白细胞介素-23在炎症性肠病的表达升高并诱导促炎细胞因子分泌

目的 通过分析白细胞介素(IL)-23p19及其受体(IL-23R)在炎症性肠病(IBD)患者中的表达,研究IL-23对IBD患者外周血T细胞激活和效应应答的影响,探讨其在疾病发生过程中的免疫病理作用.方法 收集12例克罗恩病(CD)患者、25例溃疡性结肠炎(UC)患者和20名健康者外周血和肠黏膜组织活检标本,使用免疫组化染色和逆转录(RT)-PCR分析IL-23p19表达,分离外周血单个核细胞(PBMC)和肠黏膜固有层内单个核细胞(LPMC),利用流式细胞仪检测IL-23R在CD4+、CD8+T细胞和自然杀伤(NK)细胞表面表达.体外培养PBMC,使用IL-23和抗CD3单抗体外刺激,使用酶联免疫吸附试验检测肿瘤坏死因子(TNF)-α、干扰素(IFN)-γ和白细胞介素(IL)-2分泌.结果 IBD患者炎症肠黏膜组织内IL-23p19蛋白和mRNA表达水平比健康对照者显著升高(P<0.05).IL-23R在IBD患者外周血和肠黏膜固有层组织内CD4+、CD8+T细胞和NK细胞表达水平也比健康者显著升高(P<0.05).体外培养PBMC,使用IL-23刺激,发现IL-23可显著诱导IBD患者,尤其是CD患者PBMC激活,分泌高水平TNF-α、IFN-γ和IL-2(P<0.05).结论 IL-23及其受体在IBD患者炎症肠黏膜组织中表达升高,IL-23可诱导IBD患者淋巴细胞分泌高水平的炎性介质,提示IL-23参与了肠黏膜炎症损伤,阻断IL-23生物学效应可能治疗IBD.     

英夫利西单克隆抗体对克罗恩病患者肠黏膜屏障功能修复的研究

目的探讨英夫利西单克隆抗体(IFX)对CD患者肠黏膜屏障的修复作用。方法选择2018年1月至2019年10月于上海市第十人民医院就诊的CD患者382例,均行IFX治疗,另选择103名行结肠镜检查者作为健康对照组。收集CD患者和健康对照者的一般临床资料、空腹血和肠黏膜组织样本,计算BMI,检测血红蛋白、白蛋白、ESR、CRP,以及相关炎症因子TNF-α、γ干扰素、IL-1、IL-2、IL-6、IL-8、IL-10、IL-17A表达水平及其mRNA水平。采用克罗恩病疾病活动指数(CDAI)和克罗恩病简化内镜评分(SES-CD)评价CD患者的疾病活动度。采用蛋白质印迹法分析闭锁蛋白、紧密连接蛋白-1、闭锁小带蛋白-1(ZO-1)和连接黏附分子-A(JAM-A)表达水平。通过透射电子显微镜观察肠黏膜上皮细胞。统计学分析采用t检验。结果 CD患者治疗前BMI、血红蛋白、白蛋白水平均低于健康对照组[(18.3±1.8) kg/m2比(20.2±1.2) kg/m2、(95.3±8.4) g/L比(129.2±5.7) g/L、(33.2±5.4) g/L比(50.3±3.2) g/L],差异均有统...     

IL-17BR在炎症性肠病中的表达及其临床意义

背景:炎症性肠病(IBD)目前尚无特效的治疗方式,其发病机制仍不明确。目的:探讨IL-17BR在IBD患者结肠黏膜和外周血单个核细胞(PBMC)中的表达情况及其临床意义。方法:收集40例克罗恩病(CD)患者、32例溃疡性结肠炎(UC)患者和25例健康对照者内镜结肠黏膜标本以及30例CD患者、27例UC患者和25例健康对照者的PBMC标本。采用免疫组化、流式细胞术分别检测结肠黏膜和PBMC中IL-17BR表达,并分析其与CRP、ESR以及CDAI、Mayo评分的相关性。采用ELISA法检测英夫利西单抗(IFX)治疗前后血清TNF-α浓度,并分析其与IL-17BR的相关性。结果:与正常对照者相比,CD和UC患者结肠黏膜中IL-17BR表达均显著升高(P0.05),且活动期CD和UC患者IL-17BR表达显著高于缓解期患者(P0.05)。CD、UC患者和正常对照者PBMC中IL-17BR阳性率差异相比无统计学意义(P0.05)。CD、UC患者结肠黏膜IL-17BR表达与CRP、ESR、CDAI评分、Mayo评分均呈正相关(P0.05)。IFX治疗后CD患者结肠黏膜IL-17BR表达、血清TNF-α浓度均较治疗前明显下调(P0.01),且两者呈正相关(P0.05)。结论:IBD患者结肠黏膜IL-17BR表达与炎症程度和TNF-α水平密切相关,IL-17BR可能参与IBD肠黏膜免疫应答,因此可作为反映IBD疾病活动度的指标之一。     

白细胞介素-25在炎症性肠病患者中的表达及临床意义

目的 通过检测白细胞介素(IL)-25在炎症性肠病(IBD)患者肠黏膜及血清中的表达水平,探讨其在IBD发病过程中的作用及意义.方法 收集12例溃疡性结肠炎(UC)患者、16例克罗恩病(CD)患者及13例对照者的内镜肠黏膜活检标本,采用荧光定量PCR技术检测肠黏膜内IL-25 mRNA的表达情况,免疫组化技术分析IL-25在肠黏膜中的原位表达;同期收集20例UC、24例CD患者及20名健康对照者血清标本,采用酶联免疫吸附测定(ELISA)检测血清中IL-25水平.结果 与健康对照组相比,UC及CD患者肠黏膜组织内IL-25 mRNA表达显著降低(P＜0.05),UC及CD组间的表达量差异无统计学意义(P＞0.05).免疫组化分析显示IL-25阳性细胞在正常肠黏膜固有层内有较多表达,同时黏膜内的肠上皮细胞也存在IL-25低表达,UC及CD患者肠黏膜IL-25蛋白表达量显著降低(P＜0.05),UC及CD组间的表达量差异无统计学意义(P＞0.05).ELISA显示UC及CD患者血清中IL-25表达量显著低于健康对照组(P＜0.05).结论 IL-25在IBD患者肠黏膜及血清中表达显著降低,提示IL-25表达缺陷与IBD的发生发展密切相关,IL-25有可能成为IBD治疗的新靶点.     

IL-21受体在溃疡性结肠炎中的表达及对促炎症细胞因子分泌的诱导作用

目的:探讨IL-21对UC患者外周血和肠黏膜固有层组织内淋巴细胞的免疫病理调节.方法:收集28例UC患者和22例健康成人外周血标本和肠黏膜活检标本, 分离外周血单个核淋巴细胞(PBMC)和肠黏膜固有层单个核淋巴细胞(LPMC), 利用流式细胞仪检测IL-21R在CD4+、CD8+ T细胞、B细胞和NK细胞表面表达. 培养PBMC和LPMC, 使用IL-21和抗CD3单抗体外刺激, 48 h后收集上清液,使用ELISA检测促炎症细胞因子(TNF-α、IFN-γ、IL-2)分泌, 分析IL-21在疾病发展过程中的免疫病理作用.结果:UC患者外周血和肠黏膜固有层组织内CD4+、CD8+ T细胞、CD20+ B细胞和CD56+NK细胞表达IL-21R水平比健康者显著升高(PBMC:8.42±2.14 vs 3.46±0.54, 10.35±2.17vs 5.28±2.2, 7.27±1.15 vs 2.35±0.41, 12.55±3.12 vs 5.45±1.06; LPMC:22.44±3.46 vs6.26±1.15, 24.48±4.57 vs 6.87±1.02, 16.24±3.10 vs 5.56±1.44, 23.54±4.12 vs 8.45±1.68, 均P<0.05). 体外培养PBMC或LPMC, 使用IL-21刺激, 发现IL-21可显著诱导UC患者的PBMC和LPMC激活, 并分泌高水平的TNF-α和IFN-γ(346±72 vs 120±27, 3048±426 vs1182±242; 625±113 vs 154±35, 3827±418vs 1520±304, 均P<0.05).结论:IL-21R参与肠黏膜炎症损伤, 阻断IL-21生物学效应可能治疗UC的发生.     

IL-22BP在活动期炎症性肠病患者肠黏膜中的表达及其意义

背景:白细胞介素-22结合蛋白(IL-22BP)是IL-22的内源性抑制剂,可抑制IL-22的肠道保护作用,在活动期炎症性肠病(IBD)患者肠黏膜中的表达及其意义尚未明确。目的:探讨IL-22BP在活动期IBD患者肠黏膜中的表达及其意义。方法:纳入2017年1月—2018年1月上海交通大学附属第一人民医院的25例活动期克罗恩病(CD)、36例溃疡性结肠炎(UC)患者,并以30例结肠息肉患者作为对照。评估CD、UC患者的疾病活动度,采用实时荧光定量PCR和免疫组化法分别检测黏膜中IL-22BP mRNA和蛋白表达,并分析IL-22BP蛋白表达与CD、UC疾病活动度的相关性。结果:与相应对照组相比,CD组、UC组肠黏膜中IL-22BP mRNA表达显著升高(CD:3.59±0.83对1.08±0.45,P0.001;UC:2.19±0.52对1.05±0.34,P0.001),IL-22BP蛋白表达亦显著升高(CD:6.12±2.30对1.83±1.86,P0.001;UC:5.58±2.27对2.23±1.77,P0.001)。CD、UC患者肠黏膜中IL-22BP蛋白表达与疾病活动度均呈显著正相关(r=0.649,P0.001;r=0.732,P0.001)。结论:IL-22BP mRNA和蛋白表达在活动期IBD患者肠黏膜中升高,且IL-22BP蛋白表达与IBD疾病活动度呈正相关。     

CXC趋化因子受体2和白细胞介素-8在炎症性肠病患者中的表达及其意义

背景:CXC趋化因子受体2(CXCR2)为G蛋白耦联受体超家族成员,主要参与肿瘤生长、血管形成以及炎症性疾病的发病,有研究显示其参与炎症性肠病(IBD)发病,但相关作用仍未明确。研究表明白细胞介素-8(IL-8)与CXCR1和CXCR2的相互作用在IBD发病中发挥重要作用。目的:探讨CXCR2和IL-8在IBD患者中的表达及其意义。方法:纳入武汉大学中南医院2013年10月—2014年12月收治的活动期IBD患者121例,分为克罗恩病(CD)组和溃疡性结肠炎(UC)组,选取70例同期健康体检者作为正常对照(HC)组。采用real-time PCR检测外周血和肠黏膜组织IL-8和CXCR2 mRNA表达;采用蛋白质印迹法检测肠黏膜组织CXCR2蛋白表达。结果:UC组外周血IL-8 mRNA表达水平显著高于HC组(P=0.017);CD组、UC组和HC组外周血CXCR2 mRNA表达水平无明显差异(P=0.285)。CD组、UC组和HC组肠黏膜组织IL-8 mRNA表达水平无明显差异(P=0.206);CD组和UC组肠黏膜组织CXCR2 mRNA表达水平显著高于HC组(P=0.002;P0.001),且UC组显著高于CD组(P=0.005)。UC组和CD组肠黏膜组织CXCR2蛋白表达水平高于HC组(P=0.049;P=0.080)。结论:IBD患者肠黏膜组织CXCR2 mRNA和蛋白表达均显著上调,且以UC患者为著,下调肠黏膜CXCR2表达可为治疗UC提供新靶点。IL-8主要在UC患者外周血中高表达,提示IL-8主要与UC发病相关。     

炎症性肠病患者外周血Th17细胞的变化及其临床意义

背景:Th17细胞是一类能特异性产生白细胞介素-17(IL-17)的CD4~+T细胞亚群,研究显示其参与了多种自身免疫性疾病的发病过程。目的:研究炎症性肠病(IBD)患者外周血Th17细胞比例和IL-17mRNA表达的变化,探讨Th17细胞在IBD发病机制中的意义。方法:纳入10例健康体检者(正常对照组)和53例IBD患者,其中克罗恩病(CD)25例,溃疡性结肠炎(UC)28例,以流式细胞术检测外周血Th17细胞比例,实时PCR检测外周血单个核细胞(PBMC)IL-17 mRNA表达水平。结果:IBD患者外周血Th17细胞比例明显高于正常对照组,其中活动期又明显高于缓解期,差异均有统计学意义(P0.05);其PBMC中IL-17 mRNA表达水平亦显著高于正常对照组(P0.05),但活动期与缓解期之间差异无统计学意义。结论:IBD患者外周血Th17细胞比例和IL-17 mRNA表达水平明显升高,Th17细胞参与了IBD的发生、发展过程,可能对临床疾病活动度的判断有一定意义。     

英夫利昔治疗克罗恩病的初步研究

背景：近年克罗恩病（CD）的发病率不断升高,生物制剂英夫利昔（IFX）是一种肿瘤坏死因子．d单克隆抗体,可促进CD患者黏膜的愈合。目的：观察IFX治疗CD的疗效。方法：收集2008年7月～2009年6月上海中山医院的16例CD住院患者。根据CDAI评分评估CD严重程度并将患者分为常规治疗组和IFX组．分别予美沙拉秦＋泼尼松以及IFX治疗。治疗8周后,比较两组CDAI评分、WBC、ESR、CRP和ALB水平。结果：11例轻度活动期CD患者接受常规治疗．5例中度活动期患者接受IFX治疗。治疗后．所有患者均取得临床缓解．其中2例伴肛门瘘管的轻度活动期患者经常规治疗无效后改用IFX治疗亦达到临床缓解。两组CDAI评分、ESR和CRP水平均较治疗前明显改善（P〈0．05）;IFX组CDAI评分的降低幅度高于常规治疗组,差异有统计学意义（陛0．019）。结论：IFX对CD的临床缓解优于常规治疗．并可促进常规治疗无效的肛门瘘管患者的愈合。     

免疫性血小板减少症患者外周血白细胞介素-18及其受体α链的表达

目的 通过检测新诊断的免疫性血小板减少症(ITP)患者用药前外周血IL-18和CD_3~+细胞表面IL-18受体α链(IL-18Rα)的表达,探讨IL-18和IL-18Rα在ITP发病中的作用机制.方法 以我院门诊及住院治疗的18例新诊断的ITP为研究对象,应用ELISA检测血浆中IL-18的含量,采用流式细胞术分析CD_3~+细胞和总淋巴细胞表面IL-18Rα的表达,应用RT-PCR检测外周血单个核细胞(PBMCs)中IL-18 mRNA、转录因子T-bet mRNA和GATA-3 mRNA的表达.选择15例与试验组匹配的健康志愿者作为正常对照组.结果 新诊断的ITP患者血浆中IL-18的含量为(468.57±141.62)pg/ml,显著高于对照组(P<0.05);CD_3~+细胞表面及淋巴细胞表面IL-18Rα的表达分别为(8.50±3.16)%和(9.16±2.98)%,两者均显著高于对照组(P<0.05);ITP患者PBMCs中IL-18 mRNA、T-bet mRNA和GATA-3 mRNA的表达分别为0.12±0.02、0.07±0.02和0.0039±0.0014,IL-18mRNA和T-bet mRNA显著高于对照组(P<0.05),而GATA-3 mRNA显著低于对照组(P<0.05),T-bet/GATA-3比例显著增高.结论 IL-18与ITP的发生发展有关,本研究从蛋白和基因水平说明IL-18和IL-18Rα可能上调ITP患者Th1类细胞的表达,通过调整T-bet/GATA-3的比例,恢复Th1/Th2的平衡,有望为ITP的治疗提供一个新的治疗策略.     

Blockade of the interleukin-21/interleukin-21 receptor pathway ameliorates disease in animal models of rheumatoid arthritis

OBJECTIVE: Interleukin-21 (IL-21) is a T cell-derived cytokine that modulates T cell, B cell, and natural killer cell responses. In this study, the effects of blocking IL-21 were examined in 2 rodent models of rheumatoid arthritis (RA) to determine whether IL-21 contributes to their pathologic processes. METHODS: DBA/1 mice were immunized with bovine type II collagen and then treated with murine IL-21 receptor Fc fusion protein (IL-21R.Fc), which was initiated after the onset of arthritis symptoms in 10% of the cohort. The mice were assessed 3 times per week for signs of disease, including histologic features as well as serum cytokine, Ig, and cytokine messenger RNA (mRNA) levels in the paws. In a separate experiment, Lewis rats were immunized with Freund's complete adjuvant followed by administration of IL-21R.Fc at the peak of inflammation in the joints. Rats were assessed daily for histologic features and for scoring of arthritis severity. In addition, the effects of IL-21R.Fc on the production of interferon-gamma (IFNgamma) by T cells were examined. RESULTS: Treatment of DBA/1 mice with IL-21R.Fc reduced the clinical and histologic signs of collagen-induced arthritis. Nonspecific IgG1 levels were decreased in response to treatment. The levels of IL-6 mRNA in the paws and the serum IL-6 levels were decreased after treatment with IL-21R.Fc. IFNgamma mRNA levels were increased in the paws, and the addition of IL-21R.Fc to collagen-activated lymph node cultures enhanced the levels of IFNgamma. Collagen-specific spleen cell responses in IL-21R.Fc-treated mice were observed as reduced levels of IFNgamma and increased levels of IL-6. Treatment of Lewis rats with IL-21R.Fc after induction of adjuvant-induced arthritis resulted in reversal of disease signs and improvements in histologic parameters. CONCLUSION: These findings demonstrate a pathogenic role for IL-21 in animal models of RA, and support consideration of IL-21 as a therapeutic target in human RA.     

Angiostatic activity of the antitumor cytokine interleukin-21

Interleukin-21 (IL-21) is a recently described immunoregulatory cytokine. It has been identified as a very potent immunotherapeutic agent in several cancer types in animal models, and clinical studies are ongoing. IL-21 belongs to the type I cytokine family of which other members, ie, IL-2, IL-15, and IL-4, have been shown to exert activities on vascular endothelial cells (ECs). We hypothesized that IL-21, in addition to inducing the antitumor immune response, also inhibits tumor angiogenesis. In vitro experiments showed a decrease of proliferation and sprouting of activated ECs after IL-21 treatment. We found that the IL-21 receptor is expressed on vascular ECs. Furthermore, in vivo studies in the chorioallantoic membrane of the chick embryo and in mouse tumors demonstrated that IL-21 treatment disturbs vessel architecture and negatively affects vessel outgrowth. Our results also confirm the earlier suggested angiostatic potential of IL-2 in vitro and in vivo. The angiostatic effect of IL-21 is confirmed by the decrease in expression of angiogenesis-related genes. Interestingly, IL-21 treatment of ECs leads to a decrease of Stat3 phosphorylation. Our research shows that IL-21 is a very powerful antitumor compound that combines the induction of an effective antitumor immune response with inhibition of tumor angiogenesis.     

Expression of interleukin-21 receptor, but not interleukin-21, in synovial fibroblasts and synovial macrophages of patients with rheumatoid arthritis

OBJECTIVE: To determine the role and expression of the cytokine/receptor pair interleukin-21 (IL-21)/IL-21 receptor (IL-21R) in rheumatoid arthritis (RA). METHODS: The expression of IL-21R and IL-21 was analyzed by TaqMan real-time polymerase chain reaction (PCR) and in situ hybridization of synovial biopsy samples from patients with RA and osteoarthritis (OA). Double labeling by immunohistochemistry after in situ hybridization was performed with anti-CD68 antibodies. The expression of IL-21R at the protein level was confirmed by Western blotting. Stimulation experiments were performed with recombinant IL-1beta, tumor necrosis factor alpha (TNFalpha), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGFbeta). The role of IL-21R in cartilage destruction was analyzed in the SCID mouse coimplantation model of RA. RESULTS: IL-21R was found in total RNA extracts and in synovial biopsy samples from RA patients, whereas no expression or only minimal expression was seen in samples from OA patients. Double labeling indicated that both synovial macrophages and synovial fibroblasts expressed IL-21R. Western blotting with anti-IL-21R antibodies confirmed the expression of IL-21R protein in RA synovial fibroblasts (RASFs). Of note, IL-21 was not detectable by real-time PCR and in situ hybridization in the same samples in vivo as in vitro. The level of expression of IL-21R messenger RNA (mRNA) was not altered by stimulation with IL-1beta, TNFalpha, PDGF, or TGFbeta. Interestingly, in the SCID mouse coimplantation model, RASFs did not maintain their expression of IL-21R at sites of invasion into the cartilage. Similarly, IL-21R mRNA was not expressed at sites of invasion into cartilage and bone in RA synovium. CONCLUSION: Our data demonstrate that IL-21R is expressed in RA synovium by RASFs and synovial macrophages. IL-21R is associated with the activated phenotype of RASFs independently of the major proinflammatory cytokines IL-1beta and TNFalpha, but correlates negatively with the destruction of articular cartilage and bone.     

白介素21的生物学功能与炎症性肠病的研究进展

白介素21(interleukin-21,IL-21)是一种新近发现的细胞因子,与其受体结合后可以调节B细胞的增殖,促进T细胞、NK细胞的增殖、分化并能提高NK细胞杀伤活性.炎症性肠病(inflammatory bowel disease,IBD)是一种自身免疫性疾病,其发病机制尚不明确,可能有多种因素共同参与,免疫紊乱在IBD发生发展中起着重要作用,多种细胞因子的变化与IBD有关.IL-21就是其中的一种.本文就IL-21及其与IBD关系的研究进展作一综述.     

重组人IL-21融合蛋白在大肠杆菌中的可溶性形式表达、纯化及体外对T细胞增殖的影响

目的 为重组人白细胞介素-21(rhIL-21) 的生物学活性和功能研究奠定基础.方法 构建重组表达质粒pGEX4T-2/IL-21,通过降低培养温度等优化条件于大肠杆菌中表达出可溶性形式的rhIL-21融合蛋白,纯化后以不同质量浓度作用于T细胞,观察其对T细胞增殖的影响.结果 大肠杆菌表达出可溶性形式的rhIL-21融合蛋白,纯化纯度达95%;纯化后的rhIL-21蛋白作用后T细胞增殖增加,且随rhIL-21蛋白质量浓度的增加,T细胞增殖率升高.结论 大肠杆菌中获得rhIL-21的可溶性表达形式,纯化后的蛋白对T细胞增殖有促进作用;此为rhIL-21的生物活性及功能研究奠定了基础.