补体C3d-p28增强阿尔茨海默病DNA疫苗Th2型免疫反应的研究

Enhancement of Th2 Immune Response to Alzheimer's disease DNA Vaccine by complement C3d-p28

目的 探讨补体C3d-p28作为分子佐剂,在阿尔茨海默病DNA疫苗免疫反应中的作用。方法 分别将重组质粒p（Aβ3-10）10,p（Aβ3-10）10-C3d-p28.3和空载体pc DNA3.1（＋）用肌肉注射的方法免疫8-10周龄的雌性BALB/c鼠。质粒注射前24 h,布比卡因肌肉注射诱导轻微的肌肉变性。应用ELISA方法检测血清抗Aβ抗体的滴度、抗体分型、体外脾细胞培养上清液中IL-4和IFN-γ的含量。免疫组织化学染色法检测免疫血清与转基因鼠脑内Aβ斑的结合能力。结果 重组质粒疫苗p（Aβ3-10）10仅诱导出低滴度的抗Aβ抗体,产生了Th1/Th2混合型的免疫反应。而重组质粒疫苗p（Aβ3-10）10-C3d-p28.3诱导出较高滴度的抗Aβ抗体,体外脾细胞培养上清液中IFN-γ低和IL-4高,即引起了Th2型细胞免疫反应,同时产生的抗Aβ抗体能够与双转基因鼠APP/PS1脑中沉积的Aβ斑块结合。结论 补体C3d-p28分子佐剂能够增强抗Aβ抗体的产生并且诱发Th2型的免疫反应。

Objective To discuss the effect of complement C 3d-p28 in the immune response of Alzheimer DNA vac-cine bstract.Methods Eight to ten week-old female BALB/c mice were immunized intramuscularly with recombinant plasmid p（Aβ3-10）10,p（Aβ3-10）10-C3d-p28.3 and pcDNA3.1（ ＋）.24 h prior to plasmid injection,bupivacaine was injected to induce mild muscle degeneration .ELISA was used to detect the titer of serum anti-Aβantibody ,isotypes of im-munoglobulin and levels of IL-4 and IFN-γin ex vivo splenocyte culture supernatants .Immunohistochemistry was used to detect the binding capability of antisera to Aβplaques in an APP/PS1 transgenic mouse brain .Results We have success-fully constructed two plasmid DNA vaccines：p（Aβ3-10）10 expressing ten repeats of Aβ3-10 without adjuvant and p（Aβ3-10）10-C3d-p28.3 encoding ten repeats of Aβ3-10 and three copies of C3d-p28 as a molecular adjuvant .Immunization of eight to ten week-old female BALB/c mice with p（Aβ3-10）10 vaccine induced only moderate titers of anti-amyloid-βanti-bodies and elicited a Th1/Th2 immune response.However,adding of the C3d-p28 molecular adjuvant to Aβ3-10 DNA vac-cine could generate high levels of anti-amyloid-βantibodies （P〈0.05） which bound to Aβplaques in APP/PS1 transgenic mouse brain tissue.More importantly,the vaccine elicited a predominantly IgG1 humoral response （P〈0.05）,low levels of IFN-γ（P〈0.05） and high levels of IL-4 in ex vivo cultured splenocytes （P〈0.05）,indicating a Th2-polarized cellular immune response.Conclusion Complement C3d-p28 molecular adjuvant enhances anti-amyloid-βantibody generation and induces a Th2-polarized immune response .