优化核酸疫苗对帕金森模型小鼠黑质COX-2表达的影响

Effects of optimized hα-synuclein DNA vaccine on cyclooxygenase-2 expression in substantia nigra in a murine model of Parkinson's disease

目的探讨预防性注射优化hα-syn核酸疫苗对遭受急性神经毒素损害的多巴胺能神经元的作用。方法制备优化hα-syn质粒疫苗pVAX1-IL-4/SYN-B。根据分组对C57BL小鼠双侧后肢胫骨前肌进行注射,pVAX1-IL-4/SYN-B质粒组给予α-syn核酸疫苗、pVAX1空质粒组给予空载体、PBS组给予PBS各50μl,共3次。末次注射2周后,各组小鼠均经腹腔注射MPTP(20mg/kg)制备帕金森病(PD)模型,间隔2h注射1次,共4次。在模型制备过程中观察小鼠行为学变化,评价PD模型是否制备成功。于末次MPTP注射后1、3、7d处死小鼠(n=6)。采用RT-PCR法检测不同时间点小鼠中脑黑质炎症因子COX-2 mRNA的表达,免疫组化法检测黑质COX-2和CD11b(反映小胶质细胞激活状态)的表达变化。结果各组小鼠PD模型制作成功。与pVAX1空载体组、PBS组相比,pVAX1-IL-4/SYN-B质粒组黑质区COX-2及CD11b阳性细胞明显减少(P<0.05),而前两组比较无显著差异(P>0.05)。RT-PCR检测显示,在MPTP末次注射后1、3、7d,COX-2 mRNA在各组均有表达,1d表达微弱,3d达高峰,7d各组表达均降低。同一时间点,pVAX1-IL-4/SYN-B质粒组的COX-2 mRNA表达(1、3、7d分别为0.28±0.07、0.59±0.07、0.45±0.05)与pVAX1空质粒组(1、3、7d分别为0.43±0.05、0.74±0.15、0.61±0.10)、PBS组(1、3、7d分别为0.41±0.02、0.73±0.13、0.60±0.01)相比显著降低,差异有统计学意义(P<0.01),后两组间比较差异无统计学意义(P>0.05)。结论优化hα-syn核酸疫苗预防注射能对抗炎症因子COX-2表达、抑制小胶质细胞激活,对多巴胺神经细胞具有保护作用。

Objective To explore the effects of prophylactic inoculation of optimized hα-synuclein（hα-syn） DNA vaccine on the dopaminergic neurons suffering from acute neurotoxin injury.Methods The optimized DNA vaccine,designated as pVAX1-IL-4/SYN-B,was prepared.The C57BL mice were grouped as pVAX1-IL-4/SYN-B group,pVAX1 group and PBS group.Animals in each group received 3 times of 50μl injection of plasmid pVAX1-IL-4/SYN-B,plasmid pVAX1 or PBS,respectively,in the anterior tibial muscle of either posterior limb.Two weeks after the last injection,all the mice were then given intraperitoneal injection of 20mg/kg MPTP with an attempt to reproduce the model of Parkinson’s disease（PD） 4 times with a 2h interval.The changes in behavior of mice were observed when the model was being reproduced to verify if the model was reproduced.Mice were sacrificed at 1st,3rd and 7th day（6 each） after the last injection of MPTP.The expression of COX-2 mRNA at each time point was detected by RT-PCR,and the expressive changes in COX-2 and CD11b（representing the activated state of microglia） were detected with immunohistochemistry assay.Results The PD mouse model was successfully reproduced.The number of positive COX-2 and CD11b cells was remarkably decreased in pVAX1-IL-4/SYN-B group than in pVAX1 group and PBS group,while no significant difference was found between the latter 2 groups（P〉0.05）.The results of RT-PCR showed that COX-2 mRNA expression in all the 3 groups at the 1st,3rd and 7th day after last injection of MPTP was slight on the 1st day,peak value appeared on the 3rd day,and it then declined on the 7th day.At each time point,the COX-2 mRNA expression in pVAX1-IL-4/SYN-B group（0.28±0.07,0.59±0.07 and 0.45±0.05 at day 1,3 and 7） was significantly lower（P〈0.01） than that in pVAX1 group（0.43±0.05,0.74±0.15 and 0.61±0.10） and in PBS group（0.41±0.02,0.73±0.13 and 0.60±0.01）,while no difference was found between the latter 2 groups（P〉0.05）.Conclusion Prophylactic inoculation of optimized hα-syn DNA vaccine may inhibit the expression of COX-2 and the activation of microglia,showing a protective effect on dopaminergic neurons.