降钙素基因相关肽基因修饰的成熟树突状细胞对实验性自身免疫性视神经炎的抑制作用。

Suppression of murine experimental autoimmune optic neuritis by mature dendritic cells transfected with calcitonin gene-related Peptide gene.

机构信息

Department of Ophthalmology, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan.

出版信息

Invest Ophthalmol Vis Sci. 2012 Aug 13;53(9):5475-85. doi: 10.1167/iovs.12-9935.

DOI:10.1167/iovs.12-9935

PMID:22807299

Abstract

PURPOSE

Calcitonin gene-related peptide (CGRP) exhibits prominent anti-inflammatory actions. We examined whether CGRP-transfected dendritic cells (DC) prevent the development of experimental autoimmune optic neuritis (EAON) and experimental autoimmune encephalomyelitis (EAE).

METHODS

A human CGRP-expressing plasmid was constructed, and used to transfect C57BL/6 mouse bone marrow-derived matured DC (mDC) by electroporation

METHODS

Transfection efficiency was 50% with 80% cell viability. C57BL/6 mice were immunized with myelo-oligodendrocyte glycoprotein 35-55, and injected intravenously with CGRP-expressing mDC (CGRP gene-transfected group) or mock-transfected mDC (mock-transfected group) at the induction or effector phase. EAE was diagnosed clinically and EAON was assessed histopathologically. Delayed hypersensitivity was measured. Supernatants of spleen cell cultures were assayed for cytokines using ELISA. The CD4(+)CD25(+)Foxp3(+) fraction in spleen cells was analyzed using flow cytometry.

RESULTS

For gene therapy in the induction phase, EAE developed in 50% of mice in the CGRP-transfected group compared with 80% in the mock-transfected group, and the mean pathological score for EAON was 1 in the CGRP-transfected group compared with 2 in the mock-transfected group (P < 0.05). For gene therapy in the effector phase, the mean EAE clinical score (1.5 vs. 3.0) and mean EAON pathological score (1.0 vs. 2.0) were both lower in the CGRP-transfected group compared with the mock-transfected group (P < 0.05). Delayed hypersensitivity was suppressed significantly in the CGRP-transfected group. IL-10 production by spleen cells in the CGRP-transfected group increased independent of MOG concentration, compared with the mock-transfected group. Interestingly, the proportion of CD4(+)CD25(+)Foxp3(+) cells increased significantly (P < 0.05) in the CGRP-transfected group compared with the mock-transfected group.

CONCLUSIONS

Gene therapy with CGRP-expressing mDC was effective in suppressing the development of EAON and EAE.

摘要

目的

降钙素基因相关肽（CGRP）具有显著的抗炎作用。我们研究了 CGRP 转染树突状细胞（DC）是否可以预防实验性自身免疫性视神经炎（EAON）和实验性自身免疫性脑脊髓炎（EAE）的发生。

方法

构建了一个人 CGRP 表达质粒，并通过电穿孔将其转染到 C57BL/6 小鼠骨髓来源的成熟 DC（mDC）中。

方法

转染效率为 50%，细胞活力为 80%。用髓鞘少突胶质细胞糖蛋白 35-55 免疫 C57BL/6 小鼠，并在诱导或效应阶段静脉注射 CGRP 表达 mDC（CGRP 基因转染组）或 mock 转染 mDC（mock 转染组）。临床诊断 EAE，组织病理学评估 EAON。采用 ELISA 法检测脾细胞培养上清液中细胞因子。采用流式细胞术分析脾细胞中 CD4+CD25+Foxp3+细胞群。

结果

在诱导阶段进行基因治疗时，CGRP 转染组有 50%的小鼠发生 EAE，而 mock 转染组有 80%的小鼠发生 EAE，CGRP 转染组的 EAON 平均病理评分（1 分）与 mock 转染组（2 分）相比有所降低（P<0.05）。在效应阶段进行基因治疗时，CGRP 转染组的平均 EAE 临床评分（1.5 分比 3.0 分）和平均 EAON 病理评分（1.0 分比 2.0 分）均低于 mock 转染组（P<0.05）。CGRP 转染组迟发型超敏反应明显受到抑制。与 mock 转染组相比，CGRP 转染组脾细胞 IL-10 的产生不依赖于 MOG 浓度而增加。有趣的是，与 mock 转染组相比，CGRP 转染组 CD4+CD25+Foxp3+细胞的比例明显增加（P<0.05）。