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
发布时间：2018-10-19 14:44
【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(P0.01);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(P0.01)。 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(P0.01),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(P0.01)。 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。
[Abstract]:Objective the immune system of HCC patients can not effectively remove or kill cancer cells, which is one of the important factors leading to the refractory, recurrence and metastasis of HCC. Exploring the mechanism of immune escape of hepatoma cells will provide new ideas for the treatment of liver cancer. Indole 3-dioxygenase (IDO) is the only rate-limiting enzyme that catalyzes the oxidative cleavage of tryptophan molecules outside the liver and is widely expressed in mammalian tissues and cells. It plays an important role in inducing host immune defense, inhibiting T lymphocyte immunity and anti tumor immunity, inducing maternal and fetal immune tolerance and graft immune tolerance by degrading tryptophan in local tissues. The purpose of this study was to observe the role of IDO in anti-hepatoma immune response and to provide a new theoretical basis for clinical treatment of HCC. Methods T lymphocytes and HepG2 cells were isolated from the peripheral blood of healthy people and cultured in a mixed culture. The cells were divided into two groups: control group and experimental group. The control group was a blank group. 1 methyltryptophan (1-MT), an inhibitor of IDO, and exogenous IDO; were used to detect the expression of IDOmRNA in each group. Flow cytometry was used to analyze the apoptosis rate of HepG2 cells in the mixed reaction system. The cytotoxic activity of T lymphocytes against HepG2 cells in mixed reaction system was detected by tetrathiazolium (MTT) assay. Result 1. When HepG2 cells were cultured alone, the HepG2 cells expressed IDOmRNA, and the level of IDOmRNA, expression was higher than that of T lymphocytes cultured alone. The expression level of HepG2 cells decreased with the increase of 1-MT concentration. However, with the increase of exogenous IDO concentration, there was no significant change. 2. The apoptosis rate of HepG2 cells in mixed reaction system was (3.48 卤0.34)% in control group and (7.82 卤0.41)%, (18.62 卤0.42)%, (25.32 卤0.40)% in 1-MT group, respectively. The early apoptosis rate of HepG2 cells in 1-MT group was significantly higher than that in control group. There were significant differences among the four groups (P0.01), and the rates of early apoptosis of HepG2 cells in exogenous IDO group were (3.32 卤0.35)%, (3.46 卤0.40)%, (2.75 卤0.26)%, respectively, and those in exogenous IDO group were (3.32 卤0.35)%, (3.46 卤0.40)%, (2.75 卤0.26)%, respectively. Only when exogenous IDO concentration was 5mmol/l, the early apoptosis rate of HepG2 cells was significantly higher than that of control group (P0.01). MTT experiments show that, The cytotoxic activity of T lymphocytes against HepG2 cells was (17.36 卤1.24)% in the control group and (25.48 卤1.48)%, (32.89 卤1.73)%, (42.04 卤2.16)% in the 1-MT group, respectively. The cytotoxic activity of T lymphocytes against HepG2 cells increased with the increase of 1-MT concentration. The concentrations of exogenous IDO were (17.30 卤0.52)%, (17.48 卤1.08)%, (15.74 卤0.79)%, respectively. There was also significant difference only when the exogenous IDO concentration was 5mmol/l (P0.01). Conclusion Endogenous IDO expressed by HepG2 cells and exogenous IDO at a certain concentration can inhibit the cytotoxic activity of peripheral T lymphocytes and decrease the early apoptosis rate of HepG2 cells, which may be involved in the immune escape of HCC. 1-MT, an inhibitor of IDO, can reverse this effect and may become a new target for the treatment of liver cancer.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2281449
[Abstract]:Objective the immune system of HCC patients can not effectively remove or kill cancer cells, which is one of the important factors leading to the refractory, recurrence and metastasis of HCC. Exploring the mechanism of immune escape of hepatoma cells will provide new ideas for the treatment of liver cancer. Indole 3-dioxygenase (IDO) is the only rate-limiting enzyme that catalyzes the oxidative cleavage of tryptophan molecules outside the liver and is widely expressed in mammalian tissues and cells. It plays an important role in inducing host immune defense, inhibiting T lymphocyte immunity and anti tumor immunity, inducing maternal and fetal immune tolerance and graft immune tolerance by degrading tryptophan in local tissues. The purpose of this study was to observe the role of IDO in anti-hepatoma immune response and to provide a new theoretical basis for clinical treatment of HCC. Methods T lymphocytes and HepG2 cells were isolated from the peripheral blood of healthy people and cultured in a mixed culture. The cells were divided into two groups: control group and experimental group. The control group was a blank group. 1 methyltryptophan (1-MT), an inhibitor of IDO, and exogenous IDO; were used to detect the expression of IDOmRNA in each group. Flow cytometry was used to analyze the apoptosis rate of HepG2 cells in the mixed reaction system. The cytotoxic activity of T lymphocytes against HepG2 cells in mixed reaction system was detected by tetrathiazolium (MTT) assay. Result 1. When HepG2 cells were cultured alone, the HepG2 cells expressed IDOmRNA, and the level of IDOmRNA, expression was higher than that of T lymphocytes cultured alone. The expression level of HepG2 cells decreased with the increase of 1-MT concentration. However, with the increase of exogenous IDO concentration, there was no significant change. 2. The apoptosis rate of HepG2 cells in mixed reaction system was (3.48 卤0.34)% in control group and (7.82 卤0.41)%, (18.62 卤0.42)%, (25.32 卤0.40)% in 1-MT group, respectively. The early apoptosis rate of HepG2 cells in 1-MT group was significantly higher than that in control group. There were significant differences among the four groups (P0.01), and the rates of early apoptosis of HepG2 cells in exogenous IDO group were (3.32 卤0.35)%, (3.46 卤0.40)%, (2.75 卤0.26)%, respectively, and those in exogenous IDO group were (3.32 卤0.35)%, (3.46 卤0.40)%, (2.75 卤0.26)%, respectively. Only when exogenous IDO concentration was 5mmol/l, the early apoptosis rate of HepG2 cells was significantly higher than that of control group (P0.01). MTT experiments show that, The cytotoxic activity of T lymphocytes against HepG2 cells was (17.36 卤1.24)% in the control group and (25.48 卤1.48)%, (32.89 卤1.73)%, (42.04 卤2.16)% in the 1-MT group, respectively. The cytotoxic activity of T lymphocytes against HepG2 cells increased with the increase of 1-MT concentration. The concentrations of exogenous IDO were (17.30 卤0.52)%, (17.48 卤1.08)%, (15.74 卤0.79)%, respectively. There was also significant difference only when the exogenous IDO concentration was 5mmol/l (P0.01). Conclusion Endogenous IDO expressed by HepG2 cells and exogenous IDO at a certain concentration can inhibit the cytotoxic activity of peripheral T lymphocytes and decrease the early apoptosis rate of HepG2 cells, which may be involved in the immune escape of HCC. 1-MT, an inhibitor of IDO, can reverse this effect and may become a new target for the treatment of liver cancer.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 张路英;吲哚胺2，，3-双加氧酶对HepG2细胞生物学行为影响的研究[D];山西医科大学;2011年
本文编号：2281449
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