1-甲基色氨酸联合外周血单个核细胞对人胶质瘤细胞的免疫杀伤作用

Immune killing effects of 1-methyltroptophan combined with peripheral blood mononuclear cells on human glioma cells

目的 观察吲哚胺-2,3-双加氧酶（IDO）抑制剂1-甲基色氨酸（1-MT）联合外周血单个核细胞（PBMC）对人胶质瘤细胞A172的免疫杀伤作用.方法 采用Ficoll密度梯度离心分离健康人外周血制备PBMC,根据是否联合1-MT分组.细胞计数试剂盒（CCK-8）法检测梯度1-MT（0、25、125、250、500 μmol/L;1、2、4、8、10 mmol/L）联合PBMC对A172细胞存活率影响.1-MT（500 μmol/L）联合PBMC与A172细胞共培养,流式双染术检测A172细胞凋亡率,Western blot检测A172细胞IDO蛋白表达,实时定量反转录聚合酶链反应（RT-qPCR）检测PBMC中调节T细胞叉头样螺旋转录因子（Foxp3） mRNA表达.结果 在PBMC数量一定情况下,1-MT呈剂量依赖性抑制A172细胞存活率;1-MT联合PBMC组细胞凋亡比率（45.0％）显著高于PBMC组（20.0％）及1-MT组（33.7％）;PBMC组A172细胞IDO蛋白相对表达量（0.313±0.017）明显高于1-MT联合PBMC组（0.182±0.015,P＜0.01）,1-MT、A172空白组未见明显IDO蛋白表达;PBMC组PBMC中Foxp3mRNA表达（2.764±0.180）明显高于1-MT联合PBMC组（2.189 ±0.179,P＜0.05）及PBMC空白组（1.000±0.028,P＜0.01）,1-MT联合PBMC组明显高于PBMC空白组（P＜0.01）.结论 1-MT可抑制IDO表达,下调PBMC中Foxp3转录因子表达,降低调节性T细胞增殖分化,可增强PBMC对胶质瘤细胞免疫杀伤作用.

Objective To investigate the immune killing effects of 1-methyltroptophan （1-MT）combined with peripheral blood mononuclear cells （PBMC） on human A172 glioma cells and the relative mechanism.Methods PBMC were isolated from healthy donors by Ficoll density-gradient centrifugation.A172 cells were co-cultured with or without PBMC in the presence or absence of 1-MT.Cell counting kit-8 （CCK-8） assay was used to test the inhibitory effect of 1-MT （0,25,125,250,500 μmol/L; 1,2,4,8,10 mmol/L） combined with PBMC on growth of A172 cells.Annexin V/propidium iodide （PI） staining was used to detect the effect of 1-MT （500 μmol/L） combined with PBMC on apoptosis of A172 cells.The expression of indoleamine 2,3-dioxygenase （IDO） protein in A172 cells was detected by Western blotting in different groups.The expression changes of forkhead helix transcription factor p3 （Foxp3） mRNA in PBMC were investigated by real-time quantitative polymerase chain reaction （RT-qPCR）.Results 1-MT reduced the growth rate of A172 glioma cells in a dose-dependent manner under the condition of a constant PBMC amount.The apoptosis rate of A172 cells induced by combined 1-MT and PBMC （45.0%） was significantly higher than that induced by either 1-MT （33.7%） or PBMCs （20.0%）.Western blotting analysis showed that the expression of IDO was considerably lower in 1-MT ＋ PBMC group （0.182 ±0.015） than in PBMC group （0.313 ±0.017）.1-MT group and A172 blank group showed no significant IDO expression.The mRNA level of Foxp3 was significantly higher in PBMC group （2.764 ± 0.180） than in 1-MT ＋ PBMC group （2.189 ±0.179） and PBMC blank group （1.000 ± 0.028） by RT-qPCR.Conclusion 1-MT can significantly increase the immune killing effects of PBMC by inhibiting the expression of IDO and Foxp3 and the differentiation of Treg cells in A172 cells.