【作者】 徐金环；

【导师】 张义成；

【作者基本信息】 华中科技大学 ， 内科学， 2013， 博士

【摘要】 [目的]研究IDO和IFNγ在异基因造血干细胞移植后病人中的表达情况,了解IDO活性与aGVHD的严重程度及IFN的关系,探讨IDO的活性检测在诊断aGHVD及aGHVD分级中的作用[方法]逆转录PCR法检测89例异基因造血干细胞患者和8个正常人单个核细胞中IDOmRNA的表达情况；反相高效液相色谱法检测血浆中IDO的活性；ELISA检测IFNγ的水平。[结果]在74个有aGVHD的患者中55个(74.32%)检测到IDOmRNA的表达,在26个无aGVHD患者仅有2个检测到IDOmRNA的表达,8个健康人中未检测到IDO表达；血浆IDO的活性在(?)GVHD病人远高于无aGVHD病人(4.74±3.35vs1.79±1.02；P<0.0001)或者健康人(4.74±3.35vs1.77±0.22；P<0.0001),重度aGVHD(Ⅲ/Ⅳ级)患者IDO活性高于轻度aGVHD(Ⅰ/Ⅱ级)患者(6.57±3.34vs2.46±1.41；P<0.0001).同时,在aGVHD病人血浆IFNγ的水平也是增高的(P=0.0043)；aGVHD缓解后IDO的活性下降,当aGVHD复发时,IDO的活性又会升高；血浆IDO的活性与IFN的水平相关(r=0.8288；P<0.0001),利用ROC曲线分析,IDO活性的曲线下面积高于IFNγ(0.852vs0.694),IDO的灵敏度和特异度分别是81%和78%,而IFNγ的灵敏度和特异度分别是41%和93%。[结论]aGVHD病人外周血单个核细胞有IDOmRNA表达；血浆IDO的活性增高在aGVHD病人且与aGVHD的严重程度相关；结合血浆IFN水平,IDO活性可能作为诊断和评估异基因造血干细胞移植后aGVHD的生物标志；干预IDO途径可能是一个可用的方法去治疗激素无效的aGVHD。[目的]检测吲哚胺2,3加氧酶(IDO)在aGVHD小鼠各组织器官的表达情况,探究IDO与aGVHD靶器官损伤的关系,为调节色氨酸代谢途径抑制aGVHD反应甚至诱导免疫耐受做初步探索。[方法]以BALB/c (H-2b)雌性小鼠为受者,接受致死剂量的Y射线(8.0Gy)全身照射后,输注雄性C57BL/6(H-2d)供鼠的骨髓细胞和脾细胞,同时设立同基因移植对照组,观察受鼠的体征和存活时间,进行aGVHD临床评分,aGVHD评分≥7时,处死小鼠,应用反相高效液相色谱法(HPLC)检测血浆中色氨酸(Trp)和其代谢产物犬尿氨酸(Kyn)的浓度,以及IDO的活性,用RT-PCR和免疫组化方法检测各组织中IDOmRNA与蛋白的表达水平,流式细胞学检测外周血CD4+/CD25+Treg细胞比例。[结果]异基因移植组小鼠血浆中Trp浓度明显低于同基因移植小鼠(P<0.05),而Kyn浓度高于对照小鼠(P<0.05),IDO活性明显较对照组高(P<0.01)。结肠和肺IDO mRNA表达水平高于对照小鼠(P<0.01),两组小鼠小肠IDO表达水平很低对比无明显差异,皮肤和肝脏IDO mRNA表达不明显。免疫组化结果显示：aGVHD小鼠肺脏和结肠上皮细胞和血管内皮细胞高表达IDO；小肠组织IDO的表达差别不明显；皮肤血管内皮细胞检测到IDO的表达且组织中散在IDO阳性单个核细胞；肝脏血管内皮细胞表达IDO,且汇管区周围散在分布IDO阳性的单个核细胞；对照小鼠各组织IDO的表达低或不表达。[结论]IDO的表达在aGVHD靶器官组织水平的免疫调节中有重要作用,血管内皮细胞作为最先接触到异体反应T细胞的受体细胞,参与了这一作用；调节色氨酸代谢途径可能是治疗aGVHD一个有效对策。[目的]通过吲哚胺2,3加氧酶(IDO)代谢局部色氨酸被认为是调节T细胞免疫的一个重要机制。3,4-DAA是一个人工合成的有活性的邻氨基苯甲酸酸衍生物,已经被证实对治疗Thl介导的自身免疫性疾病例如多发性硬化是有效的。我们的目的是研究3,4-DAA在治疗aGVHD的效果及它可能的作用机制[方法]构建一个小鼠的aGVHD模型,在骨髓移植后立即或aGVHD启动时,腹腔注射3,4-DAA,每只小鼠200mg/kg/day,连续用14天；定期记录aGVHD的表现和小鼠的存活情况；HE染色评估靶器官的病理损伤；反相高效液相色谱法和Elisa检测血浆中IDO的活性及细胞因子的水平。[结果]骨髓移植后给药3,4-DAA明显的降低aGVHD的严重程度和组织学评分；与对照组相比,给予3,4-DAA预防和治疗组小鼠的生存期延长,在3,4-DAA治疗组小鼠,血浆IFNγ和IL-2的水平下降,而IDO的活性剂IL-10的水平提高。与体内实验一致,在体外,3,4-DAA也能够抑制脾脏T淋巴细胞分泌IFNγ和IL-2。[结论]3,4-DAA能够减轻小鼠实验性aGVHD通过调节辅助T淋巴细胞分化,这一特性使它成为一个可能备选的药物来用于临床预防和治疗GVHD。[目的]探讨干扰素-γ(IFNγ)诱导小鼠肺微血管内皮细胞吲哚胺2,3-双加氧酶(IDO)的表达情况,及体外对T细胞增殖的影响。[方法]组织块贴壁法培养小鼠肺微血管内皮细胞,进行形态特征观察,并进行系列鉴定,经过传代纯化后,分别用不同浓度的IFNy诱导作用微血管内皮细胞,Western blot和RT-PCR分别测定未经处理的内皮细胞以及IFNγ和或VP-16处理后内皮细胞IDO基因及蛋白表达水平。反相高效液相色谱法检测细胞培养上清中色氨酸、犬尿氨酸的水平,从而检测IDO活性的水平。在体外将肺微血管内皮细胞与小鼠脾脏T淋巴细胞共培养,T淋巴细胞用羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFSE)标记,流式细胞仪测定T细胞的增殖情况,[结果]获得的内皮细胞具有规律的鹅卵石样形态,CD31相关抗原间接免疫荧光染色阳性而且在透射电镜观察到Weilel—palade小体。IFNy处理后的微血管内皮细胞IDOmRNA及蛋白表达增高,IDO活性水平随IFNy的浓度的增大逐渐提高。VP-16作用后,在IFNy刺激下,肺微血管内皮细胞表达IDO的能力减低,无IFNγ刺激,微血管内皮细胞不表达IDO。IFNγ处理后的微血管内皮细胞能够抑制T细胞的增殖(P<0.05)而未处理的内皮细胞对T细胞的增殖抑制作用不显著。[结论]IFNγ能够通过诱导IDO的表达增强内皮细胞在体外对T淋巴细胞增殖的抑制作用。更多还原

【Abstract】 [Objective] To evaluate the expression levels of IDO and interferon (IFN)y in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to identify the correlation between IDO activity, IFNy and acute graft-versus-host disease (aGVHD),[Methods] We measured IDO mRNA expression in peripheral blood mononuclear cells in89allo-HSCT patients by reverse transcriptionpolymerase chain reaction. The IDO activity in plasma was also performed by reverse-phase high-performance liquid chromatography (HPLC); plasma IFNy was detected by a standard enzyme-linked immunosorbent assay (Elisa)[Result] IDO mRNA was detected in55of74patients (74.32%) with aGVHD. Of patients without aGVHD, only2of26expressed IDO mRNA (7.69%); none of8healthy volunteers was positive for IDO expression. Plasma IDO activity was much higher in aGVHD patients than in those without aGVHD (4.74±3.35vs1.79±1.02, respectively; P <0.0001) or in healthy control subjects (4.74±3.35vs1.77±.22; P<0.0001). Patients with severe (grade Ⅲ/Ⅳ) aGVHD had much higher IDO activity than those with mild (grade Ⅰ/Ⅱ) aGVHD (6.57±3.34vs2.46±1.41; P<0.0001). Meanwhile, there was a significant increase in plasma IFNy level in aGVHD patients (P=0.0043). IDO activity decreased after alleviation of aGVHD, whereas fluctuation of plasma IDO was also observed upon the recurrence of aGVHD. Plasma IDO activity was correlated with the level of plasma IFNy(r=0.8288; P<0.0001). Using receiver-operating characteristic curves analysis, the sensitivity and specificity for evaluation of aGVHD were determined. The area under the curve of IDO activity was higher than that of IFNy(0.852vs0.694) with a sensitivity and specificity for IDO of81%and78%, respectively, whereas the sensitivity and specificity for IFNy were41%and93%, respectively.[Conclusion] IDO mRNA was expressed in blood mononuclear cells of patients with aGVHD. Plasma IDO activity was elevated in aGVHD patients and was correlated with the severity of aGVHD. In combination with plasma IFN-g, IDO activity may represent a potential biomarker for the diagnosis and evaluation of aGVHD after allo-HSCT. Intervention of the IDO pathway may also represent an alternative way to overcome steroid-resistant aGVHD. [Objective] To explore the relationship between IDO and the damage of aGVHD target organs,we detected the expression level of indoleamine2,3dioxygenase (IDO) in various organs of mice aGVHD models, Provide preliminary exploration for regulating IDO pathway may beneficial to the prevention of aGVHD and induction of tolerance.[Method] Lethally (8.0Gy) irradiated female BALB/c (H-2b) recipients were transplanted with bone marrow cells and splenic cells from male C57BL/6(H-2d) donors. The recipients were observed for the features of GVHD, established syngeneic transplant control group, observed the signs and the survival time of the mice, aGVHD clinical score, aGVHD score(?)7, the mice were sacrificed, reversed-phase high performance liquid chromatography (HPLC) measured the concentration of plasma tryptophan (Trp) and kynurenine (KYN);detected the IDOmRNA protein expression levels of the organ,by RT-PCR and immunohistochemical, flow cytometric was used to detect CD4+/CD25+T cells ratio. In peripheral blood[Results] Plasma Trp concentrationin in allogeneic transplant mouse was significantly lower than syngeneic transplant mice (P<0.05), the KYN concentration higher than that of control mice (P<0.05), IDO activity was significantly higher than control group (P<0.01). IDO mRNA expression levels of colon and lung higher than the control mice, small intestine IDO expression level in two groups mice is very low, the difference is no significant, in skin and liver, IDO mRNA expression was not obvious. Immunohistochemical results showed that:in aGVHD mice, lung and colon epithelial cells and endothelial cells highly expressed IDO; vascular endothelial cells of skin express IDO,meanwhile scattered IDO-positive mononuclear cells in skin; the scattered distribution of IDO-positive mononuclear cells in liver around blood vessels; in syngeneic transplant mice,IDO expression was very low or no expression. [Conclusion] IDO expression play an important role in the immune regulation of tissue level in aGVHD target organs, vascular endothelial cells as a first recipient cells that contact to the allo-reactive T cell were involved in this role; regulate IDO pathway maybe a effective way for treating aGVHD [Background] Local catabolism of tryptophan (Trp) by indoleamine2,3-dioxygenase (IDO) is considered an important mechanism of regulating T cell immunity.N-(3,4-dimethoxycinnamonyl) anthranilic acid (3,4-DAA) is an active synthetic anthranilic acid derivative which was proved to be effective to treat type I helper T lymphocytes (Thl) mediated autoimmune diseases such as multiple sclerosis. In this report, we investigated the effects of3,4-DAA on the acute graft versus host disease (aGVHD) following allogeneic bone marrow transplantation (allo-BMT) and its potential mechanism of action.[Methods] Using a murine aGVHD model,3,4-DAA was injected intraperitoneally at200mg/kg/day per mouse immediately after allo-BMT or at the onset of aGVHD for14consecutive days; the signs of aGVHD and the survival were recorded periodically; the histological changes of target organs were evaluated with hematoxylin-eosin staining; the IDO activity and cytokine levels in plasma were measured by reverse-phase high-performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay(ELISA), respectively.[Results] Administration of3,4-DAA after allo-BMT significantly reduced the severity and the histological score of aGVHD; The survival for mice receiving3,4-DAA prophylaxis and treatment were prolonged in comparison to the vehicle control mice. The plasma levels of IFN-y and IL-2in3,4-DAA treatment group were found to be decreased, while the IDO activity and the IL-10level elevated in these mice. In consistent with the in vivo results,3,4-DAA also inhibited IFN-y and IL-2production of spleen T lymphocytes in vitro.[Conclusions]3,4-DAA diminishes the murine experimental aGVHD through inhibiting Th1responses, this property makes it a potential alternative agent for prevention and treatment of GVHD in the clinic. [Objective] To investigate the indoleamine2,3-dioxygenase expression in pulmonary microvascular endothelial cells (PMVEC)induced by IFNy and its role in T cell proliferation[Method] Mouse PMVEC were cultivated by Tissue block method and observed of their morphological features. After series of identification and Passage purification, they were dealed with different concentrations of IFNy. Primary EC or the EC treated with IFNy or VP16was detected the IDO expression by RT-PCR and Western blot, reversed phase high-performance liquid chromatography (HPLC) was used to evaluate the the levels of tryptophan and kynurenine and the activity of IDO. after co-cultivating PMVEC and T lymphocyte derived from mice spleen, FMC was applied to detect the proliferation of T cell by labeled with carboxy fluorescein succinimidyl ester(CFSE).[Results] The EC acquired exposed the regular features of Cobblestone in morphology was positive in CD31expression with indirect immunofluorescence staining, and can be observed of the Weilel—palade globule by Transmission electron microscopy. There was higher level expression of IDOmRNA and protein in IFNy-treated PMVEC, and with the raising of the concentration of IFNy, the activity of IDO increased.After dealed with VP-16, with the stimulation of IFNy, a reduced IDO expression in PMVEC was evaluated, while no IDO expressed in without IFNy stimulation group, the PMVEC treated with IFNy inhibited the T cell proliferation, while the untreated showed no significant inhibition.[Conclusion] PMVEC could suppress T-lymphocyte responses via IDO enzyme activity in vitro.更多还原