目的探讨神经纤毛蛋白-1（Nrp-1）与膜相关转化生长因子-β（TGF-β）共表达对CD4+CD25+调节性T细胞（CD4+CD25+Treg）免疫抑制功能的影响。方法体外培养Balb/c小鼠CD4+CD25+Treg细胞，在抗CD3/CD28和脂多糖（LPS）诱导下，给予不同浓度的抗Nrp-1抗体（Ab-Nrp-1）封闭24 h后收集细胞，采用流式细胞分析仪检测细胞表面Nrp-1与膜相关TGF-β表达。分别将不同浓度Ab-Nrp-1封闭1 h后的Treg与小鼠正常CD4+CD25-T淋巴细胞共培养，观察抗CD3/CD28和LPS诱导下12、24和48 h后对CD4+CD25-T淋巴细胞增殖能力的影响。结果在抗CD3/CD28和LPS诱导下，Treg表面Nrp-1与膜相关TGF-β表达率明显增加（均P＜0.05），Treg膜相关TGF-β表达随着Ab-Nrp-1浓度的增加逐渐降低，呈明显的浓度依赖性（P＜0.05）；正常Treg与CD4+CD25-T淋巴细胞共培养后，能显著抑制CD4+CD25-T淋巴细胞增殖功能（P＜0.05），而不同浓度Ab-Nrp-1处理能逆转抑制作用，其中5μg/ml已达最大效应（P＜0.05），且24 h后作用最强，具有明显的浓度-时间依赖性（P＜0.05）。结论 Treg通过膜相关TGF-β发挥免疫抑制功能，共表达Nrp-1能够明显增强其免疫抑制作用。%Objective To investigate the effect of co-expression of neuropilin-1(Nrp-1)and transforming growth factor-β(TGF-β)on regulatory T cell(Treg)-mediated immunosuppression. Methods CD4+CD25+Tregs were isolated from the spleens of male Balb/c mice. CD4+CD25+Tregs were blocked with various doses of Nrp-1 antibody(Ab-Nrp-1,0.5,5,10 μg/ml)for 24 hours with anti-CD3/CD28 and lipopolysaccharide(LPS)stimulation, and expression of Nrp-1 and TGF-βwas determined by flow cytometry. Meanwhile,CD4+CD25+Tregs were cultured with different doses of Ab-Nrp-1 for 1 hour,and co-cultured with CD4+CD25-T cell for 12,24 and 48 hours respectively,the proliferative activity of CD4+CD25-T cells was analyzed by microplate reader. Results Compared to control group,the expressions of Nrp-1 and TGF-β were significantly increased under anti-CD3/CD28 and LPS stimulation(both P<0.05),and treatment with Ab-Nrp-1 markedly inhibited the expression of TGF-β in a dose-dependent manner(P<0.05). The normal Treg had the potential to inhibit the proliferation of CD4+CD25-T cells(P<0.05),while various doses of Ab-Nrp-1 had the ability to reverse the immunosuppressive function of CD4+CD25+Treg in a dose-and time-dependent response manner,5μg/ml has the strongest ability,expecially after 24 hours. Conclusion Treg cell plays an important role in mediating immunosuppressive response via membrane associated TGF-β,and co-expression of Nrp-1 can markedly promote the immunosuppressive function.
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