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目的探讨优化人α-突触核蛋白(humanα-synuclein,hα-syn)核酸疫苗(pVAX1-IL4/SYN-B)预防接种对慢性鱼藤酮损害小鼠的神经保护作用。方法将小鼠随机分为3组:核酸疫苗组、空质粒组、PBS对照组,在小鼠双侧后肢胫前肌部位分别注射pVAX1-IL4/SYN-B核酸疫苗,空质粒pVAX1,PBS各100μl,共免疫3次。末次注射3周后全部小鼠背部皮下注射鱼藤酮1 mg/kg,1次/d,连续注射40 d。最后一次注射后,观察小鼠行为学变化;免疫组化方法检测小鼠中脑黑质致密部α-syn表达和酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性细胞数目变化;RT-PCR检测α-syn mRNA表达情况。结果行为学观察发现,各组小鼠自由活动实验显示核酸疫苗组,空质粒组,PBS对照组小鼠移动格子数及下肢站立次数明显改善,并有显著差异(P<0.05)。免疫组化发现核酸疫苗组小鼠与空质粒组、PBS对照组相比,TH阳性细胞数增加63.27%、55.34%(P<0.05),α-syn表达减少55.68%,55.34%(P<0.05)。RT-PCR发现核酸疫苗组α-syn mRNA相对表达水平0.38±0.03较空质粒组0.77±0.05及PBS对照组0.73±0.01明显减少(P<0.05)。结论 pVAX1-IL4/SYN-B核酸疫苗能有效防止鱼藤酮神经毒素对黑质多巴胺能神经元的损害,对多巴胺能神经细胞具有保护作用。
Objective To investigate the neuroprotective effect of optimized human α-synuclein (hα-syn) nucleic acid vaccine (pVAX1-IL4 / SYN-B) on chronic rotenone-induced mice. Methods The mice were randomly divided into three groups: DNA Vaccine group, empty plasmid group and PBS control group. The mice were injected with pVAX1-IL4 / SYN-B DNA Vaccine, empty plasmid pVAX1 and PBS 100μl, co-immunization 3 times. Three weeks after the last injection, all mice were subcutaneously injected with 1 mg / kg rotenone once a day for 40 days. After the last injection, the behavioral changes of mice were observed. The expression of α-syn and the number of tyrosine hydroxylase (TH) positive cells in substantia nigra compact zone were detected by immunohistochemistry. Α-syn mRNA expression was detected. Results The results of behavioral observation showed that the free movement of mice in each group showed that the number of mobile grids and the number of standing legs of mice in the DNA vaccine group, empty plasmid group and PBS control group were significantly improved (P <0.05). Immunohistochemistry showed that the number of TH positive cells was increased by 63.27%, 55.34% (P <0.05), and the expression of α-syn was decreased by 55.68%, 55.34% (P <0.05) compared with the PBS control group ). RT-PCR results showed that the relative expression level of α-syn mRNA in the vaccine group was significantly decreased (0.38 ± 0.03 vs 0.77 ± 0.05 vs 0.73 ± 0.01 in the PBS control group, P <0.05). Conclusion The DNA vaccine of pVAX1-IL4 / SYN-B can effectively prevent rotenone neurotoxin from damaging dopaminergic neurons in substantia nigra and protect the dopaminergic neurons.