重组胶质细胞源性神经营养因子腺病毒保护小鼠中脑多巴胺能神经元

Recombinant adenovirus carrying glial cell line-derived neurotrophic factor gene protect midbrain dopaminergic neurons in mice

目的 :观察携带大鼠来源的胶质细胞源性神经营养因子 (rGDNF)的重组腺病毒在小鼠帕金森模型中有无保护中脑多巴胺 (DA)能神经元对抗神经毒素甲基 苯基 四氢吡啶 (MPTP)损伤的作用。方法 :将LacZ重组腺病毒 (Ad LacZ)通过脑立体定位仪注入小鼠单侧纹状体 ,分别于注射后 2 4h、72h、10d和 30d灌流取脑做X Gal染色 ,观察报告基因在小鼠脑内的表达情况 ;将rGDNF重组腺病毒 (Ad rGDNF)通过脑立体定位仪注入小鼠单侧纹状体。两者均于 72h后给予MPTP损伤中脑DA能神经元 ,1周后用高压液相色谱分析纹状体中DA、3,4 二羟基苯乙酸 (DOPAC)和高香草酸 (HVA)的含量。结果 :Ad LacZ注射后 2 4h表达较弱 ,72h逐渐增强 ,10d仍可见到较强表达 ,30d则逐渐减弱。LacZ基因的表达范围比较局限 ,胶质细胞和神经元都能被感染 ,并且未见明显的腺病毒载体引起的细胞毒性作用。注射Ad rGDNF侧纹状体DA含量较MPTP损伤组和MPTP +Ad LacZ组显著升高 ,而未注射侧无明显升高。此外 ,DA主要代谢产物DOPAC和HVA的含量也明显升高 ,而DOPAC/DA、HVA/DA比值则明显降低。结论 :Ad rGDNF能显著保护中脑DA能神经元对抗MPTP的损伤。

Objective: To observe the protective effects of recombinant adenovirus expressing glial cell line derived neurotrophic factor (Ad rGDNF) on the midbrain dopaminergic neurons against MPTP damage.Methods:Recombinant adenovirus carrying LacZ gene (Ad LacZ) was injected into the murine striatum using the stereotaxic apparatus. The murine brains were perfused and dissected, then stained with X Gal subsequently at 24 hours, 72 hours, 10 days and 30 days after injection. In another experiment, MPTP was injected subcutaneously into the mice 72 hours after the intrastriatal injection of Ad rGDNF (1×10 8 pfu·ml -1 ). The murine striatum was removed to evaluate the content of DA, DOPAC and HVA by HPLC ECD 1 week later. Results: X Gal staining indicated the expression of LacZ gene 24 hours after injection of Ad LacZ. Stronger expression was also detected at the end of 72 hours and 10 days, but decreased 30 days later. In addition, the LacZ gene expression was localized and detectable in both neurons and glial cells. There was no apparent cytotoxic effect induced by Ad LacZ alone. In MPTP treated mice, the content of dopamine decreased 80% compared to that in the saline treated ones. The DA content of MPTP+Ad rGDNF group on injected side was significantly higher than that of MPTP alone group and MPTP+Ad LacZ group. Changes in the content of DOPAC and HVA followed the same pattern. The ratio of DOPAC/DA and HVA/DA on injected side also decreased significantly. However, no significant change was observed on uninjected side. Conclusion: The results suggested that recombinant adenoviral vector expressing rGDNF can protect DA neurons against MPTP injury.