慢性乙型肝炎患者PB MC中H BV特异性IL-21表达及其对CD8^＋T淋巴细胞功能的影响

目的：探讨慢性乙型肝炎患者外周血单个核细胞（PBMC）中 HBV特异性IL-21的表达及其对CD8＋T淋巴细胞功能的调节作用。方法以 HBVc18-27（10μg/mL）多肽刺激46例不同临床类型慢性乙型肝炎患者PBMC。ELISA检测上清液IL-21水平。在 HBVc18-27（10μg/mL）多肽和IL-21（50 U/mL）或IL-2（100 ng/mL）培养条件下,应用 MHC-肽五聚体结合流式细胞术检测PBMC中 HBV特异性CTL细胞频数变化;免疫磁珠分离纯化免疫耐受期和免疫激活期慢性乙型肝炎患者PBMC中CD8＋T淋巴细胞,以 HBcAg（10μg/mL）预刺激CD8-PBMC 5 h后,采用Transwell 小室共培养技术,在阻断IL-21通路条件下,应用实时PCR检测CD8^＋T淋巴细胞IFN-γmRNA的表达。结果 HBV特异性IL-21在免疫控制期和免疫激活期慢性乙型肝炎患者P B MC中表达水平显著高于免疫耐受期患者,而在免疫控制期和免疫激活期两组之间无显著差异。与 PBS 对照组相比,IL-21组 HBV 特异性 CTL 细胞频数明显增高,差异有统计学意义（P＜0．01）,而与IL-2组相比差异无统计学意义。以IL-21抗体阻断IL-21通路后,CD8^＋T 淋巴细胞IFN-γmRNA 表达水平显著下降,差异有统计学意义。结论免疫控制期和免疫激活期慢性乙型肝炎患者PBMC 均可生成一定水平的 HBV 特异性IL-21,并能增强外周CD8＋T淋巴细胞的增殖和IFN-γ表达水平。     

乙型肝炎后肝硬化患者血清XCL1、IFN-γ及T细胞亚群的相关性分析

目的探讨XCL1、γ干扰素、T细胞亚群在乙肝病毒所致肝硬化中的水平变化及其临床意义。方法彩色腹部多普勒B超检查肝脏,测量脾脏厚度、门静脉宽度、有无腹水;全自动生化仪检测肝功能;采用酶联免疫吸附法(ELISA)检测血清XCL1、IFN-γ的水平;流式细胞仪检测T淋巴细胞亚群。结果正常对照组、慢性乙型肝炎组、乙型肝炎肝硬化组XCL1水平分别为(8.29±1.82)ng/mL、(13.18±2.86)ng/mL、(11.86±2.47)ng/mL,IFN-γ水平分别为(19.98±2.92)pg/mL、(37.55±6.15)pg/mL、(31.26±5.44)pg/mL。慢性乙型肝炎组、乙型肝炎肝硬化组血清XCL1、IFN-γ水平显著高于正常对照组血清XCL1、IFN-γ水平(P0.01);慢性乙型肝炎组血清XCL1、IFN-γ水平显著高于乙型肝炎肝硬化组血清XCL1、IFN-γ水平(P0.01或0.05);XCL1水平与IFN-γ水平呈正相关(r=0.457,P0.01);XCL1水平与CD4+T细胞百分比呈负相关(r=-0.339,P0.01);IFN-γ水平与CD8+T细胞百分比呈正相关(r=0.330,P0.05)。结论 XCL1通过影响IFN-γ从而导致CD4+T/CD8+T比值失衡,引起肝细胞的慢性炎症参与肝硬化进程。     

慢性乙型肝炎患者外周血CD8＋CD28＋T细胞、血清IFN-γ水平变化及意义

目的 观察慢性乙型肝炎患者外周血CD8＋ CD28＋T细胞、血清γ干扰素（IFN-γ）水平变化,并讨论其临床意义.方法 慢性乙型肝炎45例为观察组,健康体检者20例为对照组.采用双抗体夹心时间分辨荧光免疫分析法测定HBeAg,应用实时荧光定量PCR法检测外周血HBV-DNA,流式细胞术检测外周血CD8 CD28＋T细胞水平,ELISA法检测血清IFN-y水平.结果 对照组、观察组外周血CD8＋ CD28＋T细胞比例分别为12.86％±2.57％、10.74％±3.59％,两组比较P＜0.05;血清IFN-γ水平分别为（5.79±1.28）、（311.00±80.40） pg/mL,两组比较P均＜0.01.观察组Child-Pugh A、B、C级外周血CD8＋ CD28＋T细胞比例分别为7.98％±2.62％、12.37％±1.73％、13.05％±3.38％,各级间比较P均＜0.01;血清IFN-y水平分别为（320.10±86.55）、（299.95±60.84）、（307.57±88.43） pg/mL,各级间比较P均＞0.05.CD8＋ CD28＋T细胞与HBeAg、HBV-DNA相关系数分别为-0.33、-0.44,P均＜0.05;IFN-γ与CD8＋T细胞相关系数为-0.33,P＜0.05;CD28＋T细胞与IFN-γ相关系数为0.34,P＜0.05.结论 IFN-γ水平可反映HBV慢性感染者的免疫状态,CD8＋ CD28＋T细胞有助于病毒的清除.     

成人急性HBV感染后免疫指标的变化及不同转归的比较

目的 观察成人急性HBV感染后免疫指标的变化并进行不同转归比较。方法 选择29例急性乙肝患者和20例正常对照者，采用双抗体酶联分析法检测外周血细胞因子IL-2、IFN-γ、IL-4、IL-10和IL-12的水平；通过流式细胞仪观察外周血T淋巴细胞亚群变化；采用抗人T淋巴细胞单克隆抗体间接免疫荧光法观察外周血T淋巴细胞膜IFN-γ和IL-4的表达。结果29例HBeAg全部转阴，8例HBsAg未转阴。在HBsAg转阴组，病程早期和中期血清IL-2、IFN-γ、IL4及IL-12水平增高与正常组比P〈0．05，中期IL-4下降与早期比P〈0．05，晚期IL-2、IFN-γ、IL-4及IL-12下降与中期比P〈0．05；未转阴组各期IL-4水平增高与正常组比P〈0．05。转阴组早期Th1／Th2降低与对照组比P〈0．05，未转阴组各期，Th1／Th2降低与对照组比P〈0．05。未转阴组中5例入院时HBV-DNA〈10^3且IFN-γ水平明显低。两组CD4^＋／CD8^＋变化趋势不同。转阴组早期和中期IL-4膜表达高于正常对照组，中期IL-4表达低于早期，晚期IL-4表达低于中期，P值均〈0．05。未转阴组各期IL-4表达高于正常对照组，P〈0．05，各期之间IL-4表达无统计学差异。结论 IFN-γ升高与疾病恢复相关。持续高水平的IL-4表达易致慢性化。恢复Th1／Th2细胞的平衡可能是控制乙型肝炎慢性化的一条途径。CD4^＋／CD8^＋失调可能影响机体清除病毒的能力，导致感染的持续。     

复元胶囊对老年气虚血瘀证免疫功能的调节作用

目的观察复元胶囊对老年气虚血瘀证患者外周血T淋巴细胞亚群和Th1类细胞因子IFN-γ，Th2类细胞因子IL-4的影响。方法45例老年气虚血瘀证患者随机分为治疗组（24例）和对照组（21例），另设健康非老年组（20例）。治疗组给予复元胶囊、对照组给予芪参胶囊治疗，疗程60d，检测用药前后T淋巴细胞亚群、血清IFN-γ、IL-4的水平，并与对照组进行比较。结果复元胶囊能明显提高老年气虚血瘀证患者CD3^＋、CD4^＋、CD8^＋亚群及IFN-γ的水平，对CD4^＋／CD8^＋和IL-4的水平无明显影响。结论复元胶囊能增强老年气虚血瘀证患者的细胞免疫功能，改善处于劣势的Th1细胞的免疫应答。     

慢性H BV感染者外周血中Fo xp3的表达及IFN-γ分泌的变化

目的：观察不同类型 HBV 感染者和健康者 PBMC 中 Foxp3的表达和 IFN-γ含量的变化情况,探讨在HBV感染慢性化中CD4^＋CD25^＋调节性T细胞发挥的作用。方法采用RT-PCR 检测不同类型 HBV 感染者和健康人外周血中Foxp3的表达情况。应用 ELISA 的方法检测外周血血清中 IFN-γ的含量。结果急性乙型肝炎患者Foxp3的表达与健康对照组比较差异无统计学意义,而血清中 IFN-γ的含量有所增加（P＞0．05）;慢性乙型肝炎患者Foxp3的表达均明显高于健康对照组（P＜0．01）,血清中IFN-γ的含量则有所减少。结论慢性乙型肝炎患者 CD4^＋CD25^＋调节性T细胞活性较健康对照组增强,且Th1细胞活性和体液免疫均受到抑制,CD4＋CD25＋调节性T 细胞在HBV感染慢性化中起着一定的作用。     

布鲁杆菌病患者体内T细胞免疫功能研究

目的探讨布鲁杆菌病人体内T淋巴细胞及相关细胞因子的变化。方法用流式细胞术检测外周血单个核细胞中CD4^＋T细胞、CD8^＋T细胞和CD4／CD8比例;用ELISA法检测血清中IFN-γ、IL-2、IL-4和IL-5。结果试验组外周血CD4^＋T细胞和CD4／CD8比例明显高于对照组（P〈0．05）,而CD8^＋T细胞与对照组相比无显著差异（P〉0．05）;试验组血清中IFN-γ和IL-2水平明显高于对照组（P〈0．05）,而IL-4和IL-5水平与对照组相比无显著差异（P〉0．05）。结论布鲁杆菌感染后T细胞免疫主要表现为Th1功能增强,这可能对机体清除病原菌有重要意义。     

肝内IFN-γ及其分泌细胞在乙型慢加急性肝衰竭发病机制中的作用

目的通过研究肝内促炎细胞因子IFN-γ的表达及IFN-γ的分泌细胞——T淋巴细胞的数量,以明确二者在乙型慢加急性肝衰竭（acute—on—chronic liver failure,ACLF）发病机制中的作用。方法采用免疫组化方法,分析乙型ACLF患者、慢性乙型肝炎（乙肝）患者及正常对照组肝内IFN-γ的原位表达以及其分泌细胞CD4^＋T淋巴细胞与CD8^＋T淋巴细胞的数量。结果①乙型ACLF患者IFN-γ阳性分泌细胞数均明显高于慢性乙肝患者及正常对照组,差异有统计学意义（P均〈0．001）,慢性乙肝患者IFN-γ阳性分泌细胞数明显高于正常对照组,差异有统计学意义（P〈0．001）;②乙型ACLF患者肝内CD4^＋和CD8^＋T淋巴细胞数量较慢性乙肝及正常对照组明显增加（P均〈0．001）,慢性乙肝患者肝内CD4^＋和CD8^＋T淋巴细胞数量较正常对照组明显增加（P均〈0．001）;③肝内IFN-γ阳性分泌细胞数量与CD4＋T淋巴细胞和CD8^＋T淋巴细胞数量均具有明显的相关性（r=-0．896和0．885,P均〈0．001）。结论乙型ACLF患者肝内CD4^＋T淋巴细胞、CD8^＋T淋巴细胞数量的增加,其分泌的IFN-γ的增加可能参与了ACLF的发病过程。     

老年慢性阻塞性肺疾病合并2型糖尿病患者T淋巴细胞亚群免疫失衡状态及其免疫干预探讨

目的观察老年慢性阻塞性肺疾病(COPD)合并2型糖尿病(T2DM)患者T淋巴细胞亚群免疫失衡状态,并探讨针对性免疫干预。方法选取广西壮族自治区人民医院2011年1月—2014年6月收治的52例老年COPD患者,其中合并T2DM患者34例,随机分为COPD+T2DM未干预组16例和COPD+T2DM干预组18例;单纯COPD患者18例作为单纯COPD组;并选取同期体检健康的老年人20例作为对照组。单纯COPD组患者给予常规COPD对症治疗,COPD合并T2DM患者给予常规COPD及T2DM对症治疗;COPD+T2DM干预组在常规对症治疗基础上加用胸腺五肽免疫治疗,10 mg/d,皮下注射,连用14 d;COPD+T2DM未干预组不额外加用任何免疫制剂。记录对照组T淋巴细胞亚群(CD+4细胞分数、CD+8细胞分数、CD+4/CD+8细胞比值)和血清γ干扰素(IFN-γ)、白介素(IL-4)水平及IFN-γ/IL-4,比较治疗前后单纯COPD组、COPD+T2DM干预及为干预组患者CD+4细胞分数、CD+8细胞分数、CD+4/CD+8细胞比值和血清IFN-γ、IL-4水平及IFN-γ/IL-4变化。结果治疗前单纯COPD组、COPD+T2DM干预及未干预组患者CD+4细胞分数、CD+4/CD+8细胞比值均低于对照组,CD+8细胞分数均高于对照组,且COPD+T2DM干预及未干预组患者CD+4细胞分数、CD+4/CD+8细胞比值均低于单纯COPD组,CD+8细胞分数均高于单纯COPD组(P0.05);治疗后3组患者CD+4/CD+8细胞比值比较,差异无统计学意义(P0.05);治疗后COPD+T2DM干预组CD+4细胞分数高于单纯COPD组及COPD+T2DM未干预组,CD+8细胞分数低于单纯COPD组及COPD+T2DM未干预组(P0.05)。治疗前单纯COPD组、COPD+T2DM干预及未干预组患者血清INF-γ水平、INF-γ/IL-4均高于对照组,血清IL-4水平均低于对照组,且COPD+T2DM干预及未干预组患者血清INF-γ水平、INF-γ/IL-4均高于单纯COPD组,血清IL-4水平均低于单纯COPD组(P0.05);治疗后COPD+T2DM干预组血清INF-γ水平、INF-γ/IL-4低于单纯COPD组及COPD+T2DM未干预组,IL-4水平高于单纯COPD组及COPD+T2DM未干预组(P0.05)。结论老年COPD合并T2DM患者T淋巴细胞免疫功能低下,存在CD+4/CD+8细胞比值及Th1/Th2细胞比值失衡,Th细胞亚群向Th1细胞极化趋势明显,治疗中加用免疫调节剂胸腺五肽后可有效抑制Th1细胞极化趋势,改善T淋巴细胞免疫状态。     

CD4+细胞及相关细胞因子在慢性乙型肝炎患者血中的变化

目的探讨部分CD4^＋细胞及相关细胞因子在慢性乙型肝炎患者血中的变化及意义.方法用流式细胞术检测外周血单个核细胞（PBMC）CD4、CD8和CD28分子表达情况;同时检测血清白介素（IL）-2、干扰素（IFN）-γ、IL-4、IL-10和IL-12水平以及肝功能.结果与正常对照组相比,86例慢性乙型肝炎患者PBMC CD4^＋细胞比率呈降低、不变和增高3种状态（定义为3组）;而CD28分子的表达与CD4^＋细胞比率的变化趋势一致;上述血清细胞因子水平除IL-4外,其余均显著增高（P＜0.01或P＜0.05）,但3组间差异无统计学意义;肝功能检测结果显示,部分患者ALT水平有异常,且与CD4＋细胞比率呈正相关.结论慢性乙型肝炎患者血中CD4^＋T细胞比率存在降低、不变和增高的改变;与病毒相关的几种细胞因子水平升高.鉴于ALT水平与CD4^＋T细胞比率呈正相关,推测乙型肝炎患者肝细胞损伤与CD4^＋细胞中的细胞毒性T淋巴细胞（CTL）有关.     

慢性乙型肝炎患者肠源性内毒素血症与树突状细胞表型及功能的关系

目的 探讨慢性乙型肝炎(CHB)患者树突状细胞(DC)与肠源性内毒素血症(IETM)的关系.方法 CHB患者80例,健康对照者21例,采集外周血,测定血浆内毒素含量、ALT、TBil.根据血浆内毒素水平,将患者分为内毒素阳性组和阴性组.同时用重组人粒细胞巨噬细胞集落刺激因子、重组人白细胞介素-4、酪氨酸激酶受体3配体和TNF-a体外诱导、培养CHB患者DC,采用流式细胞仪检测DC表型,混合淋巴细胞反应检测DC刺激T淋巴细胞的能力,用ELISA检测DC分泌细胞因子的水平.多组间比较采用单因素方差分析.结果 CHB患者DC表达CD83、CD80、CD86和人类白细胞抗原(HLA)-DR分子的水平及诱导同种异体混合T淋巴细胞增殖的能力均明显低于健康对照组.内毒素阳性组患者表达CD83、CD80、CD86、HLA-DR水平及诱导T淋巴细胞增殖的能力分别为(8.25±3.63)%、(10.63±4.52)%、(36.61±16.16)%、(61.65±14.33)%、0.812±0.311,明显低于内毒素阴性组的(11.39±4.35)%、(13.56±5.13)%、(45.90±15.35)%、(70.35±18.89)%、1.153±0.324(F=5.123、4.213、3.714、3.323、3.125,均P＜0.05).培养至第9天,CHB患者DC分泌IL-12和IFN-γ分别为(16.99±6.74)pg/mL和(10.52±4.19)pg/mL,明显低于健康者的(44.51±14.56)pg/mL和(17.94±5.86)pg/mL.内毒素阳性组患者IL-12水平为(13.14±5.71)pg/mL,明显低于内毒素阴性组的(20.98±9.03)pg/mL(F=3.225,P=0.016).IFN-γ水平在内毒素阳性组为(9.46±3.24)pg/mL,与阴性组的(11.54±5.20)pg/mL比较,差异无统计学意义(F=2.003,P=0.076).结论 IETM是导致CHB患者体内DC功能异常的原因之一.

Abstract：

Objective To investigate the relationship between dendritic cell (DC)and intestinal endotoxemia in patients with chronic hepatitis B (CHB).Methods Peripheral blood were collected from CHB patients (n = 80)and healthy controls (n = 21 ).Plasma endotoxin (ET)levels,liver function (alanine transaminase,total bilirubin)were detected.According to plasma ET concentration,all CHB patients were divided into two groups:ET positive and ET negative.The peripheral blood mononuclear cells (PBMCs)were isolated and then cultured with recombinant human granulocyte-macrophage colony-stimulating factor ( rhGM-CSF),recombinant human interleukin-4 ( rhIL-4 ),FMS-related tyrosine kinase 3 ligand (Flt3L)and tumor necrosis factor-alpha (TNF-α)to derive DC.The phenotypic patterns were characterized by flow cytometry.The proliferation of T lymphocytes was evaluated with mixed leukocytes reaction (MLR)and the levels of IL-12 and interferon-γ (IFN-γ)produced by DC were analyzed with enzyme-linked immunosorbent assay (ELISA).Comparisons among the two groups and healthy control group were done by single factor analysis of variance.Results Compared to healthy controls,the expressions of CD83,CD80,CD86,human leucocyte antigen (HLA)-DR and the proliferation of allogeneic T lymphocytes by DC were all significantly reduced in CHB patient groups.The expressions of CD83,CD80,CD86,HLA-DR and the activation of proliferation in ET positive subjects were lower than those in ET negative subjects [CD83 (8.25±3.63)% vs(11.39±4.35)% ,CD80 (10.63±4.52)% vs (13.56±5.13)%,CD86 (36.61±16.16)% vs (45.90±15.35)%,HLA-DR (61.65±14.33)% vs (70.35±18.89)%,the activation of proliferation0.812±0.311 vs 1.153±0.324; F=5.123,4.213,3.714,3.323 and 3.125,respectively; all P＜0.05].After cultured for 9 days,the secretions of IL-12 and IFN-γ by DC were significantly lower in CHB patients than in healthy controls [IL-12 (16.99± 6.74)pg/mL vs (44.51±14.56)pg/mL,IFN-γ (10.52±4.19)pg/mL vs (17.94±5.86)pg/mL].The level of IL-12 in the ET positive group was significantly lower than that ET negative group [( 13.14 ±5.71)pg/mL vs (20.98 ± 9.03)pg/mL; F= 3.225,P = 0.016].The level of IFN-γ was not different between two groups [(9.46 ± 3.24)pg/mL vs (11.54 ± 5.20)pg/mL; F = 2.003,P =0.076].Conclusion The intestinal endotoxemia may play a role in DC dysfunction in CHB patients.     

胰岛素抵抗的慢性丙型肝炎患者外周血CD4+CD25+调节性T细胞的数量及其功能

目的 了解胰岛素抵抗的慢性丙型肝炎患者外周血CD4 +CD25+调节性T细胞(Treg)数量和功能的变化.方法 筛选40例HLA-A2+慢性丙型肝炎患者(其中20例合并胰岛素抵抗),流式细胞仪检测患者CD4+CD25+Treg细胞占外周血中CD4+T细胞的频率,液闪计数仪检测对HCV特异性CD8+T细胞增殖的抑制作用,ELISA法检测IFN-y水平.统计学处理采用t检验.结果 胰岛素抵抗的慢性丙型肝炎患者外周血CD4 +CD25+ Treg细胞占CD4+T细胞的(9.5±1.9)％,明显低于慢性丙型肝炎患者的(11.2±2.2)％(t=2.615,P＜0.05).胰岛素抵抗指数(HOMA-IR)≥4患者的CD4+CD25+ Treg细胞比例为(9.0±1.8)％,明显低于HOMA-IR＜4患者的(10.8±2.3)％(t=2.413,P＜0.05).胰岛素抵抗的慢性丙型肝炎患者CD4+CD25+ Treg细胞和去除Treg的外周血单个核细胞(PBMC)共培养上清液中IFN-y为(4 050±580) pg/mL,明显高于慢性丙型肝炎患者的(2 005±330)pg/mL(t=13.705,P＜0.01).HOMA-IR≥4患者IFN-y为(5 682±986)pg/mL,明显高于HOMA-IR＜4患者的(2 819±660) pg/mL(t=7.630,P＜0.01).结论 随着胰岛素抵抗程度加重,慢性丙型肝炎患者外周血CD4+ CD25+ Treg细胞频率减低,对HCV特异性CD8+T细胞增殖的抑制作用减弱.     

慢性乙型肝炎患者应用拉米夫定前后T淋巴细胞亚群、CD4+CD25+调节性T淋巴细胞及白细胞介素-10、干扰素-γ的变化

目的 观察慢性乙型肝炎(CHB)患者应用拉米夫定抗病毒治疗前后外周血T淋巴细胞亚群、CD4+CD25+调节性T淋巴细胞(CD4+CD25+Treg)及IL-10、IFN-γ水平的变化.方法 选择CHB患者90例,在应用拉米夫定抗病毒治疗前及治疗后52周时,用流式细胞仪检测患者外周血T淋巴细胞亚群;在治疗前及治疗后12、24、36、52周时,用流式细胞仪检测外周血CD4+CD25+Treg频率,用双抗体夹心ELISA法检测IL-10、IFN-γ水平.组间及组内总体均数比较采用方差齐性的单因素方差分析或Dunnett's检验,组内治疗前后两时段均数比较采用配对t检验.结果 在90例CHB患者中,完全应答的32例患者的CD4+T淋巴细胞、CD8+T淋巴细胞及CD4+/CD8+较治疗前升高(t=4.055、3.267、2.328,均P＜0.05),外周血CD4+CD25+Treg频率在0、12、24、36、52周时分别为(5.40±0.60)%、(4.99±0.59)%、(4.54±0.72)%、(3.86±0.95)%、(3.44±0.76)%;IFN-γ水平、IFN-γ/IL-10逐步上升,IL-10水平逐步下降.部分应答的43例患者的CD4+T淋巴细胞及CD4+/CD8+较治疗前升高(t=3.484、2.018,均P＜0.05),CD8+T淋巴细胞较治疗前无明显差异,外周血CD4+CD25+Treg频率在0、12、24、36、52周时分别为(5.65±0.60)%、(5.23±0.63)%、(4.65±0.98)%、(4.42±0.97)%、(4.32±0.82)%,IFN-γ水平、IFN-γ/IL-10升高,IL-10水平降低.无应答的15例患者的外周血CD4+T淋巴细胞、CD8+T淋巴细胞及CD4+/CD8+、外周血CD4+CD25+Treg频率及IFN-y水平、IFN-γ/IL-10、IL-10水平较治疗前无明显变化.结论 应用拉米夫定治疗过程中,获得满意应答的CHB患者的外周血CD4+CD25+Treg频率下降,CD4+/CD8+、IFN-γ/IL-10的比例升高.

Abstract：

Objective To explore the correlation between the efficacy of lamivudine (LAM)therapy and changes of T lymphocyte subsets,CD4+ CD25+ regulatory T lymphocytes (Treg),and levels of interleukin-10 (IL-10)and interferon-gamma (IFN-γ)in the peripheral blood of patients with chronic hepatitis B (CHB).Methods Ninety CHB patients were enrolled in this study.T lymphocyte subsets in the peripheral blood were detected by flow cytometry at baseline and week 52 of LAM therapy.The frequencies of CD4+ CD25+ Treg in the peripheral blood were detected by flow cytometry and levels of IL-10 and IFN-γ were detected by enzyme-linked immunosorbent assay (ELISA)at baseline,week 12,24,36 and 52.The comparisons of overall means between groups and within groups were done by analysis of variance or Dunnett's test.The comparison of means before and after LAM therapy was done by paired t test.Results In 32 complete-responders of 90 CHB patients,the proportions of CD4+ T lymphocytes,CD8+ T lymphocytes and CD4+/CD8+ ratio were increased significantly after LAM therapy (t=4.055、3.267、2.328,all P＜0.05); the frequencies of CD4+CD25+ Treg at baseline,week 12,24,36 and 52 were (5.40±0.60)%,(4.99±0.59)%,(4.54± 0.72)%,(3.86±0.95)% and (3.44±0.76)%,respectively; the levels of IFN-γ,IFN-γ/IL-10 ratio were increased,while the IL-10 level was decreased after LAM therapy.In 43 partial-responders,the proportion of CD4+T lymphocytes and ratio of CD4+/CD8+ were increased after LAM therapy (t= 3.484,2.018,both P＜0.05); the proportion of CD8+ T lymphocytes was not changed significantly after therapy; the frequencies of CD4+ CD25+ Treg at baseline,week 12,24,36 and 52 were (5.65±0.60)%,(5.23±0.63)%,(4.65±0.98)%,(4.42±0.97)% and (4.32±0.82)%,respectively;IFN-γ level,IFN-γ/IL-10 ratio were increased,while IL-10 level was decreased.In 15 non-responders,the proportion of T lymphocyte subsets,the frequency of CD4+ CD25+ Treg,the levels of IFN-γ and IL-10 were not changed significantly after LAM treatment.Conclusions In CHB patients who have achieved response after LAM therapy,the frequency of CD4+ CD25+ Treg in the peripheral blood is decreased,while ratios of CD4+/CD8+ and IFN-γ/IL-10 in the peripheral blood are increased.     

羟氯喹抑制紫外线诱导的系统性红斑狼疮CD4+T细胞白细胞介素-10和干扰素-γ的表达

目的 研究紫外线对系统性红斑狼疮(SLE)CD4+T细胞因子的影响和羟氯喹的抑制作用.方法 选择SLE 30例,健康对照10名.磁珠分选SLE患者和健康人的CD4+T细胞,紫外线311 nm窄谱中波紫外线暴露,加入羟氯喹共培养,酶联免疫吸附试验(ELISA)检测培养上清白细胞介素(IL)-10和干扰素-γ的表达水平.采用t检验进行统计学分析.结果 SLE患者CD4+T细胞IL-10表达高于健康对照[(27±4)和(18±3) pg/ml,P=0.011];经45、100 mJ/cm2紫外线暴露后,SLE活动患者CD4+T细胞IL-10表达升高[(27±4)和(77±42) pg/ml,(40±18)和(77±42) pg/ml,P=0.022,P=0.048],经100 mJ/cm2紫外线暴露后,活动患者CD4+T细胞IL-10表达高于稳定患者[(77±42)和(24±4)pg/ml,P=0.029];羟氯喹降低SLE活动患者CD4+T细胞IL-10和干扰素-γ表达[(2.6±4.0)和(17.9±2.3)pg/ml,P=0.018,P=-0.017)];羟氯喹降低经45,100 mJ/cm2紫外线暴露后SLE活动患者T细胞IL-10表达[(40±18)和(22±6)pg/ml,(77±42)和(21±5) pg/ml,P=0.037,P=0.04];羟氯喹降低经100 mJ/cm2紫外线暴露的SLE活动和稳定患者T细胞干扰素-γ表达[(18±3)和(13±14) pg/ml,(19±7)和(12±5) pg/ml,P=0.013,P=0.049].结论 紫外线加重SLE患者体内Th1/Th2细胞因子的比例失衡;羟氯喹抑制了紫外线诱发SLE患者干扰素-γ和IL-10的表达.

Abstract：

Objective To explore the role of hydroxychloroquine (HCQ) in ultraviolet B (UVB)- induced expression of interleukin (IL)-10 and interferon (IFN)-γ from CD4+T cells in patients with systemic lupus erythematosus (SLE). Methods Thirty patients with SLE and 10 healthy controls were enrolled in the study. CD4+ T cells were isolated using magnetic beads from SLE patients and healthy controls. HCQ was added in culture media before and after irradiation with UVB 311 nm narrow band ultraviolet B (NB-UVB). The levels of IL-10 and IFN-γ in the supernatant were detected with enzyme-linked immunosorbent (ELISA). Comparisons between groups were performed by t-test. Results The level of IL-10 was higher in SLE patients [(27±4) pg/ml] than that in healthy controls [(18±3) pg/ml, P=0.011]. After exposure of CD4+T cells to UVB in 45 or 100 mJ/cm2 dosages, the level of IL-10 was increased significantly in patients with active disease (P=0.022, P=0.048). After exposure of CD4+T cells to UVB in 100 mJ/cm2 dosages, the levels of IL-10 was higher in patients with active disease [(77±42) pg/ml] than patients with stable disease [(24± 4) pg/ml, P=0.029]. When CD4+ T cell were cultured with HCQ, IL-10 and IFN-γ levels in patients with active disease [(2.6±4.0), (17.5±2.3) pg/ml] were decreased significantly (P=0.018, P=0.017). HCQ reversed UVB-induced IL-10 expression in active SLE patients after exposure of CD4+T cells to UVB in 45 or 100 mJ/cm2 dosages (P=0.037, P=0.04). HCQ also reversed UVB-induced IFN-7 expression in active SLE patients and stable SLE patients after exposure to CD4+T cells with UVB in 100 mJ/cm2 dosages (P=0.013, P= 0.049). Conclusion UVB can aggravate the imbalance of Th1 and Th2 cytokines. HCQ inhibits UVB-induced IL-10 and IFN-7 expression of CD4+T cells in patients with SLE, especially in patients with active disease.     

拉米夫定联合苦参素治疗慢性乙型肝炎患者外周血T细胞亚群和细胞因子的变化

目的观察拉米夫定联合苦参素治疗对慢性乙型肝炎患者血清细胞因子水平的影响和T细胞亚群的变化。方法 86例慢性乙型肝炎患者被随机分为治疗组42例和对照组44例,治疗组给予拉米夫定联合苦参素治疗,对照组单用拉米夫定治疗,在48周末观察结果。采用ELISA双抗体夹心法检测IL-2、IL-6、IL-10和IFN-γ水平;采用FACScalibur流式细胞仪检测外周血T细胞亚群。结果在治疗48周末,治疗组和对照组谷丙转氨酶复常率分别为90.2%和83.3%(P>0.05);治疗组HBeAg转阴率、HBeAg/HBeAb转换率和HBV DNA转阴率分别为57.1%、35.3%和88.5%,显著高于对照组的30.2%、19.8%和67.1%(P<0.05);治疗组和对照组IL-2水平分别为181.1±26.1pg/ml和86.9±10.7pg/m(l P<0.05),IFN-γ水平分别为47.5±4.3pg/ml和27.8±4.4pg/m(l P<0.05),IL-6水平分别为13.2±5.8pg/ml和27.1±6.4pg/m(l P<0.05),IL-10水平分别为109.7±32.1pg/ml和169.5±36.2pg/m(l P<0.05);治疗组和对照组CD4+T淋巴细胞百分率分别为37.2±5.8%和27.8±6.4%(P<0.05),CD8+T淋巴细胞百分率分别为20.8±5.2%和26.3±6.2%(P<0.05),CD4+/CD8+比值分别为1.56±0.3和1.34±0.2(P<0.05)。结论拉米夫定联合苦参素治疗可以纠正慢性乙型肝炎患者Th1和Th2型细胞因子紊乱,改善T细胞亚群中各成分的比值,对慢性乙型肝炎患者细胞免疫有一定的调节作用,因而具有治疗作用。     

抗病毒治疗对慢性丙型肝炎CD4^＋CD25^＋调节性T细胞频率和功能的影响

目的研究抗病毒治疗前后慢性丙型肝炎患者CD4^＋CD25^＋调节性T细胞（Treg）频率和功能的变化。方法筛选HLA—A2阳性慢性丙型肝炎患者31例，给予聚乙二醇化干扰素α-2a（相对分子质量为40000）180μg每周1次皮下注射，联合口服利巴韦林。分别在治疗前和治疗结束随访24周时应用流式细胞仪检测患者CD4^＋CD25^＋ Treg细胞占外周血CD4^＋T细胞的频率，应用液闪计数仪检测其对HCV特异性CD8^＋T细胞增殖的抑制作用，ELISA法检测细胞培养上清γ干扰素（IFN-γ）水平的变化情况。结果治疗结束随访24周，患者外周血CD4^＋CD25^＋ Treg细胞频率为（9．6±3．0）％，明显低于治疗前的（11．0±2．3）％（t=2．028，P〈0．05）；持续病毒学应答（SVR）组CD4^＋CD25^＋ Treg细胞频率为（8．9±2．7）％，明显低于未获得SVR患者组的（10．4±2．3）％（t=3．324，P〈0．01）。抗病毒治疗后CD4^＋CD25^＋ Treg细胞抑制HCV特异性CD8^＋T细胞增殖的作用减弱。治疗后患者IFN-γ水平为（3959±577）pg／ml，明显高于治疗前的（1965±326）pg／ml（t=16．1，P〈0．01）；获得SVR患者组IFN-γ（6824±568）pg／ml，明显高于未获得SVR患者组的（2219±286）pg／ml（t=29．853，P〈0．001）。结论慢性丙型肝炎患者随着HCV RNA水平的下降，CD4^＋CD25^＋Treg细胞频率降低，抑制HCV特异性CD8^＋T细胞增殖的作用减弱。     

血清白介素1和γ干扰素的检测对肺结核的诊断价值

目的探讨血清白介素1(IL-1)和γ干扰素(IFN-γ)的检测在肺结核诊断中的意义。方法采用酶联免疫法(ELISA)检测42例肺结核患者和27例健康人血清中IL-1和IFN-γ的水平。结果与健康人组相比较,肺结核组血清中IL-1[(78.21±27.13)pg/mL vs(8.80±6.94)pg/mL]和IFN-γ[(56.85±20.62)pg/mL vs(29.11±31.52)pg/mL]的水平均明显升高(P<0.05,P<0.01)。结论血清IL-1和IFN-γ的测定有助于肺结核患者的诊断。     

Cytokines are early diagnostic biomarkers of graft-versus-host disease in liver recipients

BACKGROUND: Graft-versus-host disease(GVHD) is associated with high mortality. Early diagnosis is essential to start treatment and to improve outcomes. Because of the inflammatory nature, we hypothesis that cytokine profile of patients with GVHD may serve as diagnostic markers. The present study was to evaluate the role of cytokine profile in the diagnosis of GVHD.METHODS: An immunoassay was used to detect 29 cytokines simultaneously in the serum; the measuring sensitivity of all cytokines was pg/m L. Healthy subjects undergoing annual routine physical examinations served as negative controls; 23 patients with hepatocellular carcinoma(HCC) who had undergone liver transplantation(the LT group) comprised the test subjects. A total of 22 kidney recipients with biopsyconfirmed GVHD(the RT group) were included for comparison. HCC patients with radical surgery(the HCC group, n=22) served as positive control. The liver contents of the three cytokines, IL-2, IL-18, and IFN-γ, were detected with immunohistochemistry. Serum granzyme B and perforin were measured by flow cytometry.RESULTS: Of the 29 cytokines, the levels of IL-2 and IL-18 were increased significantly in liver recipients with GVHD compared with healthy controls(P0.05). The serum levels of these three cytokines in the healthy, HCC, LT, and RT groups were IL-2: 0.90±0.02, 4.14±0.61, 5.10±0.89, and 1.48 ±0.09 pg/m L; IL-18: 80.61±9.35, 109.51±10.93, 230.11±12.92, and 61.98±7.88 pg/m L; IFN-γ: 24.06±3.88, 24.84±3.21, 40.37±5.88, and 15.33±4.72 pg/m L, respectively. Immunohistochemistry showed that these 3 cytokines expressions in the liver were parallel to the serum cytokine. After standard anti-GVHD treatment, the expressions of IL-2, IL-18, and IFN-γ were decreased in the liver(P0.05). Serum granzyme B and perforin were significantly increased in GVHD patients(P0.05). CONCLUSIONS: IL-2, IL-18 and IFN-γ were from liver and might serve as biomarkers for monitoring GVHD development and the effects of anti-GVHD treatment. Granzyme B and perforin may play a role in increasing IL-2, IL-18, and IFN-γ levels in GVHD patients.     

类风湿关节炎患者外周血Th17细胞的检测及意义

目的 探讨Th17细胞在类风湿关节炎(RA)患者外周血中的水平及意义.方法 分离RA患者和健康者外周血单个核细胞(PBMC),免疫磁珠阴选CD4~+T细胞,用或不用非特异性刺激剂(A-CD3、A-CD28),然后加佛波酯/离子霉素(PMA/Ion),经固定,透膜处理进行细胞内染色,流式细胞术(FCM)检测CD4~+T细胞内白细胞介素(IL)-17~+/干扰素(IFN)-γ~+、IL-17~+/IL-6~+水平.分组:①健康对照组;②RA病情稳定组;③RA病情活动组.结果 免疫磁珠阴选CD4~+T细胞纯度>90%.RA病情活动组IL-17表达水平(1.54±0.41)显著高于RA病情稳定组(0.70±0.21,P<0.01),二者又显著高于健康对照组(0.42±0.12,P<0.01).用A-CD3、A-CD28、IL-23刺激后,CD4~+T细胞IL-17胞内表达水平较无刺激组显著增加(P<0.05).CD4~+T细胞IFN-γ表达水平呈现与IL-17表达相似的特点,而RA患者与健康对照组IL-6表达水平差异无统计学意义.RA患者IL-17胞内表达水平与IFN-γ、IL-6无显著相关.结论 RA患者外周血CD4~+T细胞中存在异常增高的Th17细胞,且其水平与病情活动相关,有望作为判断RA患者病情活动的指标之一.