吲哚胺2，3-双加氧酶在T淋巴细胞对肝癌细胞发生免疫应答中的作用
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摘要
目的肝癌患者的免疫系统不能有效地清除或杀伤癌细胞,是导致肝癌难以治愈、复发和转移的重要因素之一。探索肝癌细胞免疫逃逸的机制,将为肝癌的治疗提供新的思路。吲哚胺2,3-双加氧酶(IDO)是肝脏以外唯一可催化色氨酸分子氧化裂解的限速酶,在哺乳动物的组织和细胞中广泛表达。它通过降解局部组织中的色氨酸,在诱发宿主免疫防御、抑制T淋巴细胞免疫和抗肿瘤免疫、诱导母胎免疫耐受和移植物免疫耐受中均发挥重要的代谢性免疫调节作用。本研究通过观察IDO在抗肝癌免疫应答中的作用,为临床治疗肝癌提供新的理论依据。
方法从健康人的外周血中分离出T淋巴细胞和HepG2细胞进行混合培养,分为对照组和实验组进行比较,对照组为空白组,实验组中分别加入IDO的抑制剂1-甲基色氨酸(1-MT)和外源性IDO;用RT-PCR检测每组中细胞IDOmRNA的表达水平,流式细胞仪分析混合反应体系中HepG2细胞的凋亡率,四噻唑蓝(MTT)实验检测混合反应体系中T淋巴细胞抗HepG2细胞的细胞毒活性。
结果1.单独培养HepG2细胞,不表达IDOmRNA,而单独培养的T淋巴细胞表达微量的IDOmRNA;混合培养后,HepG2细胞表达IDOmRNA,而且表达水平较单独培养的T淋巴细胞高,其表达水平随着1-MT浓度的增高而降低,而随着外源性IDO浓度的增高而无明显变化。
2.流式细胞仪分析混合反应体系中HepG2细胞的凋亡率,对照组中HepG2细胞的早期凋亡率为(3.48±0.34)%,1-MT组(浓度分别为1.25 mmol/l,2.5 mmol/l,5mmol/l)中分别为(7.82±0.41)%,(18.62±0.42)%,(25.32±0.40)%,加入1-MT组的HepG2细胞的早期凋亡率明显高于对照组,四者比较有显著性差异(P<0.01);外源性IDO组(浓度分别为0.05 mmol/l,0.5 mmol/l,5mmol/l)中分别为(3.32±0.35)%,(3.46±0.40)%,(2.75±0.26)%,只有当外源性IDO浓度为5mmol/l时HepG2细胞早期凋亡率与对照组比较才有统计学意义(P<0.01)。
3. MTT实验显示,对照组中T淋巴细胞抗HepG2细胞的细胞毒活性为(17.36±1.24)%,1-MT组(浓度分别为1.25 mmol/l,2.5 mmol/l,5mmol/l)中分别为(25.48±1.48)%、(32.89±1.73)%、(42.04±2.16)%,四者比较有显著性差异(P<0.01),并且T淋巴细胞抗HepG2细胞的细胞毒活性随着1-MT浓度的增高而升高;外源性IDO组(浓度分别为0.05 mmol/l,0.5 mmol/l,5mmol/l)中分别为(17.30±0.52)%,(17.48±1.08)%,(15.74±0.79)%,同样也是只有当外源性IDO浓度为5mmol/l时与对照组比较才有统计学意义(P<0.01)。
结论HepG2细胞表达的内源性IDO和一定浓度的外源性IDO均可抑制外周T淋巴细胞发挥抗HepG2细胞的细胞毒活性作用,降低HepG2细胞的早期凋亡率,它们可能参与了肝癌的免疫逃逸;而IDO的抑制剂1-MT却可以逆转这种作用,有望成为治疗肝癌的新靶点。
Objects Indoleamine 2,3-dioxygenase(IDO)catalyzes the rate-limiting step of tryptophan degradation along the kynurenine pathway , and both the reduction in local tryptophan concentration and the production of immunomodulatorytryptophan metabolites contribute to the immunosuppressive effects of IDO. Rencent studies have focused on immunoregulatory role of IDO in mononuclear cells. To seek the new way of curing hepatocellular carcinoma, we investigated the role of IDO in anticancer immune responses.
Methods T-lymphocytes freshly isolated from healthy people and HepG2 cell were cocultured, and IDO inhibitor (1-methyl-tryptophan,1-MT) or exogenous IDO was added to make an intervening experiment. The level of IDO mRNA expression in HepG2 cell cocultured with T-lymphocyte was detected by RT-PCR;the apoptosis rate of HepG2 cell cocultured with T-lymphocyte was analyzed by flow cytometer; the cytotoxicity of T-lymphocyte against HepG2 cell was examined by MTT assay.
Results 1.In HepG2 cell studied, IDO mRNA was not expressed under normal culture conditions. A low level of IDO mRNA expression was detectable in T-lymphocyte under normal culture conditions. The IDO mRNA was expressed exclusively when HepG2 cell was cultured with T-lymphocyte. The level of IDO mRNA expression in HepG2 cells cultured with T-lymphocyte was especially higher than that in na?ve T-lymphocyte. And the level of IDO mRNA expression was faintly induced after addition of 1-MT, but that was not altered by exogenous IDO.
2.The earlier apoptosis rate of HepG2 cell in control group was (3.48±0.34)% and 1-MT(1.25 mmol/l,2.5 mmol/l,5mmol/l) group was respectively(7.82±0.41)%,(18.62±0.42)%,(25.32±0.40)%, the earlier apoptosis rate of HepG2 cell was significantly higher in cocultured condition with 1-MT group than without 1-MT group; the exogenous IDO (0.05 mmol/l,0.5 mmol/l,5mmol/l) group was respectively(3.32±0.35)%,(3.46±0.40)%,(2.75±0.26)%, it was considered statistically significant compared with control group when the concentration of exogenous IDO was 5mmol/l.
3.The cytotoxicity of T-lymphocyte against HepG2 cell in control group was (17.36±1.24)%,and 1-MT(1.25 mmol/l,2.5 mmol/l,5mmol/l) group was respectively(25.48±1.48)%,(32.89±1.73)%,(42.04±2.16)%, there was a significant increase in the cytotoxicity of T-lymphocyte against HepG2 cell in cocultured condition with 1-MT group than without 1-MT group; the exogenous IDO (0.05 mmol/l,0.5 mmol/l,5mmol/l) group was respectively(17.30±0.52)%,(17.48±1.08)%,(15.74±0.79)%, it was considered statistically significant compared with control group when the concentration of exogenous IDO was 5mmol/l, too.
Conclusions These results suggest that IDO from HepG2 cell and some concentration of exogenous IDO can suppress the cytotoxicity of T-lymphocyte against HepG2 cell and the earlier apoptosis rate of HepG2 cell , which may be contributing to hepatocellular carcinoma immune escape , but 1-MT can reverse the immunoregulatory role. In the future, it maybe become a new target for treatment of hepatocellular carcinoma.
方法从健康人的外周血中分离出T淋巴细胞和HepG2细胞进行混合培养,分为对照组和实验组进行比较,对照组为空白组,实验组中分别加入IDO的抑制剂1-甲基色氨酸(1-MT)和外源性IDO;用RT-PCR检测每组中细胞IDOmRNA的表达水平,流式细胞仪分析混合反应体系中HepG2细胞的凋亡率,四噻唑蓝(MTT)实验检测混合反应体系中T淋巴细胞抗HepG2细胞的细胞毒活性。
结果1.单独培养HepG2细胞,不表达IDOmRNA,而单独培养的T淋巴细胞表达微量的IDOmRNA;混合培养后,HepG2细胞表达IDOmRNA,而且表达水平较单独培养的T淋巴细胞高,其表达水平随着1-MT浓度的增高而降低,而随着外源性IDO浓度的增高而无明显变化。
2.流式细胞仪分析混合反应体系中HepG2细胞的凋亡率,对照组中HepG2细胞的早期凋亡率为(3.48±0.34)%,1-MT组(浓度分别为1.25 mmol/l,2.5 mmol/l,5mmol/l)中分别为(7.82±0.41)%,(18.62±0.42)%,(25.32±0.40)%,加入1-MT组的HepG2细胞的早期凋亡率明显高于对照组,四者比较有显著性差异(P<0.01);外源性IDO组(浓度分别为0.05 mmol/l,0.5 mmol/l,5mmol/l)中分别为(3.32±0.35)%,(3.46±0.40)%,(2.75±0.26)%,只有当外源性IDO浓度为5mmol/l时HepG2细胞早期凋亡率与对照组比较才有统计学意义(P<0.01)。
3. MTT实验显示,对照组中T淋巴细胞抗HepG2细胞的细胞毒活性为(17.36±1.24)%,1-MT组(浓度分别为1.25 mmol/l,2.5 mmol/l,5mmol/l)中分别为(25.48±1.48)%、(32.89±1.73)%、(42.04±2.16)%,四者比较有显著性差异(P<0.01),并且T淋巴细胞抗HepG2细胞的细胞毒活性随着1-MT浓度的增高而升高;外源性IDO组(浓度分别为0.05 mmol/l,0.5 mmol/l,5mmol/l)中分别为(17.30±0.52)%,(17.48±1.08)%,(15.74±0.79)%,同样也是只有当外源性IDO浓度为5mmol/l时与对照组比较才有统计学意义(P<0.01)。
结论HepG2细胞表达的内源性IDO和一定浓度的外源性IDO均可抑制外周T淋巴细胞发挥抗HepG2细胞的细胞毒活性作用,降低HepG2细胞的早期凋亡率,它们可能参与了肝癌的免疫逃逸;而IDO的抑制剂1-MT却可以逆转这种作用,有望成为治疗肝癌的新靶点。
Objects Indoleamine 2,3-dioxygenase(IDO)catalyzes the rate-limiting step of tryptophan degradation along the kynurenine pathway , and both the reduction in local tryptophan concentration and the production of immunomodulatorytryptophan metabolites contribute to the immunosuppressive effects of IDO. Rencent studies have focused on immunoregulatory role of IDO in mononuclear cells. To seek the new way of curing hepatocellular carcinoma, we investigated the role of IDO in anticancer immune responses.
Methods T-lymphocytes freshly isolated from healthy people and HepG2 cell were cocultured, and IDO inhibitor (1-methyl-tryptophan,1-MT) or exogenous IDO was added to make an intervening experiment. The level of IDO mRNA expression in HepG2 cell cocultured with T-lymphocyte was detected by RT-PCR;the apoptosis rate of HepG2 cell cocultured with T-lymphocyte was analyzed by flow cytometer; the cytotoxicity of T-lymphocyte against HepG2 cell was examined by MTT assay.
Results 1.In HepG2 cell studied, IDO mRNA was not expressed under normal culture conditions. A low level of IDO mRNA expression was detectable in T-lymphocyte under normal culture conditions. The IDO mRNA was expressed exclusively when HepG2 cell was cultured with T-lymphocyte. The level of IDO mRNA expression in HepG2 cells cultured with T-lymphocyte was especially higher than that in na?ve T-lymphocyte. And the level of IDO mRNA expression was faintly induced after addition of 1-MT, but that was not altered by exogenous IDO.
2.The earlier apoptosis rate of HepG2 cell in control group was (3.48±0.34)% and 1-MT(1.25 mmol/l,2.5 mmol/l,5mmol/l) group was respectively(7.82±0.41)%,(18.62±0.42)%,(25.32±0.40)%, the earlier apoptosis rate of HepG2 cell was significantly higher in cocultured condition with 1-MT group than without 1-MT group; the exogenous IDO (0.05 mmol/l,0.5 mmol/l,5mmol/l) group was respectively(3.32±0.35)%,(3.46±0.40)%,(2.75±0.26)%, it was considered statistically significant compared with control group when the concentration of exogenous IDO was 5mmol/l.
3.The cytotoxicity of T-lymphocyte against HepG2 cell in control group was (17.36±1.24)%,and 1-MT(1.25 mmol/l,2.5 mmol/l,5mmol/l) group was respectively(25.48±1.48)%,(32.89±1.73)%,(42.04±2.16)%, there was a significant increase in the cytotoxicity of T-lymphocyte against HepG2 cell in cocultured condition with 1-MT group than without 1-MT group; the exogenous IDO (0.05 mmol/l,0.5 mmol/l,5mmol/l) group was respectively(17.30±0.52)%,(17.48±1.08)%,(15.74±0.79)%, it was considered statistically significant compared with control group when the concentration of exogenous IDO was 5mmol/l, too.
Conclusions These results suggest that IDO from HepG2 cell and some concentration of exogenous IDO can suppress the cytotoxicity of T-lymphocyte against HepG2 cell and the earlier apoptosis rate of HepG2 cell , which may be contributing to hepatocellular carcinoma immune escape , but 1-MT can reverse the immunoregulatory role. In the future, it maybe become a new target for treatment of hepatocellular carcinoma.
引文
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