SUPPRESSION OF EXPERIMENTAL AUTOIMMUNE UVEORETINITIS BY NEUROPEPTIDE GENE-TRANSFECT-CELLS

目的：探讨降钙素基因相关肽(CGRP)诱导的成熟树突状细胞(DC)对小鼠实验性自身免疫性葡萄膜视网膜炎(EAU)的抑制作用。C57BL/6小鼠骨髓细胞与GM-CSF共培养6d后，用AutoMACS纯化分化后的CD11~+未成熟DC。未成熟DC进一步与脂多糖(1μg/ml)共同培养过夜，然后与人视黄样受体结合蛋白残基1-20(hIRBP-p)加CGRP或转化生长因子-β2。用弗氏S佐剂中的hIRBP-p和百日咳毒素免疫小鼠，诱导EAU。同时，将hIRBP-p免疫的小鼠静脉注射经CGRP或转化生长因子-β2培养的成熟DC。免疫后第19天检测hIRBP-p特异性迟发型超敏反应。免疫后第21天处死动物，病理评估EAU的程度。注射含有CGRP或转化生长因子-β2和hIRBP-p的成熟DC可显著抑制EAU和抗原特异性的Dh。对照组EAU发生率为87%(13/15只EAU小鼠)，TGF-B2培养组EAU发生率为60%(9/15只EAU鼠)，CGRP培养组EAU发生率为28%(5/18只EAU鼠)。在接下来的实验中，我们用电穿孔的方法制备了CGRP基因转染树突状细胞。结果表明，CGRP基因转导的DC可抑制IRBP-p的EAU，并抑制其对DHs的增殖。结果表明，成熟DC与CGRP及CGRP基因转导的DC共同培养，对EAU的发生、发展具有免疫抑制作用，且抑制作用强于单独应用转化生长因子-β2的DC。

To investigate the role of the suppression in murine experimental autoimmune uveoretinitis (EAU) by mature dendritic cells (DC) cultured with calcitonin gene-related peptide (CGRP). Bone marrow cells derived from C57BL/6 mice were cultured with GM-CSF for six days, and differentiated CD11^+ immature DC were purified by autoMACS. Immature DC were further cultured with LPS (1 μg/ml) overnight, and then with human interphotoreceptor retinoid binding protein (IRBP) residues 1-20 (hIRBP-p) in the presence of CGRP or TGF-B2. EAU was induced by immunization with hIRBP-p in Freund' s adjuvant and pertussis toxin in mice. At the same time, hIRBP-p-immunized mice were injected the mature DC cultured with CGRP or TGF-B2 intravenously. On day 19 after immunization, hIRBP-p-specific delayed hypersensitivity (DH) were measured. On day 21 after immunization, animals were sacrificed, and assessed the extent of EAU pathologically. EAU and antigen-specific DH was predominantly suppressed by injection of mature DC cultured with CGRP or TGF-B2 and hIRBP-p. Incidence of EAU on control group was 87% (13 of 15 EAU mice), TGF-B2 culture group was 60% (9 of 15 EAU mice), and CGRP culture group was 28% (5 of 18 EAU mice). On the next experiments, we prepared CGRP gene-transfect-DC by electroporation methods. As the results, CGRP gene-transfect-DC can suppress EAU, and suppress DH spepific for IRBP-p. It was demonstrated that mature DC cultured with CGRP and CGRP gene-transfect-DC play an immunosuppressive role in development of EAU, which were more effective than those cultured with TGF-B2.