吲哚胺2,3-双加氧酶基因转染对肝癌细胞凋亡的影响及相关机制研究

Effects of Hepatocellular Carcinoma Cells' Apoptosis and the Related Mechanisms after Indoleamine 2,3-Dioxygenase Gene Transfection

目的:通过细胞培养和在体实验探讨吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)基因转染后对肝癌细胞凋亡的影响及相关细胞免疫机制的研究。方法:提取健康人外周血中的T细胞利用细胞培养和基因转染技术将T细胞和肝癌细胞混合培养。实验分为6组:根据是否加入D-1-MT分为未干预组和干预组,每组根据培养细胞的不同又分为T细胞与HepG2细胞组、T细胞与pcDNA3.1-HepG2细胞组、T细胞与pcDNA3.1-IDO-HepG2细胞组。于混合培养2天后应用流式细胞术、MTT法检测各组中HepG2细胞的凋亡情况和T细胞抗HepG2细胞的细胞毒活性。在混合培养5天后应用流式细胞术检测调节性T细胞(Regulatory T cell,Treg)的比例。并建立人肝癌细胞小鼠模型,用流式细胞仪检测荷瘤小鼠外周血中Treg细胞的比例。结果:1.混合培养2天后,转染IDO基因的肝癌细胞其凋亡率和T细胞抗HepG2细胞的细胞毒活性均明显降低,分别为(1.65±0.14)%和(35.00±2.20)%(p<0.05);加入1-MT干预后,以上指标均明显高于干预前,且干预前后比较有明显的统计学意义(P<0.05)。2.混合培养5天后,IDO-HepG2细胞组Treg细胞的比例明显升高(10.53±1.05)%,与其余两个未干预组比较有统计学意义(p<0.05);加1-MT干预后,Treg细胞比例均明显降低(p<0.05)。3.转染IDO的荷瘤小鼠模型中外周血Treg细胞比例明显升高(15.33±1.18)%,与其余两组比较有统计学意义(p<0.05)。结论:1.IDO可能通过增加调节性T细胞的比例来抑制肝癌细胞(HepG2细胞)的凋亡和T细胞的免疫毒性功能。1-MT可抑制IDO的这种作用。2.在体实验证实IDO的过量表达可提高外周血Treg细胞的比例。

Objective：To explore after indoleamine-2,3-dioxygenase（IDO）gene transfection the influence of the hepatocellular carcinoma cells’ apoptosis and the related cellular immune mechanisms by cell culture and in vivo.Methods： By cell culture and gene transfection technology,T-lymphocytes freshly isolated from healthy people and hepatocellular carcinoma cells were cocultured.The experiments were divided into six groups： T-lymphocytes and HepG2 cells group,T-lymphocytes and pcDNA3.1-HepG2 cells group,T-lymphocytes and pcDNA3.1-IDO-HepG2 cells group,and the three intervention groups which were added to IDO inhibitor 1-MT（1-methyl-tryptophan） on the basis of the above three groups.After two days of combine reaction,the apoptosisrate of HepG2 cell and the cytotoxicity of T-lymphocyte against HepG2 cell were examined by flow cytometer and MTT assay.After five days in mixed culture,the percentage of regulatory T cells（Treg） were analyzed by flow cytometer.Through establishment of the mouse model of human liver cancer cells,the percentage of Treg cells in peripheral blood of mouse was analyzed by flow cytometer.Results： 1.After two days of combine reaction,the apoptosis rate of HepG2 cell and the cytotoxicity of T-lymphocyte against HepG2 cell in IDO-HepG2 group were significantly lower than which in pcDNA3.1-HepG2 and HepG2 groups.They were respectively（1.65 ±0.14） % and（35.00±2.20）%（p〈0.05）.With 1-MT groups,the above indexes were significantly higher than before the intervention（p〈0.05）.2.After five days in mixed culture,the percentage of Treg cells in IDO-HepG2 group was significant higher and that was（10.53±1.05）%,it was considered statistically significant compared with the control group without 1-MT.In adding 1-MT groups,it decreased significantly（p〈0.05）.3.In the mouse model of human liver cancer cells,the percentage of Treg cells in peripheral blood of IDO-HepG2 group increased significantly,that is（15.33 ±1.18）% and compared with the other two groups was statistically significant（p〈0.05）.Conclusion： 1.Though increasing the percentage of Treg cells in T-lymphocytes,IDO can suppress the apoptosis rate of hepatocellular carcinoma cells and the cytotoxicity of T-lymphocyte.1-MT can reverse the role of IDO.2.In vivo test,it can be confirmed that over-expression of IDO can increase the proportion of Treg cells in peripheral blood.