补体C3d-p28对阿尔茨海默病DNA疫苗的免疫增强作用

目的探讨补体C3d-p28作为分子佐剂,在阿尔茨海默病DNA疫苗基因免疫中的作用。方法在第1、8、22、43、64、85、106、127天,将重组质粒p(Aβ3-10)10、p(Aβ3-10)10-C3d-p28.3和pcDNA3.1(+)肌肉注射于APP/PS1双转基因鼠后腿股四头肌内。疫苗接种前、自第2次注射开始每次接种后7天取鼠眶静脉血共8次,以ELISA法检测抗Aβ抗体的滴度和分型;第8次(最后1次)眶静脉取血后进行6d的Morris水迷宫实验,通过定位航行和空间探索实验评估小鼠空间学习记忆能力。水迷宫实验结束后处死小鼠,以ELISA法检测小鼠脾细胞培养上清液中白细胞介素4(IL-4)和干扰素γ(IFN-γ)水平,免疫组化染色法检测小鼠脑内Aβ斑的表达。结果 p(Aβ3-10)10-C3d-p28.3组抗Aβ抗体水平高于p(Aβ3-10)10组[(55.03±8.93)μg/mLvs.(27.32±7.69)μg/mL,t=-4.455,P0.05],p(Aβ3-10)10-C3d-p28.3组抗体类型主要是IgG1型[(50.64±6.96)μg/mL],明显高于p(Aβ3-10)10组[(14.15±3.16)μg/mL,P0.05]。与p(Aβ3-10)10组比较,p(Aβ3-10)10-C3dp28.3组Morris水迷宫实验平均逃避潜伏期变短、穿越平台次数和穿越平台所在象限停留的时间比例明显增多(均P0.05);脾细胞培养上清液中IL-4水平增高[(110.22±18.12)pg/mL vs.(170.12±22.16)pg/mL,P0.05]、IFN-γ水平减低[(800.12±80.11)pg/mL vs.(640.12±70.53)pg/mL,F=6.152,P0.05];脑内沉积的Aβ斑块明显减少(P0.05)。结论补体C3d-p28分子佐剂使p(Aβ3-10)10-C3d-p28.3抗Aβ抗体的产生增加、Th2型免疫反应增强,转基因鼠空间学习记忆能力提高。

Immunoenhancement of complement C3d-p28 on Alzheimer's disease DNA vaccine

Objective To discuss the effect of complement C3d-p28 in the genetic immunization of Alzheimer's disease DNA vaccine.Methods On day 1 , 8 , 22 , 43 , 64 , 85 , 106 , 127 , the recombinant plasmid p (Aβ3-10) 10 ,recombinant plasmid p (Aβ3-10 ) 10-C3d-p28.3 and pcDNA3.1 ( + ) were injected intramuscularly into the musculi quadriceps femoris of APP /PS1 double transgenic mouse hind leg. Before the vaccination and 7 days after the second injection ,the orbital vein blood was taken 8 times. ELISA was used to detect the titer of serum anti-Aβ antibody ,isotypes of immunoglobulin. After the eighth orbital vein blood collection ,Morris water maze test was conducted to evaluate the spatial learning and memory ability of the mice through the positioning navigation and space exploration tests for 6 days. The mice were killed at the end of the water maze test ,and the contents of IL-4 and IFN-γ in the supernatant of spleen cell culture were detected by ELISA. The β-amyloid plaques in mouse brains were detected by immunohistochemistry. Results The titers of anti-Aβ antibodies in the p (Aβ3-10) 10-C3d-p28.3 group were higher than those in the p (Aβ3-10) 10 group [ (55.03 ± 8.93) μg/mL vs. (27.32 ± 7.69) μg/mL , t= - 4.455 , P< 0.05] . The type of antibody in the p (Aβ3-10) 10-C3d-p28.3 group was mainly IgG1 type [ (50.64 ± 6.96) μg /mL ] , apparently higher than p (Aβ3-10) 10 group [ (14.15 ± 3.16) μg/mL , P< 0.05] . In the Morris water maze ,the p (Aβ3-10) 10-C3d-p28.3 group showed shorter escape latency and spent significant more time in the target quadrant and crossed the platform significantly more often than p (Aβ3-10) 10 immunized mice. In the p (Aβ3-10) 10-C3d-p28.3 group , the content of IL-4 in the supernatant of spleen cell culture was higher [ (170.12 ± 22.16) pg/mL , P< 0.05] , the content of IFN-γ was lower [ (640.12 ± 70.53) pg/mL , F = 6.152 , P < 0.05 ] ,and the deposition of amyloid burden in hippocampal and cortex was significantly reduced (P< 0.05) ,compared with the p (Aβ3-10) 10 group. Conclusions Complement C3d-p28 molecular adjuvant enhanced the production of anti A β antibody of recombinant plasmid p (Aβ3-10) 10-C3d-p28.3 , enhanced the Th2 type immune response ,and improved the spatial learning and memory ability of transgenic mice