pVAX1-IL4/SYN-B核酸疫苗对鱼藤酮慢性帕金森病小鼠的治疗作用

The thera peutic effects of human α-synuclein DNA vaccine in chronic rotenone mouse model of Parkinson's disease

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对鱼藤酮慢性帕金森病小鼠的治疗效果。方法采用鱼藤酮1 mg/kg给予背部皮下注射,1次/d,连续注射40 d,建立慢性帕金森病小鼠模型。建模后小鼠随机分为3组:核酸疫苗组、空质粒组、PBS组(n=8)。于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,每次间隔3周。末次免疫后2周,观察小鼠行为学变化;免疫组化方法检测小鼠脑黑质致密部α-syn表达和酪氨酸羟化酶阳性细胞数目变化;RT-PCR检测α-syn mRNA表达情况。结果行为学观察各组小鼠自由活动实验显示,核酸疫苗组与空质粒组及PBS组小鼠移动格子数和下肢站立次数均有明显改善(P<0.05)。免疫组化检测发现核酸疫苗组小鼠与空质粒组、PBS组相比,TH阳性细胞数增加51.43%、59.00%(P<0.05),α-syn表达减少45.64%、43.28%,(P<0.05)。RT-PCR检测发现核酸疫苗组α-syn mRNA表达水平(0.37±0.17)较空质粒组(0.80±0.01)及PBS组(0.74±0.05)明显减少(P<0.05)。结论 hα-syn核酸疫苗有较强改善帕金森病小鼠行为学的作用;能有效改善鱼藤酮神经毒素对黑质多巴胺能神经元的损害,能对帕金森病小鼠产生较好的免疫治疗作用。

Objiective To investigate the immuotherapeutic effects of optimized human α-synuclein DNA vaccine on rotenone-induced chronic mouse model of Parkinson＇s disease（PD）.Methods Chronic rotenone mouse PD model was established by subcutaneous injection of low dose（1 mg / kg） rotenone daily for 40 consecutive days.Those animals were then randomly divided into three groups： the optimized human α-synuclein DNA vaccine group（pVAX1-IL4 / SYN-B）、the empty plasmid control group and the PBS control group.Animals were immunized by injections of 100 μL volume of either the pVAX1-IL4 / SYN-B,vacant vector plasmid or PBS at anterior tibial muscles of hind leg daily for three consecutive days.The immunization was repeated three times at three week intervals.The behavioral changes were recorded two weeks after final immunization.The expression of tyrosine hydroxylase（TH） and α-syn in substantia nigra（SN） was assayed by using immunohistochemistry and α-syn mRNA was detected by RT-PCR.Results Animals in the DNA vaccine group showed significant improvements in average ambulation grids（P 0.05P）and frequency of rearing（P 0.05） compared with control group.Compared with the empty plasmid control group,the DNA vaccine increased the TH positive cells up to 51.43% and decreased the α-syn expression in SN up to 45.64%.Compared with the PBS control group,the DNA vaccine increased the TH positive cells up to 59.00%（P 0.05） and decreased the α-syn expression in SN up to 43.28%（P 0.05）.Compared to the empty plasmid control group and the PBS control group,the α-syn mRNA level of the DNA vaccine group was decreased significantly（P 0.05）.Conclusions Optimized human α-synuclein DNA vaccine（pVAX1-IL4 / SYN-B） significantly improves the behavior function and attenuate rotenone-induced neuron damage in mouse model of PD.