转化生长因子β_1诱导的耐受性树突状细胞及其机制

TGF-β_1 induced the tolerant dendritic cells and the mechanism

目的探讨耐受性树突状细胞(DC)的诱导及其机制。方法以20ng/ml转化生长因子β_1(TGF-β_1)与C57BL/6小鼠来源的树突状细胞系(DC2.4)共培养6d,诱导耐受性DC(TGFβ- DC);以脂多糖(LPS)刺激DC 48 h即为成熟DC(LPS-DC);以特异性针对免疫球蛋白样受体B(PIR- B)的小RNA干扰片段(PIR-B siRNA)转染TGF-BC(si-DC)。应用半定量逆转录聚合酶链反应和流式细胞仪测定各细胞表面PIR-A/B共同的胞外段PIR的表达、PIR-A/B及其mRNA的表达,以Balb/c小鼠脾淋巴细胞为反应细胞进行混合淋巴细胞反应(MLR),观察各组DC刺激反应细胞增殖的能力,同时以酶联免疫吸附试验测定MLR上清液中γ干扰素(IFN-γ)的水平。结果TGFβ-DC的PIR-B mRNA表达明显上调,PIR-A mRNA的表达明显下调(P<0.05),而LPS-DC的PIR-B mRNA表达明显下调,PIR-A mRNA表达明显上调(P<0.05),二者表面PIR的表达均增加,但差异无统计学意义(P>0.05);转染PIR-B siRNA后,TGFβ-DC的PIR-B mRNA表达明显下调,细胞表面的PIR表达也受到明显抑制(P<0.05)。正常DC2.4细胞可以刺激异基因淋巴细胞增殖;LPS-DC刺激异基因淋巴细胞增殖的能力明显增强,其反应体系中的IFN-γ水平明显升高;TGFβ-DC刺激淋巴细胞增殖的能力受到明显抑制,其反应体系中的IFN-γ水平明显下降;而转染PIR-B siRNA后,TGFβ-DC刺激淋巴细胞增殖的能力得到明显恢复,其反应体系中的IFN-γ水平明显回升。结论TGF-β_1可诱导产生耐受性DC,其高度表达免疫抑制性受体PIR-B,上调PIR-B的表达可能是TGF-β_1诱导产生耐受性DC的分子机制之一。

Objective To investigate the induction of tolerant dendritic cells （DCs） and its mechanism in mice. Methods The dendritic cell line derived from C57BL/6 mouse, DC2. 4 cells were co-cultured with TGF-β1 （20 ng/ml） to induce the tolerant DCs （TGFβ-DC） and stimulated wihh lipopolysaccharide （LPS） for 48 h to induce the mature DCs （LPS-DC）. Special small interference RNA molecule （siRNA） of PIR-B was chemically synthesized and was transfected into TGFβ-DC by lip2000 （si-DC）. The expression of paired immunoglobin receptor A/B （PIR-A/B） in DC2. 4 cells was measured by semi-quantitative RT-PCR and flow cytometry （FCM）. The allogeneic stimulating capacity of DCs was measured by mixed lymphocyte reaction （MLR） using ^3H-thymidine incorporation test with the Balb/c spleen cells as reaction cells. The concentration of IFN-γ in supernatants of MLR from distinct groups was tested by enzyme linked immunosorbent assay （ELISA）. Results TGF-β1 up-regulated the PIR-B mRNA expression and down-regulated the PIR-A mRNA expression （P〈0.05）,on the contrary,LPS down-regulated the PIR-B mRNA expression and up-regulated the PIR-A mRNA expression （P〈0. 05）. The expression of PIR was increased in both TGFβ-DC and the LPS-DC groups, but no significance was found between TGFβ-DC and the LPS-DC. The PIR-B mRNA expression was reduced after the siRNA transfection and the PIR receptor expression in the DC2. 4 cells was also inhibited （P〈0. 05）. The normal DCs could stimulate the allogenetic lymphocytes proliferation. LPS-DC enhanced the alloactived T cell proliferation and IFN-γ secretion was increased in the supernatants of reaction. TGF-DC inhibited alloactived T cell proliferation and down-regulated the IFN-γ secretion.TGFβ-DC could restore the T cell proliferation and the IFN-γ secretion was ascended in MLR after transfecting the siRNA molecules of PIR-B. Conclusion TGFβ-DC is tolerant DCs with higher PIR-B expression and up-regulated PIR-B expression may be one of molecular mechanisms for tolerant DCs induced by TGF-β1 in mice.