兰州大学机构库 >学院待认领
抑制吲哚胺2,3-双加氧酶活性促进慢性粒细胞白血病源树突状细胞的功能
Alternative TitleInhibition of indoleamine 2,3-dioxygenase activity promotes function of dendritic cells derived from chronic myeloid leukemia
许思娟
Thesis Advisor张连生
2009-05-26
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword慢性粒细胞性白血病 吲哚胺2, 3-二氧化酶 树突状细胞 1-甲基色氨酸
Abstract一 目的 树突状细胞(dendritic cell, DC) 因其强大的抗原提呈功能和激活抗原特异性CTL( cytotoxic T lymphocytes)反应,成为肿瘤免疫治疗的重要载体。以DC为基础诱导的特异性抗肿瘤免疫治疗也成为肿瘤生物治疗领域的研究热点,白血病细胞来源的DC因携带自身肿瘤抗原在白血病免疫治疗中倍受关注。研究发现白血病来源的DC与正常DC相比,其表面分子表达相似,但抗原提呈功能较弱。吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)是色氨酸分解代谢所必需的限速酶,在体内分布比较广泛。由于IDO的活化,使得游离的色氨酸减少以及色氨酸代谢产物的增加,导致细胞周期停滞、T细胞无应答,以及诱导调节性T细胞(CD4+CD25+ regulatory T cell ,Treg)的产生。已证实IDO有免疫负调节作用并参与肿瘤免疫逃逸。随着慢性粒细胞性白血病源树突状细胞(dendritic cells derived from chronic myeloid leukemia,CML-DCs)的成熟,IDO表达明显上调,因此我们设想能否通过抑制CML-DCs中IDO的活性以增强CML-DCs的免疫刺激功能。 二 方法 采集初治慢性粒细胞性白血病(CML)慢性期患者的骨髓,分离单个核细胞(mononuclear cells, MNCs),采用人rhGM-CSF和rhIL-4细胞因子组合先将CML-MNCs诱导分化为不成熟DC(imDC),再加用TNF-α进一步使其成熟(mDC)。RT-PCR检测CML-DCs中的IDO- mRNA的表达情况。流式细胞仪检测CML-DCs免疫表型。在有或无IDO抑制剂1-甲基色氨酸(1-MT)作用下,分别以不成熟CML-DCs(imDC)和成熟CML-DCs(mDC)为刺激细胞,完全缓解期(complete remission,CR)CML患者外周T淋巴细胞为反应细胞建立混合淋巴细胞反应体系,ELISA法检测CML-DCs上清液IL-12水平,MTT法检测CML-DCs刺激自体T淋巴细胞的增殖能力。 三 结果 慢性粒细胞性白血病骨髓单个核细胞经体外扩增后,具有典型的树突状形态特征,经染色体检查证实了其慢粒源性。RT-PCR检测成熟DCs和不成熟DCs中IDOmRNA的表达,显示随着CML-DCs的诱导分化和成熟,IDOmRNA的表达不断增高(P<0.01);经TNF-α诱导的DCs的免疫表型除CD1a外,CD80、CD86、CD83、HLA-DR的表达明显上调(P<0.05),上述表达不受1-MT的影响。应用1-MT抑制IDO活性后的不成熟DCs 和成熟DCs,其分泌IL-12水平均明显增高(P<0.05,P<0.01),且激发自体T淋巴细胞增殖的能力均明显增强(P<0.05,P<0.01)。 四 结论 本实验结果显示,随着慢性粒细胞白血病源性树突状细胞诱导和分化的成熟,具有免疫负调节作用的IDO其表达不断升高。抑制慢性粒细胞性白血病源性树突状细胞中IDO的活性可提高慢性粒细胞白血病源性树突状细胞的IL-12分泌水平且可增强其对自体T细胞增殖的刺激能力。说明IDO在DC负性调节中起着极为重要的作用。抑制IDO活性的策略将使CML-DCs疫苗发挥更强的抗白血病效应,对临床克服微小残留病、防止复发并最终治愈白血病有着深远的理论和实践意义,为白血病生物治疗提供了全新的思路和方法。
Other AbstractObjective Dendritic cells (DCs) are the most powerful profession antigen-presenting cells (APCs) in vivo and play a crucial central role in the outcome of several immune responses. DCs have been successfully used in clinical trails to induce tumor-specific immunity. Although the DCs derived from leukemia patients have the similar morphology and immunophenotype with DCs from normal individuals, the immunological function are weaker. Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the catabolism of tryptophan along the kynurenine pathway, which is widely distributed in the body. IDO enzymatic activity results in the local depletion of tryptophan and a local increase in the concentration of downstream metabolites. The decrease in tryptophan and the local increase in tryptophan metabolites can cause cell cycle arrest, anergy induction in responding T cells,and differentiation of new Tregs from uncommitted CD4+T cells. IDO has been confirmed negative regulation of immune and participated in tumor immune escape. Expressions of IDO were obviously higher after DC maturation. To investigate the expression of indoleamine 2,3-dioxygenase (IDO) in dendritic cells derived from chronic myeloid leukemia(CML-DCs) and to study the influence of IDO activity inhibition on the function of CML-DCs. Methods After inspection of Hematology, bone marrow, chromosome and molecular, a clear diagnosis of chronic myeloid leukemia. Bone marrow mononuclear cells (BMMNCs) were isolated from CML patients by density gradient centrifugation and were cultured in RPMI-1640 culture medium supplemented with recombinant human (rh) GM-CSF and rhIL-4.The expressions of IDO mRNA in dendritic cells derived from chronic myeloid leukemia were detected by RT-PCR. The phenotypes of CML-DCs were analyzed by flow cytometry. The immature CML-DCs (imDC) and the mature CML-DCs (mDC) were used as stimulating cells and autologous T-lymphocytes were used as reactive cells for a mixed lymphocyte reaction system.IL-12 concentrations were detected by ELISA kits; Mixed lymphocyte reaction were analyzed by MTT assay. Results It was demonstrated that DCs derived from bone marrow mononuclear cells of CML displayed the typical morphology of DCs. Expressions of co-stimulatory molecules on DCs, such as CD80, CD86, CD83 and HLA-DR, except for CD1a, were obviously higher after maturation (P<0.05) and were not be influenced by 1-methyltryp tophan (1-MT). Inhibition IDO activity in mature and imma...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222336
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
许思娟. 抑制吲哚胺2,3-双加氧酶活性促进慢性粒细胞白血病源树突状细胞的功能[D]. 兰州. 兰州大学,2009.
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