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目的探讨Neurturin(NTN)基因转染Vero细胞后的表达情况,为恒河猴帕金森病(PD)基因治疗打下基础。方法将表达载体pcDNA3-hNTN转染Vero细胞,挑选稳定表达克隆,用RT-PCR及免疫荧光分析鉴定,并对表达的人NTN蛋白进行了体外生物学活性测定。结果pcDNA3-hNTN质粒转染Vero细胞后获得了稳定表达克隆且有目的蛋白hNTN表达,表达的hNTN在体外能够促进培养的鸡胚背根神经节长出神经突起,并能促进体外培养的胚胎中脑多巴胺能神经元的存活。结论获得了能稳定表达NTN蛋白的Vero细胞株,其表达的hNTN对体外培养的中脑多巴胺能神经元具有保护作用,为移植并进行PD猴基因治疗动物实验打下了基础。
Objective To investigate the expression of Neurturin (NTN) gene in Vero cells and lay a foundation for the gene therapy of Parkinson disease (PD) in rhesus monkeys. Methods The expression vector pcDNA3-hNTN was transfected into Vero cells. Stably expressing clones were selected and identified by RT-PCR and immunofluorescence analysis. The expressed human NTN protein was assayed for biological activity in vitro. Results The pcDNA3-hNTN plasmid was successfully transfected into Vero cells and expressed hNTN protein. The expressed hNTN could promote neurite outgrowth in vitro and in vitro of embryos The survival of brain dopaminergic neurons. Conclusion The Vero cell line stably expressing NTN protein was obtained. The expressed hNTN could protect mesencephalic dopaminergic neurons in vitro and lay a foundation for transplantation and gene therapy animal experiments.