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

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、500 U/ml)的IFN-γ诱导作用DC后,实时聚合酶链反应( Real-time PCR)测定DC中IDO mRNA的相对表达水平,Westemblot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光学显微镜下观察,具有典型的树枝状突起.0X62表达率达到80％以上,CD80、CD86的阳性表达也在80％左右；DC的IDOmRNA(2 -△△Ct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-γ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.

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

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-specific molecules and surface molecules CD80 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.Allogeneic 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 CD80 and CD86 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 lymphocyte 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.