【摘要】： 目的肝癌患者的免疫系統(tǒng)不能有效地清除或殺傷癌細(xì)胞,是導(dǎo)致肝癌難以治愈、復(fù)發(fā)和轉(zhuǎn)移的重要因素之一。探索肝癌細(xì)胞免疫逃逸的機(jī)制,將為肝癌的治療提供新的思路。吲哚胺2,3-雙加氧酶(IDO)是肝臟以外唯一可催化色氨酸分子氧化裂解的限速酶,在哺乳動(dòng)物的組織和細(xì)胞中廣泛表達(dá)。它通過(guò)降解局部組織中的色氨酸,在誘發(fā)宿主免疫防御、抑制T淋巴細(xì)胞免疫和抗腫瘤免疫、誘導(dǎo)母胎免疫耐受和移植物免疫耐受中均發(fā)揮重要的代謝性免疫調(diào)節(jié)作用。本研究通過(guò)觀察IDO在抗肝癌免疫應(yīng)答中的作用,為臨床治療肝癌提供新的理論依據(jù)。 方法從健康人的外周血中分離出T淋巴細(xì)胞和HepG2細(xì)胞進(jìn)行混合培養(yǎng),分為對(duì)照組和實(shí)驗(yàn)組進(jìn)行比較,對(duì)照組為空白組,實(shí)驗(yàn)組中分別加入IDO的抑制劑1-甲基色氨酸(1-MT)和外源性IDO;用RT-PCR檢測(cè)每組中細(xì)胞IDOmRNA的表達(dá)水平,流式細(xì)胞儀分析混合反應(yīng)體系中HepG2細(xì)胞的凋亡率,四噻唑藍(lán)(MTT)實(shí)驗(yàn)檢測(cè)混合反應(yīng)體系中T淋巴細(xì)胞抗HepG2細(xì)胞的細(xì)胞毒活性。 結(jié)果1.單獨(dú)培養(yǎng)HepG2細(xì)胞,不表達(dá)IDOmRNA,而單獨(dú)培養(yǎng)的T淋巴細(xì)胞表達(dá)微量的IDOmRNA;混合培養(yǎng)后,HepG2細(xì)胞表達(dá)IDOmRNA,而且表達(dá)水平較單獨(dú)培養(yǎng)的T淋巴細(xì)胞高,其表達(dá)水平隨著1-MT濃度的增高而降低,而隨著外源性IDO濃度的增高而無(wú)明顯變化。 2.流式細(xì)胞儀分析混合反應(yīng)體系中HepG2細(xì)胞的凋亡率,對(duì)照組中HepG2細(xì)胞的早期凋亡率為(3.48±0.34)%,1-MT組(濃度分別為1.25 mmol/l,2.5 mmol/l,5mmol/l)中分別為(7.82±0.41)%,(18.62±0.42)%,(25.32±0.40)%,加入1-MT組的HepG2細(xì)胞的早期凋亡率明顯高于對(duì)照組,四者比較有顯著性差異(P0.01);外源性IDO組(濃度分別為0.05 mmol/l,0.5 mmol/l,5mmol/l)中分別為(3.32±0.35)%,(3.46±0.40)%,(2.75±0.26)%,只有當(dāng)外源性IDO濃度為5mmol/l時(shí)HepG2細(xì)胞早期凋亡率與對(duì)照組比較才有統(tǒng)計(jì)學(xué)意義(P0.01)。 3. MTT實(shí)驗(yàn)顯示,對(duì)照組中T淋巴細(xì)胞抗HepG2細(xì)胞的細(xì)胞毒活性為(17.36±1.24)%,1-MT組(濃度分別為1.25 mmol/l,2.5 mmol/l,5mmol/l)中分別為(25.48±1.48)%、(32.89±1.73)%、(42.04±2.16)%,四者比較有顯著性差異(P0.01),并且T淋巴細(xì)胞抗HepG2細(xì)胞的細(xì)胞毒活性隨著1-MT濃度的增高而升高;外源性IDO組(濃度分別為0.05 mmol/l,0.5 mmol/l,5mmol/l)中分別為(17.30±0.52)%,(17.48±1.08)%,(15.74±0.79)%,同樣也是只有當(dāng)外源性IDO濃度為5mmol/l時(shí)與對(duì)照組比較才有統(tǒng)計(jì)學(xué)意義(P0.01)。 結(jié)論HepG2細(xì)胞表達(dá)的內(nèi)源性IDO和一定濃度的外源性IDO均可抑制外周T淋巴細(xì)胞發(fā)揮抗HepG2細(xì)胞的細(xì)胞毒活性作用,降低HepG2細(xì)胞的早期凋亡率,它們可能參與了肝癌的免疫逃逸;而IDO的抑制劑1-MT卻可以逆轉(zhuǎn)這種作用,有望成為治療肝癌的新靶點(diǎn)。
[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 鹵0.34)% in control group and (7.82 鹵0.41)%, (18.62 鹵0.42)%, (25.32 鹵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 鹵0.35)%, (3.46 鹵0.40)%, (2.75 鹵0.26)%, respectively, and those in exogenous IDO group were (3.32 鹵0.35)%, (3.46 鹵0.40)%, (2.75 鹵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 鹵1.24)% in the control group and (25.48 鹵1.48)%, (32.89 鹵1.73)%, (42.04 鹵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 鹵0.52)%, (17.48 鹵1.08)%, (15.74 鹵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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 張路英;吲哚胺2，，3-雙加氧酶對(duì)HepG2細(xì)胞生物學(xué)行為影響的研究[D];山西醫(yī)科大學(xué);2011年

本文編號(hào)：2281449