过表达 BTLA 的树突状细胞对小鼠 EAE 免疫耐受作用的初步研究

摘要

Objective To investigate the regulating effect of dendritic cells (DC)over-expressing B-and T-lymphocyte attenuator (BTLA)upon the immune tolerance of mouse models with experimental autoim-mune encephalomyelitis (EAE).Methods The recombinant lentiviral vector carrying the target gene of BTLA was constructed.Bone marrow-derived DCs were isolated from the C57BL/6 mice,cultured and transfected at a multiplicity of infection (MOI)of 1 5 to construct DCs over-expressing BTLA.The C57BL/6 mouse models with EAE model were established.During the peak of onset,the spleen cells were isolated,and CD4 + T lym-phocytes were separated by magnetic beads and co-cultured with DCs over-expressing BTLA.The Th1 -type cy-tokine IFN-γand Th2-type cytokine IL-1 0 were quantitatively detected by ELISA.Results The recombinant LPP-Mm26645-Lv203-400 lentiviral vector carrying mouse BTLA gene was successfully constructed.DCs were transfected and cultured for 5 d at a MOI of 1 5.DCs over-expressing BTLA were detected after 96 h.The peak of onset was observed in C57BL/6 mice at 21 -24 d after MOG35-55 peptide immunization.CD4 +T lymphocytes and DCs over-expressing BTLA were co-cultured.ELISA demonstrated that the expression level of IFN-γwas significantly down-regulated,whereas the expression level of IL-1 0 was considerably up-regulated in the co-cul-ture system (both P <0.05).Conclusion Over-expressing of BTLA can enhance the tolerance of DCs,yield specific antigen immune tolerance to EAE via reversing Th1 /Th2 imbalance and negatively regulate the immune response of EAE.%目的：探讨过表达 B 和 T 淋巴细胞衰减子（BTLA）的树突状细胞（DC）对 C57BL／6小鼠实验性自身免疫性脑脊髓炎（EAE）免疫反应的调节作用。方法构建携带目的基因 BTLA 的重组慢病毒载体，以感染复数15转染原代分离培养的 C57BL／6小鼠骨髓源性的 DC 从而在 DC 上过表达 BTLA。构建 C57BL／6小鼠 EAE 模型，发病高峰时分离脾细胞并从中磁珠分选出 CD4＋T 淋巴细胞并与过表达 BTLA 的 DC 共培养，以 ELISA 法检测共培养体系中辅助性 T 淋巴细胞 Th1类细胞因子干扰素-γ和 Th2类细胞因子 IL-10的含量。结果成功构建携带 BTLA 基因的重组慢病毒 LPP-Mm26645-Lv203-400，以感染复数15转染培养5 d 后的 DC，96 h 后 DC 上有 BTLA 过表达。采用 MOG35-55多肽免疫 C57BL／6小鼠后，21～24 d 出现发病高峰，分选出的 CD4＋T 淋巴细胞与过表达 BTLA 的 DC共培养72 h，ELISA 法检测共培养体系中干扰素-γ分泌明显减少，IL-10分泌明显增多（P 均＜0.05）。结论过表达 BTLA 可以增强 DC 的耐受性，通过逆转 Th1／Th2失衡，对 EAE 产生特异性抗原免疫耐受，负性调节 EAE 的免疫反应。