摘要
目的 观察基因转染后高表达吲哚胺2,3-双加氧酶（IDO）的树突细胞（DC）及色氨酸代谢产物（TC）对CD4＋T细胞增殖的抑制作用.方法 将IDO基因转染小鼠DC,实时定量聚合酶链反应（Real-time PCR）及免疫细胞化学检测IDO表达.设DC、空转染DC、转染DC、转染DC＋1-甲基色氨酸（1-MT）、DC ＋TC、转染DC＋ TC 6组,混合淋巴细胞培养检测各组对同种异系CD4＋T细胞增殖的影响.结果 转染组IDO mRNA表达水平（0.1550±0.0124）较DC组（0.1197±0.0463）及空转染组（0.1152±0.0260）显著增高（P＜0.05）.免疫细胞化学检测显示转染DC胞质内出现阳性染色.转染组刺激指数（SI）（2.8640±0.1459）较DC组（3.3040±0.2394）及空转染组（3.2340±0.1862）显著降低（P＜0.05）.转染DC＋1-MT组SI（3.1900±0.2906）与DC组及空转染组差异无统计学意义（P＞0.05）.转染DC＋ TC组中犬尿氨酸（KYN）、3-羟基犬尿氨酸（3-HK）、3-羟邻氨基苯甲酸（3-HAA）的最小有效抑制浓度（分别为100、100和50 μmol/L）较DC＋ TC组（分别为200、200和100 μmol/L）明显降低.在最小有效抑制浓度下,转染DC＋ TC组SI（分别为2.4930±0.1555、2.4924±0.1737、2.4662±0.2482）较转染组显著降低（P＜0.05）.结论 转染DC可表达有功能的IDO.转染DC与TC可协同抑制CD4＋T细胞增殖.
Objective To demonstrate the inhibitory effect of indoleamine 2, 3-dioxygenase （IDO） -transfected dendritic cells （DCs） and Tryptophan catabolites on proliferation of CIM T lympho cytes. Methods IDO eDNA was obtained and inserted into eukaryotic expression vector pEGFP-N1 to con struct the recombinant expression plasmid pEGFP-NI-IDO. The recombinant plasmid was transfected into mouse DCs by GenePORTER 3000. IDO expression level was detected by using real-time quantitative poly merase chain reaction （Real-time PCR） and immunocytochemistry. The transfected DCs with or without Tryptophan catabolites and 1-mythotryptophan were co-cultured with allogeneic CD4 ± T lymphocytes. Stim- ulation index （SI） was analysed by mixed lymphocyte reaction （MLR）. Results pEGFP-NI-IDO was successfully generated and verified by sequencing. IDO mRNA expression level was significantly higher in pEGFP-Nl-IDO-transfected DCs group （0. 1550 ± 0. 0124） than that in DCs group （0. 1 t97 ± 0. 0463 ） or pEGFP-Nl-transfected DCs group （0. 1152 ± 0. 0260）. Positive staining was found in the pEGFP-N1- transfected DCs by immunocytoehemistry. SI in pEGFP-Nl-IDO-transfeeted DCs group （2. 8640 ± 0. 1459） was significantly lower than that in non-transfected group （3. 3040 ± 0. 2394） , pEGFP-Nl-transfected DCs group （3. 2340 ± 0. 1862） and 1-methyl-tryptophan （1-MT） addition group （3. 1900± 0. 2906）. The in- hibitory concentrations of Tryptophan catabolites in pEGFP-Nl-IDO-transfeeted DCs group were lower sig- nificantly than those in non-transfected group or pEGFP-Nl-transfected DCs group. Conclusion pEGFP- Nl-IDO-transfected DCs can express functional IDO. The pEGFP-Nl-IDO-transfected DCs and tryptophan eatabolites both can inhibit the proliferation of CD4T lymphocytes.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第11期2278-2280,共3页
Chinese Journal of Experimental Surgery
