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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 investigate the effect of indoleamine 2,3-dioxygenase (IDO) gene transfection on hepatocellular carcinoma cell apoptosis and related cellular immune mechanisms through cell culture and in vivo experiments. Methods: T cells from peripheral blood of healthy people were extracted. T cells and hepatoma cells were mixed by cell culture and gene transfection. The experiment was divided into 6 groups: According to whether D-1-MT was added into the untreated group and the intervention group, each group was divided into T cell and HepG2 cell group according to different cultured cells. T cell and pcDNA3.1-HepG2 cell group , T cells and pcDNA3.1-IDO-HepG2 cell group. After 2 days of mixed culture, the apoptosis of HepG2 cells and the cytotoxic activity of T cells against HepG2 cells were detected by MTT assay. Flow cytometry was used to detect the proportion of regulatory T cells (Tregs) after 5 days of mixed culture. A mouse model of human hepatocellular carcinoma was established and the proportion of Treg cells in peripheral blood of tumor-bearing mice was detected by flow cytometry. After mixed culture for 2 days, the apoptosis rate of hepatoma cells transfected with IDO gene and the cytotoxic activity of T cells against HepG2 cells were significantly lower (1.65 ± 0.14% vs (35.00 ± 2.20)% p <0.05). After adding 1-MT, the above indexes were significantly higher than those before intervention, and there was significant statistical significance before and after intervention (P <0.05). After mixed culture for 5 days, the proportion of Treg cells in IDO-HepG2 cells increased significantly (10.53 ± 1.05)%, compared with the other two non-intervention groups (p <0.05) , Treg cells were significantly lower (p <0.05). The percentage of Treg cells in peripheral blood of IDO-bearing mice was significantly increased (15.33 ± 1.18)%, which was statistically significant compared with the other two groups (p <0.05). IDO may inhibit the apoptosis of hepatoma cells (HepG2 cells) and the immunotoxicity of T cells by increasing the proportion of regulatory T cells. 1-MT inhibits this effect of IDO. In vivo experiments confirmed IDO overexpression can increase the proportion of peripheral blood Treg cells.