hTH,然后转染至 SH SY5Y细胞系,利用RT PCR鉴定和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能神经元共培养,免疫细胞化学检测观察多巴脂能神经元的数目和生长状态。　结果　(1)双转基因工程细胞可防止多巴脂能神经元退变死亡,与对照组比较,神经元数目最多提高了 2 8 倍(P<0 .01);(2)工程细胞可抵抗N 甲基 苯基吡啶(MPP+)对多巴脂能神经元的毒性损伤作用,与对照组比较,细胞数目增加了83 .6%(为7 6倍,P<0. 01)。　结论　首次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,对多巴脂能神经元有防、治兼顾的保护作用。更多还原

【Abstract】 Objective To construct genetically engineered cells that secrete human TH and GDNF at the same time by stable co-transfection pcDNA3.0/hTH and pcDNA3.1/hGDNF in SH-SY5Y cells and study their effects on the gene therapy of Parkinson′s disease (PD). Methods pcDNA3.0/hTH and pcDNA3.1/hGDNF were constructed by inserting human TH and GDNF cDNA into pcDNA3.0 and pcDNA3.1, respectively. SH-SY5Y cells were transfected with pcDNA3.0/hTH and pcDNA3.1/hGDNF and the positive cell clones of human TH and GDNF cDNA engineered cells could be identified by RT-PCR. The engineered cells were co-cultured with primary dopaminergic neurons in mouse. The number and growth condition of primary dopaminergic neurons were examined by Immunocytochemistry. Results The number of primary dopaminergic neurons protected by double-gene engineered cells increased at least by 2.8 times in comparison with the control cells(P<0.01), and those cells against MPP+ toxicity increased by 83.6%(7.6 times, P<0.01). Conclusions The engineered cells showed highly effective and stable expression of both TH and GDNF at the same time. They may play protective roles by combining prevention and treatment of DA neurons from damage.更多还原