纳米金介导的GMCSF-NgR-PirB双靶向融合核酸疫苗的制备及其对损伤脊髓的免疫治疗作用与机制研究

Immunization with nucleic acid vaccine is an effective therapeutic strategy for central nervous system injury developed in recent years, with strong targeting and weak harmful autoimmune response. More importantly, it can overcome the blood brain barrier and micro osmotic pump damage. Myelin inhibitory factors (MAIs) suppress the regeneration of central nervous system injury mainly through their co-receptor NgR and PirB. Based on this, the construction of NgR-PirB dual targeted nucleic acid vaccine, and intervention simultaneously by two key targets through vaccination may effectively reverse MAIs mediated inhibition signals, and promote nerve regeneration. However, the improvement of the immune response potency induced by nucleic acid vaccine needs the help of immune adjuvant. GM-CSF not only has the effect of immunological adjuvant, can also activate stem cells, and inhibit the formation of glial scar. As a novel immune adjuvant, gold nanoparticles (GNPs) can overcome the side effects of liposomes and other commonly used adjuvants. This study is firstly to construct a GM-CSF, NgR and PirB co-expression eukaryotic plasmid, after conjugated with gold nanoparticles, form a dual targeted fusion nucleic acid vaccine. Then, the animals will be vaccinated and the antibody titer of anti-serum will be detected. In order to observe the effects of vaccination on the spinal cord injured animals, the effects of antiserum on neurite outgrowth will be observed in vitro, the effects of the repair of spinal cord injury will be observed in vivo, and the mechanisms are to be further explored. The present study, will provide a new method and theory for the treatment and pharmaprojects of the injured spinal cord and other central nervous system.

核酸疫苗接种靶向性强，有害自身免疫反应弱，还可克服血脑屏障及微渗透泵损伤，是一种极具应用潜力的中枢神经损伤治疗手段。髓磷脂抑制因子（MAIs）主要通过其共同受体NgR及PirB抑制损伤中枢神经的再生。因此，构建NgR-PirB双靶向核酸疫苗，通过疫苗接种同时干预两个关键靶点，可能会有效逆转MAIs介导的神经再生抑制信号，促进神经再生。但核酸疫苗免疫应答效力的提高需要免疫佐剂的参与。GM-CSF不仅具有免疫佐剂效应，还可激活干细胞、抑制胶质瘢痕形成。纳米金（GNPs）作为一种新型免疫佐剂可有效克服脂质体等常规佐剂的毒副作用。本研究拟构建GM-CSF、NgR及PirB共表达真核载体，纳米金包被后形成双靶向融合核酸疫苗，免疫动物并检测抗血清中抗体效价，体外观察抗血清对培养神经元突起生长的影响，体内观察其对损伤脊髓的修复作用并初步探讨其作用机制，为中枢神经损伤治疗及药物研发提供新的方法和理论依据。

核酸疫苗接种靶向性强，有害自身免疫反应弱，还可克服血脑屏障及微渗透泵损伤，是一种极具应用潜力的中枢神经损伤治疗手段。髓磷脂抑制因子（MAIs）主要通过其共同受体NgR及PirB抑制损伤中枢神经的再生。因此，构建NgR-PirB双靶向核酸疫苗，通过疫苗接种同时干预两个关键靶点，可能会有效逆转MAIs介导的神经再生抑制信号，促进神经再生。但核酸疫苗免疫应答效力的提高需要免疫佐剂的参与。GM-CSF不仅具有免疫佐剂效应，还可激活干细胞、抑制胶质瘢痕形成。纳米金（GNPs）作为一种新型免疫佐剂可有效克服脂质体等常规佐剂的毒副作用。本研究首先构建了GM-CSF、NgR及PirB共表达真核载体，以纳米金包被后形成双靶向GMCSF-NgR-PirB融合核酸疫苗，免疫动物并检测抗血清中抗体效价，体外观察抗血清对培养神经元突起生长的影响，体内观察其对损伤脊髓的修复作用并初步探讨其作用机制。结果表明，成功构建了共表达GM-CSF、NgR及PirB的双靶向融合真核表达载体，并在此基础上经GNPs包被后成功制备了高效价的GMCSF-NgR-PirB双靶向融合核酸疫苗。纳米金介导的GMCSF-NgR-PirB双靶向融合核酸疫苗免疫产生的抗血清体外对神经元突起生长具有促进作用；体内能促进损伤脊髓的再生修复及功能的改善。其机制可能是通过免疫产生的NgR、PirB抗血清有效逆转了内源性NgR、PirB介导的轴突再生抑制信号进而达到促进神经再生的目的。该研究为中枢神经损伤治疗及药物研发提供新的方法和理论依据。