不同浓度γ干扰素诱导大鼠脾脏来源树突状细胞表达吲哚胺2，3-双加氧酶的变化及对T淋巴细胞增殖的影响

Inhibition of T-cell proliferation by induction of IDO in spleen-derived dendritic cells of rats

目的观察不同浓度γ干扰素（IFN-γ）作用下大鼠脾脏来源的树突状细胞（Dc）中吲哚胺2，3-双加氧酶（IDO）mRNA和蛋白表达的变化及IDO对同种异体T淋巴细胞增殖的影响。方法应用细胞因子体外诱导培养大鼠脾脏来源DC，应用流式细胞仪测定大鼠DC特异性分子OX62和表面分子CD80、CD86的表达。分别用不同浓度（0、100、300、500U／m1）的IFN-γ诱导作用DC后，实时聚合酶链反应（Real-timePCR）测定DC中IDOmRNA的相对表达水平，Westernblot检测DC中IDO蛋白在Dc中的表达水平。同种异体混合淋巴细胞反应（MLR）检测不同浓度IFN-γ诱导后的DC对同种异体T淋巴细胞增殖的影响。结果体外诱导培养的DC光学娩微镜下观察，具有典型的树枝状突起。OX62表达率达到80％以上，CD80、CD86的阳性表达也在80％左右；DC的IDOmRNA（2-ΔΔCt值分别为：1．010±0．094、1．340±0．114、1．700±0．087、2．080±0．150）和蛋白的相对表达量（分别为-0．861±0．612、1．155±0．059、1．308±0．068、1．755±0．118）随着IFN．7作用浓度的增大逐渐增大，分别为：不同浓度组间差异有统计学意义（P〈0．05）；各组IFN-γ作用后DC与对照组比较，T淋巴细胞的增殖率显著降低（P〈0．05）；且随着IFN-γ作用浓度的增大T淋巴细胞的增殖率逐渐降低，A值分别为：0．458±0．041、0．423±0．030、0．349±0．019、0．312±0．036，各组间差异有统计学意义（P〈0．05）。结论采用改良的培养黏附法体外获得纯度较高的大鼠脾脏来源的DC；IFN-γ可以诱导大鼠脾脏来源DC表面活性IDO的表达增加，减弱脾脏DC对同种T细胞增殖的刺激能力．

Objective To investigate the expression chenges of indoleamine 2,3-dioxygenase （IDO） mRNA and protein in dendritic cells （DCs） derived from rat spleen in vitro as well as the effect of IDO on proliferation of allogeneic T lymphocytes in terms of different concentrations of interferon （IFN）-γ. Methods We first extracted DCs from the spleen of rats, then measured the expression of DC-speeific molecules and surface molecules CDSO and CD86 in rats by flow cytometry. DCs were induced by IFN-γ at differenct concentrations （0, 100, 300, 500 U/ml）. Real-time polymerase chain reactio （PCR） and Western blotting were used to detect the relative expression levels of IDO mRNA and protein in DCs. Allo- geneic mixed lymphocyte reaction （MLR） was applied to test the effect of DCs induced with different concentrations of IFN-γ on allogeneic T lymphocyte proliferation. Results The results showed that the spleenderived DCs from rats cultured for 10 days had a typical dendritic morphology, whose expression rate of OX62 was above 80% and positive expression rate of CDS0 and CD96 was more than 75% and 90% respectively. The expression levels of IDO mRNA （ 1. 010 ±0. 094, 1. 340 ±0. 114, 1. 700 ±0. 087, 2. 080 ±0. 150） and protein （0. 861 ±0. 612, 1. 155 ±0. 059, 1. 308 ±0. 068, 1. 755 ±0. 118） were increased gradually with the increase of concentrations of IFN-γ, which showed significant difference between any two groups. As compared with the control group, the groups induced by the IFN-γ had a decrease in T lympho- cyte proliferation of DCs （P 〈 0. 05）. Besides, with the increased concentration of IFN-γ, the rate of T lymphocyte proliferation was decreased gradually and there was significant difference between ant two groups （P 〈 0. 05 ）. Conclusion The IFN-γ can induce the increased expression of IDO in spleen-derived DCs from rats, and the decreased stimulation capacity of spleen DCs to the antigen-specific T cell response.