IDO和FoxP3表达与宫颈癌发生和病理进程关联的分析

目的研究讨论FoxP3表达和吲哚胺2,3-双加氧酶与宫颈癌发生和病理进程关联。方法选取2005年²013年患者标本,50例实验组和20例对照组。其中实验组包含10例(CINⅠ2013年患者标本,50例实验组和20例对照组。其中实验组包含10例(CINⅠ^Ⅱ级组),10例CINⅢ级组,宫颈癌30例(宫颈癌组)。应用SPSS 16.0统计分析软件,且P<0.05时,数据具有统计学意义。结果实验结果表明,不论是CIN组还是对照组DIO和FoxP3都明显的低于宫颈癌组。且IDO的阳性表达式与宫颈癌FIGO分期、宫颈癌FIGO组织学分级相关;FoxP3的阳性表达只与FIGO分期相关。结论 IDO和FoxP3对于宫颈癌的表达结果普遍大于实验组和对照组,可见IDO、FoxP3与宫颈癌的发展和发生有着密切的关联     

shRNA沉默IDO基因的表达对裸鼠卵巢癌种植瘤生长的影响

目的 研究短发夹RNA(shRNA)沉默吲哚胺2,3-双加氧酶(IDO)基因表达对裸鼠卵巢癌种植瘤生长的影响.方法 shRNA沉默IDO基因表达质粒和空白质粒分别稳定转染至人卵巢癌细胞株SKOV-3中,应用Western blot检测IDO在SKOV-3、SKOV-3/Mock和SKOV-3/shIDO细胞中的表达,MTT检测肿瘤细胞体外生长速度,观察各组裸鼠体内卵巢癌种植瘤的生长速度.结果 IDO蛋白在SKOV-3和SKOV-3/Mock细胞中有表达,而在SKOV-3/shIDO细胞中表达明显被抑制.SKOV-3、SKOV-3/Mock和SKOV-3/shIDO细胞在体外生长速度无统计学差异(P＞0.05).与SKOV-3和SKOV-3/Mock细胞比较,SKOV-3/shIDO细胞接种的肿瘤生长缓慢(P＜0.05).结论 应用shRNA沉默IDO基因表达质粒稳定转染SKOV-3细胞,对卵巢癌细胞体外生长速度无影响,但可抑制裸鼠皮下卵巢癌种植瘤的生长.因此,IDO有望作为卵巢癌基因治疗的新靶点.     

基于靶标IDO1、TDO的抑制剂研究进展

吲哚胺2,3-双加氧酶1(IDO1)和色氨酸2,3-双加氧酶(TDO)催化色氨酸代谢的第一步也是限速步骤,与肿瘤免疫耐受和患者的不良预后相关,已经成为肿瘤免疫治疗的重要靶标.到目前为止,有9个IDO1抑制剂、3个IDO1/TDO双靶点抑制剂进入临床试验.本篇综述从药物化学角度总结了IDO1抑制剂、TDO抑制剂、IDO1...     

分子嵌合MHC-Ⅰ基因对小鼠DC反应的研究

目的:构建分子嵌合主要组织相容性复合体(MHC)-Ⅰ基因小鼠骨髓造血干细胞,并探讨其诱导脾脏T细胞对异基因小鼠树突状细胞(DC)反应的机制。方法:密度梯度法分离培养BALB/c小鼠骨髓造血干细胞。构建携带C57BL/6小鼠MHC-Ⅰ基因慢病毒载体(病毒感染组),携带无意义基因慢病毒载体(阴性对照组)。分别感染BALB/c小鼠骨髓造血干细胞,构建分子嵌合细胞。分别取病毒感染组、阴性对照组及未加入病毒的空白对照组造血干细胞输注BALB/c小鼠后7d,获取脾脏T淋巴细胞,分别与C57BL/6小鼠DC进行混合淋巴细胞培养,测定刺激指数。结果:成功体外分选及培养BALB/c小鼠骨髓造血干细胞。病毒感染组C57BL/6小鼠MHC-Ⅰ蛋白表达率可达98.17%。单向混合淋巴细胞培养结果显示,C57BL/6小鼠DC对输注病毒感染组细胞后BALB/c小鼠脾脏T细胞刺激指数明显降低(P<0.01)。结论:输注分子嵌合MHC-Ⅰ基因造血干细胞后,小鼠脾脏T细胞对异基因小鼠DC反应明显减低。     

The Research on Chimeric MHC-Ⅰ Gene Responsing to Allogeneic Dendritic Cells

目的:构建分子嵌合主要组织相容性复合体(MHC)-Ⅰ基因小鼠骨髓造血干细胞,并探讨其诱导脾脏T细胞对异基因小鼠树突状细胞(DC)反应的机制.方法:密度梯度法分离培养BALB/c小鼠骨髓造血干细胞.构建携带C57BL/6小鼠MHC-Ⅰ基因慢病毒载体(病毒感染组),携带无意义基因慢病毒载体(阴性对照组).分别感染BALB/c 小鼠骨髓造血干细胞,构建分子嵌合细胞.分别取病毒感染组、阴性对照组及未加入病毒的空白对照组造血干细胞输注BALB/c小鼠后7d,获取脾脏T淋巴细胞,分别与C57BL/6小鼠DC进行混合淋巴细胞培养,测定刺激指数.结果:成功体外分选及培养BALB/c小鼠骨髓造血干细胞.病毒感染组C57BL/6小鼠MHC-Ⅰ蛋白表达率可达98.17％.单向混合淋巴细胞培养结果显示,C57BL/6小鼠DC对输注病毒感染组细胞后BALB/c小鼠脾脏T细胞刺激指数明显降低(P＜0.01).结论:输注分子嵌合MHC-Ⅰ基因造血干细胞后,小鼠脾脏T细胞对异基因小鼠DC反应明显减低.     

吲哚胺2，3-双加氧酶的表达与宫颈癌发生和病理进程的关系研究

目的：探讨吲哚胺2,3-双加氧酶（IDO）的表达情况与女性宫颈癌的发生与病理进程的相关性。方法选取宫颈癌手术治疗后的石钠组织标本75例（宫颈癌组）,宫颈上皮瘤变Ⅰ级标本30例（CINⅠ级组）、宫颈上皮瘤变Ⅱ~Ⅲ级标本30例（CINⅡ~Ⅲ级组）、体检收集的正常宫颈标本30例（正常组）,采用免疫组织化学法检测各组样本中的 IDO 阳性表达率,并追踪观察 IDO 表达与宫颈癌患者病理及生存与后的关系。结果宫颈癌组标本的 IDO 阳性表达率显著的高于 CINⅠ级组、CINⅡ~Ⅲ级组和正常组（ P <0.05）;CINⅡ~Ⅲ级组 IDO 阳性表达率显著的高于正常组（ P <0.05）。低分化宫颈癌患者、Ⅲ+Ⅳ期宫颈癌患者、发生淋巴结转移的宫颈癌患者的 IDO 阳性率表达显著的增高,IDO 阳性表达与患者的分化程度、FIGO 分期、淋巴结转移具有一定的关系（P <0.05）。经过36个月的随访,IDO 阳性表达53例患者中有7例死亡、2例失访,IDO 阴性表达患者22例中有3例死亡,1例失访,IDO 表达阳性与阴性患者的3年病死率差异无统计学意义（ P >0.05）;IDO 阴性表达患者的3年生存中位时间35.4个月与 IDO 阳性表达患者的34.2个月差异无统计学意义（ P >0.05）。结论 IDO 在宫颈癌组织中高表达,与宫颈癌的分化程度、FIGO 分期、淋巴结转移具有一定的关系,与患者的3年生存预后关系不显著,需进一步延长随访时间进行观察。     

IDO抑制剂的研究进展

吲哚胺2,3-双加氧化酶（indoleamine 2,3-dioxygenase, IDO）是肝脏以外唯一的催化色氨酸沿犬尿氨酸途径分解代谢的限速酶。IDO可通过降低微环境中色氨酸的浓度而达到抑制病原微生物增殖的作用; IDO与神经系统疾病也密切相关,它能降低5-羟色胺的水平而导致抑郁, 也可造成脑中喹啉酸等具有神经毒性的代谢产物的累积; IDO在抑制T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。IDO已被证实与阿尔茨海默病、白内障、癌症等多种人类重大疾病密切相关,因此IDO抑制剂作为重要的药物受到日益广泛的关注。本文综述近年来IDO抑制剂的研究进展。     

共载多柔比星和IDO1 siRNA的高渗透性纳米粒增强肿瘤免疫治疗

肿瘤免疫治疗存在两个严重障碍:(1)免疫抑制肿瘤微环境(immunosuppressive tumor microenvironment,ITM)和肿瘤的低免疫原性的存在,严重降低肿瘤的免疫应答;(2)致密而复杂的病理生理屏障严重限制了实体瘤的深部给药。化疗药物多柔比星(doxorubicin, DOX)诱导的肿瘤免疫原性细胞死亡(immunogenic cell death, ICD)是增强肿瘤免疫活性的有效方法。但是ICD作用后细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)分泌的干扰素-γ(interferon-γ, IFN-γ)会增加吲哚胺2,3-双加氧酶1 (indoleamine 2,3-dioxygenase 1, IDO1)蛋白的表达,其能够增强ITM。而IDO1 siRNA的协同作用会降低IDO1蛋白的表达,调节肿瘤免疫抑制微环境,调节ITM,从而增强DOX的ICD作用。本文利用pH敏感材料PLD [poly(ethylene glycol)-poly-L-lysine-2,3-dimethylmaleic anhydride, mPEG-...     

吲哚胺2,3-双加氧酶1(IDO1)抑制剂的研究进展

吲哚胺2,3-双加氧酶1 (indoleamine 2,3-dioxygenase 1,IDO1)是代谢色氨酸转化为犬尿氨酸的限速酶.其在肿瘤组织中过表达,造成肿瘤微环境中的色氨酸耗竭,从而抑制T细胞功能,介导肿瘤的免疫逃逸,是潜在的肿瘤免疫治疗靶点.目前,多个IDO1抑制剂处于临床研究阶段.本文主要介绍IDO1介导肿...     

FasL转染小鼠树突状细胞诱导异系T淋巴细胞凋亡的研究

目的:制备稳定表达FasL蛋白的小鼠骨髓源树突状细胞(DC)并探讨其体外诱导异系T淋巴细胞凋亡的机制。方法:采用培养基选择法体外培养小鼠骨髓源DC,脂质体法转染FasL基因至小鼠成熟DC,流式细胞仪检测转染前后FasL蛋白的表达。混合淋巴细胞培养,四甲基偶氮唑蓝(MTT)法检测DC对异系T淋巴细胞增殖的影响,流式细胞仪检测诱导异系T淋巴细胞凋亡的能力。结果:体外培养可获得成熟的小鼠骨髓源DC,转染FasL基因的DC较未转染DCFasL蛋白表达明显升高,混合淋巴细胞培养结果显示,FasL转染后DC对异系T淋巴细胞刺激指数明显下降(P<0.01),且诱导T淋巴细胞凋亡的能力明显增加(P<0.01)。结论:培养基选择法可收获大量骨髓源DC,具有典型形态,高表达MHCⅡ和共刺激分子,所获骨髓源DC能激活初始型T细胞免疫应答。脂质体转染FasL基因至小鼠骨髓源DC,可以使DC高表达FasL蛋白。转染FasL的DC体外刺激异系T淋巴细胞增殖的能力下降,且能明显诱导T淋巴细胞凋亡。     

Discovery of Imidazopyridines as Potent Inhibitors of Indoleamine 2,3-Dioxygenase 1 for Cancer Immunotherapy

Indoleamine 2,3-dioxygenase 1 (IDO1) has been identified as a target for small-molecule immunotherapy for the treatment of a variety of cancers including renal cell carcinoma and metastatic melanoma. This work focuses on the identification of IDO1 inhibitors containing replacements or isosteres for the amide found in BMS-986205, an amide-containing, IDO1-selective inhibitor currently in phase III clinical trials. Detailed subsequently are efforts to identify a structurally differentiated IDO1 inhibitor via the pursuit of a variety of heterocyclic isosteres, leading to the discovery of highly potent, imidazopyridine-containing IDO1 inhibitors.     

N1-Fluoroalkyltryptophan Analogues: Synthesis and in vitro Study as Potential Substrates for Indoleamine 2,3-Dioxygenase

l-tryptophan through the kynurenine pathway, thereby exerting immunosuppressive properties in inflammatory and tumoral tissues. The syntheses of 1-(2-fluoroethyl)-tryptophan (1-FETrp) and 1-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-tryptophan, two N¹-fluoroalkylated tryptophan derivatives, are described here. In vitro enzymatic assays with these two new potential substrates of hIDO show that 1-FETrp is a good and specific substrate of hIDO. Therefore, its radioactive isotopomer, 1-[¹⁸F]FETrp, should be a molecule of choice to visualize tumoral and inflammatory tissues and/or to validate new potential inhibitors.     

An overview on the methods of determining the activity of Indoleamine 2, 3-Dioxygenase 1

Abstract

Immune escape plays an important hallmark of cancer. The tryptophan-catabolizing enzyme indoleamine 2, 3-dioxygenase 1 (IDO1) is involved in the immune escape mechanism of human tumours, the expression of which is associated with a poor prognosis in cancer and correlated with tumour progression clinically. Therefore, finding the inhibitors targeting IDO1 may be an impellent approach that can reverse the complicated processes in tumour immune escape and induce an antitumor response as well. Here, we summarised and compared some methods of determining the activity of IDO1 and they can be used to screen IDO1 inhibitors as well, including HPLC, fluorescence detection, cell-based assay, NFK GreenScreen^TM and absorbance assay. Each determining method possesses special advantages and disadvantages, so we need to have a comprehensive view of these methods and select the most suitable one for further research.     

脐带血与外周血来源树突细胞体外刺激特异抗白血病T细胞免疫应答

目的：比较脐血（CB）及外周血（PB）单个核细胞（MNC）体外诱导生成树突状细胞（DC）的产量;并将此DC负载白血病抗原,检测两者诱导的特异性细胞毒T细胞（CTL）对白血病细胞的杀伤活性。方法：取CB及健康成人PB各8份,以淋巴细胞分离液分离获得MNCs,培养体系中加入重组人粒单核细胞集落刺激因子（rhGM-CSF）,重组人白细胞介素4（rhIL-4）和肿瘤坏死因子α（TNF-α）,并在培养第5天,加入HL-60细胞经反复冻融提取的抗原,致敏DC。培养过程中,观察树突状细胞形态,流式细胞仪（FCM）检测表型。培养第12天收获成熟DC,CB组分别与8例PB来源T淋巴细胞共培养;PB组与自体T淋巴细胞共培养,诱导产生白血病特异性CTL,乳酸脱氢酶法（LDH法）测定溶靶细胞活性。结果：（1）CB与PB来源DC均表现出成熟DC典型形态和免疫表型特征,2组DC相关分化抗原CD1a、CD83、CD86、CD80和人类白细胞相关抗原DR（HLA-DR）的表达较培养前增高,2组比较差异无统计学意义（P〉0.05）。（2）CB组DC产量高于PB组,差异有统计学意义（P〈0.01）。（3）以白血病抗原冲击的DC所刺激的T淋巴细胞对白血病靶细胞显示出明显的杀伤活性,按照各效靶比进行两者的比较,差异无统计学意义（P〉0.05）。结论：利用细胞因子体外诱导CB及PB来源MNCs均能产生大量成熟DC,且功能相同;前者DC产量更高,来源丰富,可能成为急性白血病免疫治疗中DC的丰富来源。     

苦参碱抑制人卵巢癌细胞株HO-8910PM增殖及其机制研究

目的 研究苦参碱对人高转移卵巢癌细胞系HO-8910PM增殖的影响及其作用机制.方法 以MTT法和台盼蓝拒染法检测苦参碱对人高转移卵巢癌HO-8910PM细胞增殖的影响;流式细胞术检测药物作用后该细胞细胞周期的改变.结果 苦参碱能明显抑制细胞的增殖,苦参碱作用后出现细胞周期的改变,S期细胞比例明显增加(0.4977±0.0358),与对照组相比(0.2980±0.0421)P＜0.05.结论 苦参碱能抑制高转移性人卵巢癌细胞HO-8910PM的增殖能力,其抗肿瘤细胞增殖的能力与细胞周期改变有关.     

The Targeting of Indoleamine 2,3 Dioxygenase ‐Mediated Immune Escape in Cancer

The era of immunotherapies was unleashed in 2010 with the Food and Drug Administration (FDA) approval of the first therapeutic vaccine sipuleucel‐T as a standard treatment for metastatic prostate cancer. Next, the first immune‐activating anticytotoxic lymphocyte antigen‐4 (CTLA‐4) antibody ipilimumab exhibiting ‘immune checkpoint blockade’ was approved by FDA and European Medical Agency (EMA) for the treatment of patients with metastatic melanoma. New generations of immune checkpoint blockading antibodies targeting programmed cell death 1 (PD‐1) and its ligand (PD‐L1) are now under intense investigation in metastatic melanoma (MM) and non‐small‐cell lung cancer (NSCLC), and impressive clinical results are anticipated. Despite these successes, only a fraction of patients become clinical responders to therapy. Thus, to improve the selection of patients likely to respond, scrutinizing different immune parameters during treatment is essential. In the summary of this PhD thesis, we investigated changes in immune parameters and their possible correlation with clinical efficacy in patients with MM during treatments with the standard chemo‐ and immunotherapies, temozolomide (TMZ) and interferon‐α2b/interleukin‐2 (IFN‐α/IL‐2) immunotherapy. The overall aim was to assess changes in frequency and absolute counts of different immune cell subsets before and after treatment and correlate to clinical benefit. Furthermore, the thesis covers a finalized, clinical phase 1 study in patients with NSCLC testing a peptide vaccination with a HLA‐A2‐restricted epitope derived from indoleamine 2,3 dioxygenase (IDO). The overall aim in this trial was to evaluate safety and tolerability of IDO as an anticancer vaccine target in patients with NSCLC and to assess whether immunity correlated to clinical response.     

丁酸钠抑制人鼻咽癌上皮细胞CNE2中吲哚胺双加氧酶表达的体外研究

目的:研究丁酸钠(NaB)抑制人鼻咽癌上皮细胞CNE2中吲哚胺双加氧酶(IDO)表达的分子机制。方法:体外培养CNE2细胞,采用蛋白印迹法、免疫组化法检测不同浓度NaB对重组人干扰素γ(IFN-γ)诱导后CNE2细胞中IDO的表达及NaB作用不同时间对核转录因子STAT1磷酸化的影响;利用pSTAT1-GFP转染CNE2细胞,观察NaB对STAT1核内分布的影响;采用蛋白印迹法检测组蛋白去乙酰化酶抑制剂CAY10398对IDO表达的影响;采用免疫沉淀法检测NaB对转染细胞的STAT1质粒的乙酰化作用。结果:随NaB或CAY10398浓度增加,CNE2细胞中IDO表达均减少;随NaB作用时间延长,IFN-γ诱导下的STAT1磷酸化水平降低;NaB使IFN-γ诱导的细胞核内STAT1绿色荧光蛋白减少;NaB能明显增强转染和未转染细胞中STAT1的乙酰化作用(P<0.001)。结论:NaB抑制CNE2细胞IDO表达的机制可能与其乙酰化STAT1、降低STAT1的磷酸化和核内转移有关。     

丹皮酚体外抑制TGF-β诱导的成纤维细胞增殖的研究

目的:观察丹皮酚影响下离体成纤维细胞的增殖情况,为预防因成纤维细胞增殖引起的一系列病理过程提供理论依据。方法:取10只健康Wistar大鼠用于培养成纤维细胞,将离体大鼠腹膜成纤维细胞平均分为4组,分别加入细胞培养液RPMI-1640,RPMI-1640 TGF-β,RPMI-1640 丹皮酚,RPMI-1640 TGF-β 丹皮酚。测定各培养液中成纤维细胞增殖情况,并进行比较。结果:单独使用丹皮酚不影响成纤维细胞的增殖(P>0.05),单独使用TGF-β可以刺激成纤维细胞活化进入增殖状态(P<0.01),加入丹皮酚的干预后成纤维细胞的增殖率明显下降(P<0.01)。结论:TGF-β可以促进成纤维细胞活化,丹皮酚有抑制活化的成纤维细胞增殖的作用,应用丹皮酚可能在一定程度上减轻病理状态下成纤维细胞对人体的损害。     

Indoleamine 2,3-dioxygenase as a modifier of pathogenic inflammation in cancer and other inflammation-associated diseases

Chronic inflammation underlies the basis for development and progression of cancers and a variety of other disorders, but what specifically defines its pathogenic nature remains largely undefined. Recent genetic and pharmacological studies in the mouse suggest that the immune modulatory enzyme indoleamine 2,3-dioxygenase (IDO), identified as an important mediator of immune escape in cancer, can also contribute to the development of pathology in the context of chronic inflammatory models of arthritis and allergic airway disease. IDO-deficient mice do not display spontaneous disorders of classical inflammation and small molecule inhibitors of IDO do not elicit generalized inflammatory reactions. Rather, in the context of a classical model of skin cancer that is promoted by chronic inflammation, or in models of inflammation-associated arthritis and allergic airway disease, IDO impairment can alleviate disease severity. Here we offer a survey of preclinical literature suggesting that IDO functions as a modifier of inflammatory states rather than simply as a suppressor of immune function. We propose that IDO induction in a chronically inflamed tissue may shape the inflammatory state to support, or in some cases retard, pathogenesis and disease severity.