老年早期糖尿病肾病患者血清炎症因子、外周血NFκ-B活性变化及阿托伐他汀的治疗作用

目的观察老年早期糖尿病肾病（DN）患者血清炎症因子水平、外周血NF-κB活性的变化,并探讨阿托伐他汀对DN患者的治疗作用。方法根据是否合并DN将96例2型糖尿病患者分为糖尿病（DM）组30例、DN组66例。DN组随机分为DM常规治疗组（DN1组）31例、DM常规治疗＋阿托伐他汀治疗组（DN2组）35例,并选择30例健康体检者作为对照组（NC组）。分别观察各组患者高敏C反应蛋白（hs-CRP）、TNF-α、IL-6、IL-1β及外周血NF-κB p65的磷酸化水平。结果 NC组、DM组、DN组血清hs-CRP、TNF-α、IL-6、IL-1β水平及外周血NF-κBp65活性逐渐升高（P〈0.01或〈0.05）。阿托伐他汀治疗后NF-κB p65活性及hs-CRP、TNF-α、IL-6、IL-1β明显下降（P〈0.01或〈0.05）。结论阿托伐他汀可抑制老年早期DN患者外周血NF-κB p65的活性,并可降低血清hs-CRP、TNF-α、IL-6、IL-1β等炎症因子的水平,显著改善患者炎症状态。     

肝内CD68阳性细胞及TNF-α在慢加急性乙型肝炎肝衰竭发病机制中的作用

目的通过研究肝内CD68＋和TNF-α阳性细胞的表达,来探讨它们在乙型肝炎慢加急性肝衰竭（ACLF）发病机制中的作用。方法采用免疫组化法检测乙型肝炎ACLF患者、慢性乙型肝炎（CHB）患者及正常对照人群肝内TNF-α的表达以及其分泌细胞CD68＋细胞的数量。结果ACLF患者肝内TNF-α阳性分泌细胞平均数为90/高倍视野（hpf）,明显高于CHB患者及正常对照组（分别为12/hpf和5/hpf）,差异均非常显著（P〈0.001）;ACLF患者肝内CD68＋细胞平均数为94/hpf,明显高于CHB患者及正常对照组（分别为48/hpf和15/hpf）,差异均非常显著（P〈0.001）;TNF-α阳性分泌细胞与CD68阳性细胞数量具有显著的正相关（r=0.866,P〈0.001）。结论乙型肝炎ACLF患者肝内CD68＋细胞数量明显增加,其分泌的TNF-α也明显增加,推测TNF-α可能参与了乙型肝炎ACLF的发病过程。     

实验性结肠炎大鼠内毒素-Toll样受体4-核因子κB信号通路的变化及益生菌的作用

目的探讨和分析实验性结肠炎大鼠血浆内毒素水平的变化及结肠Toll样受体4（TLR4）和核因子κB（NF-κB）表达及意义,并分析益生菌的作用。方法30只雄性W istar大鼠均分为正常对照组（NC组）、模型对照组（UC组）和益生菌治疗组（PC组）;UC和PC组建立2,4,6-三硝基苯磺酸（TNBS）实验性结肠炎大鼠模型,PC组大鼠给予双歧三联活菌悬液（2.2×10^9CFU/只）治疗,1次/d,共4周。光镜下观察肠黏膜炎性反应并评分;鲎试验检测各组大鼠血浆内毒素水平;W estern印迹法和实时荧光定量PCR法检测大鼠结肠TLR4和NF-κB p65蛋白及mRNA的表达,分析其变化及益生菌的作用。结果PC组炎性反应评分较UC组明显降低（P〈0.05）,但高于NC组（P〈0.01）;PC组血浆内毒素明显低于UC组（P〈0.05）,但高于NC组（P〈0.01）;UC组TLR4和NF-κB p65的蛋白及mRNA表达明显高于PC组和NC组（P〈0.05、0.01）,PC组高于NC组（P〈0.01）。结论内毒素-TLR4-NF-κB信号通路参与了大鼠实验性结肠炎的发生和发展,减少内毒素的产生、降低大鼠结肠TLR4和NF-κB表达可能是益生菌减轻大鼠结肠炎症的机制之一。     

沙格列汀对ox-LDL诱导的血管内皮细胞损伤及miR-590/TLR4/NF-κB表达的影响

目的探讨沙格列汀对ox-LDL诱导的血管内皮细胞损伤及miR-590/TLR4/NF-κB表达的影响。方法培养人脐静脉内皮细胞(HUVEC)并分为对照组、ox-LDL组、沙格列汀组、沙格列汀+miR-590抑制物组、NC模拟物组、miR-590模拟物组、NC抑制物组、miR-590抑制物组。检测细胞的增殖活力、细胞中miR-590/TLR4/NF-κB表达量及培养基中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、细胞间黏附分子1(ICAM-1)、血管细胞黏附分子1(VCAM-1)的含量。结果 ox-LDL组细胞中TLR4、NF-κB p65表达量及培养基中TNF-α、IL-1β、ICAM-1、VCAM-1的含量均明显高于对照组,细胞增殖活性及细胞中miR-590的表达量明显低于对照组;沙格列汀组细胞中TLR4、NF-κB p65表达量及培养基中TNF-α、IL-1β、ICAM-1、VCAM-1的含量均明显低于ox-LDL组,细胞增殖活性及细胞中miR-590的表达量明显高于ox-LDL组;沙格列汀+miR-590抑制物组细胞中TLR4、NF-κB p65表达量及培养基中TNF-α、IL-1β、ICAM-1、VCAM-1的含量均明显高于沙格列汀组,细胞增殖活性及细胞中miR-590的表达量明显低于沙格列汀组;miR-590模拟物组细胞中TLR4、NF-κB p65的表达量及培养基中TNF-α、IL-1β、ICAM-1、VCAM-1的含量均明显低于NC模拟物组,miR-590抑制物组细胞中TLR4、NF-κB p65的表达量及培养基中TNF-α、IL-1β、ICAM-1、VCAM-1的含量均明显高于NC抑制物组。结论沙格列汀能够通过miR-590/TLR4/NF-κB通路减轻ox-LDL诱导的血管内皮细胞损伤。     

慢性乙型肝炎患者HBVDNA载量与PMBC分泌IFN-γ、IL-6水平相关性研究

目的探讨不同HBVDNA载量慢性乙型肝炎（CHB）患者经拉米夫定治疗前后外周血单核细胞（PM-BC）分泌IFN-γ、IL-6水平变化规律及其临床意义。方法血清HBVDNA载量采用荧光定量聚合酶链反应（FQ-PCR）测定,PMBC分泌IL-6、IFN-γ水平采用双抗体夹心酶联免疫吸附试验（DAS-ELISA）测定。结果治疗前CHB患者IFN-γ水平显著低于正常对照组（P〈0.01）,IL-6水平显著高于正常对照组（P〈0.01）;拉米夫定治疗后HBV高、中载量组IFN-γ水平较治疗前显著升高（P〈0.01和P〈0.05）,IL-6水平较治疗前显著降低（P〈0.01和P〈0.05）,治疗后中、低载量组IFN-γ水平仍低于正常对照组（P〈0.01）,IL-6的水平高于正常对照组（P〈0.01）;患者INFγ水平变化与HBVDNA载量呈负相关（r=0.89,P〈0.001）,IL-6水平变化与HBVDNA载量和ALT呈正相关（分别为：r=0.92,P〈0.001;r=0.74,P〈0.001）。结论不同HBVDNA载量CHB患者经拉米夫定治疗应答与IFN-γ、IL-6水平程度有关,IFN-γ和IL-6的含量能较好的反映TH1/TH2的水平,且可以作为CHB疗效参考指标。     

细胞因子在乙肝相关性肝衰竭患者中的表达意义

[目的]观察细胞因子水平在乙肝相关性肝衰竭患者中的水平变化,探讨其表达意义。[方法]选取于2012年8月~2015年7月来我院就诊的乙肝相关性慢加急性肝衰竭(ACLF)患者48例、慢性乙型肝炎(CHB)患者48例和健康对照患者20例。检测3组患者IL-6、IL-8、IL-10、TNF-α、IFN-γ水平,检测3组患者各项临床指标进行分析。[结果]3组间IL-6、IL-8和TNF-α水平分析可见,ACLF组明显高于CHB组,差异有统计学意义(P0.001);CHB组明显高于对照组,差异有统计学意义(P0.01);ACLF组明显高于对照组,差异有统计学意义(P0.001)。IL-10、IFN-γ在3组间差异无统计学意义。死亡组的MELD评分、IL-6、TNF-α水平明显高于对照好转组,差异有统计学意义(P0.05)。IL-8水平在2组间比较差异无统计学意义。IL-6、TNF-α水平与MELD评分明显呈正相关(r=0.487、0.402,P=0.013、0.037)。其他细胞因子与MELD评分、ALT、AST等临床指标间无明显相关性。[结论]细胞因子在HBV相关ACLF患者的疾病发生、发展过程中有重要作用,其中,IL-6、TNF-α可能与患者的预后有关,促进患者疾病的发生发展。     

乙型肝炎病毒抑制巨噬细胞TLR3、Mda-5和RIG-I表达

目的探讨乙型肝炎病毒（HBV）与巨噬细胞上模式识别受体（PRR）的相互作用。方法以不同载量HBV刺激佛波酯（PMA）诱导的单核源巨噬细胞,实时定量PCR检测刺激2、4、12及24h后巨噬细胞Toll样受体3（TLR3）、黑色素瘤分化相关基因5（Mda-5）、视黄酸诱导基因I（RIG-I）表达;以poly I：C刺激与HBV共培养12h的巨噬细胞,酶联免疫吸附试验（ELISA）测定poly I：C刺激4h后培养上清液中β干扰素（IFNβ-）的分泌水平。结果不同病毒载量组巨噬细胞TLR3表达在2、4、12、24h较对照组下调,且高病毒载量组表达低于低病毒载量组（F值分别为123.64、24.50、6.69、7.61,P均〈0.01）;Mda-5和RIG-I在上述4个时间点的表达为高病毒载量组低于低病毒载量组（F值分别为36.44、48.58、46.59、12.77;67.68、74.54、18.18、17.76;P均〈0.01）。IFNβ-的表达为：阳性对照组〉低病毒载量组〉高病毒载量组〉阴性对照组（F=78.42,P〈0.01）,表达量分别为（497.2±43.7）、（294.2±48.4）、（92.5±12.3）、（40.4±9.9）pg/mL。结论 HBV能抑制巨噬细胞TLR3、Mda-5和RIG-I表达,致IFN-β分泌下降,可能是其逃逸机体天然免疫的机制之一。     

干扰素治疗前后慢性乙型肝炎患者外周血单核细胞Toll样受体4水平的变化

目的 探讨慢性乙型肝炎(CHB)患者干扰素治疗前后外周血单核细胞Toll样受体(TLR)4水平的变化及其意义.方法 用流式细胞仪检测30例健康人、65例CHB患者外周血单核细胞表面TLR4的表达,ELISA法检测上述患者血清白细胞介素(IL)-6的水平,鲎试验检测血清内毒素.结果 CHB患者组与正常对照组比较,外周血单核细胞TLR4表达水平、血清IL-6及内毒素水平均升高,具有显著性差异(P＜0.05,P＜0.01,P＜0.01).患者应用干扰素针治疗12周时TLR4水平及IL-6水平均下降,但无显著性差异(P＞0.05),内毒素水平下降较明显(P＜0.05);治疗24周时,患者TLR4水平、血清IL-6水平及内毒素水平均明显下降,与治疗前比较有显著性差异(P＜0.05,P＜0.01,P＜0.01).结论 外周血单核细胞TLR4表达水平与CHB的发病及治疗转归有关,且TLR4水平与IL-6及内毒素水平密切相关.     

阿德福韦酯体外对慢性乙型肝炎患者树突状细胞功能的影响

目的:探讨体外阿德福韦酯(adefovir dipivoxil,ADV)对慢性乙型肝炎(chronic hepatitis B,CHB)患者外周血树突状细胞(dendritic cell.DC)功能的影响.方法:分离CHB患者及健康自愿者外周血单核细胞(Mo),在含rhGM-CSF rhIL-4及不同浓度ADV(20、100、500μg/L)培养条件下制备单核细胞源DC(MoDC).在倒置显微镜下观察DC的形态:FACS分析DC的表型;MTT法测定DC刺激同种异体T细胞增殖的能力;ELISA检测DC培养上清中IL-12 p40 p70的含量.结果:与CHB未处理组比较,CHB ADV处理组DC形态分化明显,激发初始T细胞增殖的能力加强.上清液中分泌的IL-12 p40 p70增多;在CHB ADV处理组(100μg/L),CDla 、CD83 、CD866和MHC-DR DC的数量均最多,显著高于CHB未处理组(43.5±5.7 vs 20.6±2.8.34.64±1.9 vs 16.7±3.4.40.94±2.8 vs 25.8±6.6,66.94±5.4 vs 40.74±4.2,均P＜0.05),但低于健康对照组,三组间差异具有统计学意义(P＜0.05).结论:ADV可以增强CHB患者外周血MoDC的免疫功能,提示其可能通过调节DC的功能而参与免疫应答,发挥间接抗病毒作用.     

连续性血液净化治疗老年多器官功能障碍及对机体免疫功能的影响

目的观察连续性血液净化（CBP）治疗老年多器官功能障碍综合征（MODS）及对机体免疫功能的影响。方法18例老年MODS患者，CBP治疗72h。在治疗0，24，48，72h时，测定单核细胞人类白细胞DR抗原（HLA—DR）的表达情况判断其抗原呈递功能（流式细胞仪），ELISA法测定细胞因予水平（TNF-α、IL-6、IL-10）；同时观察外周血单核细胞计数。结果18例患者在接受CBP治疗时，（1）单核细胞分泌功能：治疗前单核细胞分泌活跃，治疗后该组单核细胞分泌TNF-α、IL-6和IL-10均明显减少（P〈0．05）。（2）单核细胞抗原呈递功能：与正常人相比，MODS患者单核细胞表达HLA—DR都明显降低（P〈0．001）。治疗后明显改善（P〈0．01）。（3）单核细胞数量：治疗前，与正常指标比较单核细胞数量均明显降低，治疗后单核细胞数量明显上升接近正常水平。CBP治疗后，血浆TNF-α和IL-6水平较治疗前明显降低（P〈0．01）。（4）7例死亡患者IL-10较其他患者明屁升高，HLA—DR的表达则明显低下，CBP治疗后无明显改变。结论（1）CBP能明显改善老年MODS患者单核细胞功能，重建机体免疫系统内稳状态。（2）HLA-DR持续低表达和高血浆IL-10水平者预后差。（3）对免疫过度激化伴血浆细胞因子明硅升高者，CBP显示了是好的清除效果。     

Correlation of the expression of toll-like receptors in monocyte-derived dendritic cells with prognosis of chronic severe hepatitis B

OBJECTIVE: This study aims to measure the expression of toll‐like receptors (TLR) in monocyte‐derived dendritic cells (MoDC) from chronic severe hepatitis B (CSHB), to assess the contribution of TLRs in CSHB. METHODS: Peripheral blood was collected from 40 CSHB patients, 30 chronic hepatitis B (CHB) patients, and 30 healthy individuals who served as healthy controls (HCs). Purified monocytes were isolated by a combination of Histopaque‐1.077 and CD14 Microbeads. MoDCs were induced with granulocyte macrophage colony‐stimulating factor and interleukin‐4 for 6 days from CD14⁺ monocytes. The expression of TLRs in MoDC was measured using real‐time PCR and flow cytometry. RESULTS: The expressions of TLR‐1, ‐2, ‐7 were significantly higher in MoDC of CSHB than that of HCs, of which the level of TLR‐3 was decreased. Particularly in CSHB patients, the TLR‐3 expression was further decreased compared to CHB patients. In non‐survival CSHB patients, TLR‐3 level was significantly decreased, while TLR‐2 expression was dramatically increased. Linear correlation analysis demonstrated significant correlations between TLR‐3 level and disease severity markers (total bilirubin, prothrombin activity, creatinine, white blood cell count, and maximum volume of ascitic fluid) in individual CSHB patients. CONCLUSIONS: TLR‐2 and TLR‐3 may be involved in the pathogenesis of CSHB, and TLR‐3 may influence the prognosis of CSHB.     

Toll-like receptor 2 is overexpressed in Familial Mediterranean fever patients and is inhibited by colchicine treatment

Aim: To study the role of Toll-like receptor (TLR) 2 in Familial Mediterranean fever (FMF) inflammatory process.Methods: TLR2 expression on monocytes of FMF attack-free patients (n = 20) and the effect of sera of FMF patients with an acute attack (n = 9) on TLR2 expression on monocytes of healthy donors were studied by flow cytometry (FACS). TLR2 expression was also studied in THP-1 cells, and TLR2 downstream signaling was studied by ELISA for the secretion of IL-1β and pro-inflammatory cytokines or by western blotting to measure nuclear factor (NF)-κB.Results: FMF attack-free patients had increased CD14 + TLR2+ cell count as compared to healthy donors. High-dose colchicine treatment (≥2 mg/d) inhibited this increased expression in FMF patients. Colchicine in vitro also inhibited TLR2 expression on THP-1 cells. Sera from FMF patients with an acute attack induced TLR2 expression by both monocytes of healthy donors and THP-1 cells as well as pro-inflammatory cytokine secretion by healthy monocytes, while colchicine inhibited this induction. Pam2CSK4 increased interleukin-1β (IL-1β) secretion by peripheral blood mononuclear cells (PBMCs) of healthy donors, and this activation was inhibited by colchicine. THP-1 cells presented elevated NF-κB expression when cultured with Pam2CSK4, whereas colchicine inhibited this elevation.Conclusions: TLR2 activation was upregulated in monocytes of FMF patients, and colchicine inhibited this upregulation both in -vitro and in -vivo. This indicates that elevated expression of TLR2 promotes the production of pro-inflammatory cytokines, which may contribute to uncontrolled inflammation in FMF.     

慢加急性乙型肝炎肝衰竭患者肝组织IFN-γ、TNF-α和IL-10的表达

目的研究乙型肝炎慢加急性肝衰竭（ACLF）患者肝内促炎细胞因子和抗炎细胞因子的表达特性。方法采用免疫组化法检测乙型肝炎ACLF患者、慢性乙型肝炎患者和正常人肝组织IFN-γ、TNF-α和IL-10的表达。结果在ACLF患者肝组织IFN-γ和TNF-α阳性细胞数均明显高于慢性乙型肝炎患者和正常对照组（P均〈0.001）;ACLF患者、慢性乙型肝炎患者和正常人肝组织IL-10的表达无统计学差异（P〉0.05）;肝内IFN-γ表达与TNF-α具有明显的相关性（r=0.886,P〈0.001）。结论乙型肝炎ACLF患者因肝内促炎细胞因子和抗炎细胞因子表达的失衡而发生了肝损伤。     

乙型肝炎病毒感染者单核细胞来源的树突状细胞中维甲酸诱导基因-Ⅰ的表达及功能

目的 探讨HBV感染后维甲酸诱导基因-Ⅰ(RIG-Ⅰ)在单核细胞来源的树突状细胞(MoDC)中的表达和功能,并了解RIG-Ⅰ和MoDC在HBV感染疾病进程中的作用.方法 采集28例HBV感染者外周血,21例为慢性乙型肝炎(CHB)患者,7例为急性乙型肝炎(AHB)患者;18例健康志愿者作为对照.采用CD14免疫微珠分离纯化外周血CD14~+单核细胞,用粒-巨噬细胞集落刺激因子(GM-CSF)和IL-4联合诱导培养7 d成为树突状细胞(DC),用水泡性口炎病毒(VSV)感染刺激RIG-Ⅰ活化.在不同时间点收集细胞抽提RNA,实时定量PCR检测RIG-Ⅰ、干扰素启动子刺激基因1(IPS-1)和IFN-β的表达.正态分布数据采用方差分析,连续性变量两组间比较采用Mann-Whitney U检验,多组间比较采用Kruskal-Wallis检验.结果 VsV感染活化RIG-Ⅰ后,CHB、AHB组和健康对照组的RIG-Ⅰ mRNA表达水平在16 h(2.44±2.03,19.54±3.15.21.48±8.39;F=7.451.P=0.002)和24 h(2.68±2.93,10.31±3.88,14.01±5.04;F=7.908,P=0.001)时差异均有统计学意义;IPS-1在CHB和AHB组中的表达在16 h(2.05±1.08,1.99±1.56)和24 h(2.27±2.16,3.24±1.21)均高于健康对照组(16 h:0.60士0.31,F=7.246,P=0.003;24 h;1.08±0.73,F=13.598,P=0.01).CHB组中IFN-β水平在16 h和24 h均低于AHB组和健康对照组,差异有统计学意义.结论 RIG-Ⅰ和IPS-1在HBV感染者MoDC中的表达明显异常,提示RIG-Ⅰ信号通路可能被HBV干扰,RIG-Ⅰ和MoDC的功能缺陷在HBV感染和慢性化中发挥重要作用.     

慢性乙型重型肝炎患者外周血单个核细胞衍生的树突状细胞表型与功能

目的 通过检测慢性乙型重型肝炎(CSHB)患者外周血单个核细胞(PBMC)衍生的树突状细胞(MoDC)经聚肌胞苷酸刺激后表型变化及细胞因子分泌情况,了解MoDC的免疫调节功能及其在慢性乙型肝炎(CHB)重症化中的作用.方法 采集CSHB组患者37例、CHB组患者20例和健康对照者20例的外周血,分离PBMC,免疫磁珠细胞分选法获得纯化的PBMC,体外培养诱导为未成熟树突状细胞(iDC),聚肌胞苷酸刺激为成熟树突状细胞(mDC).流式细胞术检测iDC及mDC细胞表面人类白细胞DR抗原(HLA-DR)、CD83、CD86、CD80的平均荧光强度(MFI);ELISA测定聚肌胞苷酸刺激后12、24、48 h的MoDC培养上清液中IL-12、IL-6、TNF-α分泌水平.多组间比较采用单因素方差分析,并进行方差齐性检验.结果 3组iDC表面HLA-DR、CD83、CD86、CD80的MFI比较,差异无统计学意义.CSHB组患者mDC表面HLA-DR、CD83、CD86、CD80的MFI与CHB组、健康对照组比较,显著低下(F值分别为59.73、13.95、34.80和73.02,均P＜0.05).聚肌胞苷酸刺激12、24、48 h时IL-12分泌量为:健康对照组＞CHB组＞CSHB组(F值分别为151.34、126.65、72.76,均P＜0.05),其中24 h时分泌量最高,分别为(48.2±7.6)、(56.7±11.8)、(97.8±16.2)ng/L.IL-6在上述3个时间点的分泌量为:CSHB组＞CHB组＞健康对照组(F值分别为92.50、86.89、64.57,均P＜0.05),其中12 h分泌量最高,分别为(1698.3±340.4)、(965.8±231.7)、(697.8±213.6)ng/L.TNF-α在上述3个时间点的分泌量为:CSHB组＞CHB组＞健康对照组(F值分别为58.66、122.36、44.73,均P＜0.05),24 h表达量分别为(19 672.7±4214.7)、(9946.1±2586.5)、(6659.2±955.8)ng/L.结论 CSHB患者MoDC成熟障碍,表现为IL-12分泌低下,而IL-6及TNF-α分泌亢进,可能是加剧肝脏炎性反应致重型肝炎的重要因素.     

Knockdown of myeloid differentiation protein-2 reduces acute lung injury following orthotopic autologous liver transplantation in a rat model

BackgroundAcute lung injury (ALI) is a serious complication that commonly occurs during orthotopic liver transplantation (OLT). Toll-like receptor 2/4 (TLR2/4) are the main membrane receptors that respond to inflammatory stimuli and mediate NF-kappa B (NF-κB) signal pathway. We previously showed that TLR2/4 expression on monocytes and serum cytokine levels were increased in patients with ALI induced by OLT. Myeloid differentiation protein-2 (MD-2) expresses the functional domains that combines TLRs and play a key regulatory role in TLRs activation. Therefore, we hypothesized that blocking MD-2 would inhibit the TLR2/4-mediated inflammatory response and lessen ALI induced by liver transplantation.MethodThirty-two Sprague Dawley (SD) rats were randomly divided into four groups. One group received a sham operation (Group S), and the other three groups underwent orthotopic autologous liver transplantation (OALT) 48 h after intratracheal administration of saline (Model group; Group M), non-targeting siRNA (negative siRNA control group; Group NC) or siRNA against MD-2 (intervention group; Group RNAi). Lung pathology, lung water content, PaO₂, and expression levels of MD-2, TLR2/4, NF-κB, TNF-α, IL-1β and IL-6 were assessed 8 h after OALT.ResultsIn Groups M and NC, OALT produced marked lung pathology with decreased PaO₂ levels and increased MD-2, TLR2/4 gene and protein expression levels. Furthermore, the nuclear translocation of the NF-κB P65 subunit, was increased, as were lung concentrations of TNF-α, IL-1β and IL-6. The pathology of ALI and the severity of the above biochemical changes induced by OALT were significantly reduced in the group treated with MD-2 siRNA.ConclusionMD-2 gene knock-down attenuated the increase in TLR2/4 activation and reduced ALI after OALT.     

乙型肝炎慢加急性肝功能衰竭患者免疫功能抑制与疾病严重程度的研究

目的 探讨乙型肝炎慢加急性肝功能衰竭(ACLF)患者体内免疫功能抑制与疾病严重程度的关系.方法 收集上海公共卫生临床中心2009年8月至2010年4月住院治疗的乙型肝炎ACLF患者27例(ACLF组),活动性慢性乙型肝炎患者28例(CHB组)和健康志愿者8名(对照组)的临床资料及外周血标本.以APACHEⅢ评分及肝性脑病程度作为反映疾病严重程度的量化指标.应用流式细胞术检测患者外周血中T淋巴细胞亚群的绝对计数和单核细胞表面人类白细胞抗原(HLA)-DR的表达量.采用CBA试剂盒中检测患者血浆中促炎性细胞因子及抑炎性细胞因子[干扰素(IFN)-γ、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-2、IL-4、IL-10]的水平.采用SPSS 16.0软件进行数据统计.结果 与CHB组和对照组相比,ACLF组患者抑炎性细胞因子IL-10含量显著增加(Z=-4.279,U=124,P＜0.01;Z=-3.871,U=9.5,P=0.0001),促炎性因子IFN-γ、TNF-α、IL-2、IL-4则处于检测值下限;外周血中单核细胞表面HLA-DR表达量显著下调(Z=-4.714,U=98,P＜0.01;Z=-4.086,U=4,P＜0.01),HLA-DR表达量和APACHEⅢ评分存在显著负相关(R2=0.2667,P=0.0167);适应性免疫细胞CD4+T淋巴细胞绝对数量减少(Z=-4.411,U=116,P＜0.01;Z=-3.575,U=17,P=0.004).结论 乙型肝炎ACLF患者的免疫系统存在单核细胞功能抑制、CD4+T细胞耗竭及高含量抑炎性细胞因子的免疫紊乱状态,单核细胞HLA-DR表达量持续下降是反映疾病严重程度的指标.

Abstract：

Objective To explore the association of immune suppression with the severity of HBV-related acute-on-chronic liver failure(ACLF) patients.Methods From August 2009 to April 2010 in Shanghai Public Health Clinical Center, the peripheral blood samples and clinical data of 27 HBV-related ACLF patients (ACLF group), 28 patients wit h chronic active hepatitis B (CHB group)and 8 healthy individuals (Control group) were collected.APACHE Ⅲ score and the grade of hepatic encephalopathy were as quantitative index to evaluate the severity degree of the disease.The absolute counts of the subsets of T lymphocytes and human leukocyte antigen (HLA)-DR expression on the surface of monocyte in patients' peripheral blood were examined by flow cytometry, the proinflammatory cytokines and anti-inflammatory cytokines(IFN-γ, TNF-α, IL-2, IL-4, IL-10) in patients' plasma were detected by cytometric bead array (CBA) kit.The data was analyzed with SPSS 16.0 software.Results Compared with CHB group and control group, the level of anti-inflammatory eytokire IL-10 markedly increased ir HBV-related ACLF patients (Z= -4.279 ,U= 124, P＜0.01;Z= - 3.871, U= 9.5, P= 0.0001 ), however the level of pro-inflammatory cytokines IFN-γ、 TFN-α、IL-2 、 IL-4 in plasma were at low limit of detectable value.Meanwhile the expression quantity of HLA-DR on the peripheral blood monocytes significantly down-regulated (Z= -4.714, U= 98, P＜0.01;7= - 4.086, U = 4, P＜ 0.01), and there was negative correlation between HLA-DR expression quantity and APACHE Ⅲ score (R2 =0.2667, P=0.0167).In addition, the absolute counts of CD4+T lymphocytes in adaptive immune cells significantly decreased (Z= -4.411, U= 116, P＜0.01; Z=-3.575, U= 17, P= 0.0004).Conclusions The immune system of HBV-related ACLF patients displays immune dysfunction like monocyte function inhibition; CD4+ T lymphocytes depletion and high level of anti-inflammatory eytokines, the persistent down-regulation of the HLA-DR expression on monocyte is an indicator for the severity of disease.     

慢性乙型重型肝炎患者外周血树突状细胞核因子-κB及其激活因子的表达及意义

目的 探讨慢性乙型重型肝炎(CSHB)患者外周血单个核细胞衍生的树突状细胞核因子(NF)-κB、肿瘤坏死因子(TNF)α、白细胞介素(IL)-6与疾病严重程度及预后的相关性.方法 采集37例CSHB患者、20例慢性乙型肝炎(CHB)患者和20例健康对照者的外周血,分为CSHB组、CHB组与对照组并分别用密度梯度离心法分离外周血单个核细胞,用免疫磁珠细胞分选法获得纯化的单核细胞,体外培养6 d为未成熟的树突状细胞,用聚肌胞刺激48 h为成熟的树突状细胞(mDC).用实时定量PCR检测mDC NF-κB p50表达;用酶联免疫吸附试验测定培养mDC上清液中TNF α、IL-6的分泌水平.两组间比较采用t检验,3组间比较采用One-Way ANOVA分析法,相关分析采用直线相关分析法.结果 3组研究对象mDC表达NF-κB p50,表达量CSHB组为2.63±0.94、CHB组为2.03±0.39、对照组为1.41±0.40,3组比较,F=19.01,P<0.01,差异有统计学意义.mDC分泌TNF α,CSHB组、CHB组、对照组分别为(15 317.69±4124.90)pg/ml、(9670.29±3654.68)pg/ml、(6547.43±1027.20)pg/ml,3组比较,F=45.77,P<0.01,差异有统计学意义; IL-6分泌量分别为(1423.78±375.14)pg/ml、(862.68±93.68)pg/ml、(567.26±167.04)pg/ml,3组比较,F=67.60,P<0.01,差异有统计学意义.3组研究对象mDCNF-κB p50表达与TNF α、IL-6水平呈正相关(r=0.52,P<0.01;r=0.65,P<0.01).CSHB组mDC表达TNF α、IL-6与凝血酶原活动度呈负相关(r=-0.41,P=0.01;r=-0.40,P=0.01),与ALT、总胆红素、HBV DNA无相关性(r=-0.03,P=0.85,r=0.01,P=0.93,r=0.01,P=0.95;r=-0.09,P=0.58,r=0.16,P=0.34,r=0.09,P=0.59).CSHB 存活组与死亡组mDC表达TNF α、IL-6差异无统计学意义(t=0.42,P=0.67;t=0.76,P=0.45).结论 CSHB患者树突状细胞衍生的NF-κB及其激活产物TNF α、IL-6表达增加,可能与CSHB疾病严重程度相关.     

Hepatitis C virus-infected hepatocytes extrinsically modulate dendritic cell maturation to activate T cells and natural killer cells

Dendritic cell maturation critically modulates antiviral immune responses, and facilitates viral clearance. Hepatitis C virus (HCV) is characterized by its high predisposition to persistent infection. Here, we examined the immune response of human monocyte-derived dendritic cells (MoDCs) to the JFH1 strain of HCV, which can efficiently replicate in cell culture. However, neither HCV RNA replication nor antigen production was detected in MoDCs inoculated with JFH1. None of the indicators of HCV interacting with MoDCs we evaluated were affected, including expression of maturation markers (CD80, 83, 86), cytokines (interleukin-6 and interferon-beta), the mixed lymphocyte reaction, and natural killer (NK) cell cytotoxicity. Strikingly, MoDCs matured by phagocytosing extrinsically-infected vesicles containing HCV-derived double-stranded RNA (dsRNA). When MoDCs were cocultured with HCV-infected apoptotic Huh7.5.1 hepatic cells, there was increased CD86 expression and interleukin-6 and interferon-beta production in MoDCs, which were characterized by the potential to activate NK cells and induce CD4+ T cells into the T helper 1 type. Lipid raft-dependent phagocytosis of HCV-infected apoptotic vesicles containing dsRNA was indispensable to MoDC maturation. Colocalization of dsRNA with Toll-like receptor 3 (TLR3) in phagosomes suggested the importance of TLR3 signaling in the MoDC response against HCV. CONCLUSION: The JFH1 strain does not directly stimulate MoDCs to activate T cells and NK cells, but phagocytosing HCV-infected apoptotic cells and their interaction with the TLR3 pathway in MoDCs plays a critical role in MoDC maturation and reciprocal activation of T and NK cells.     

Insulin reduces LPS-induced lethality and lung injury in rats

Insulin is a main glucose homeostatic hormone in the body. Previous reports showed that insulin also exerted anti-inflammatory actions and attenuated systemic inflammatory response. Here, we observed the effects and the underlying mechanisms of insulin on lipopolysaccharide (LPS)-induced acute lung injury (ALI). As revealed by survival study, insulin reduced mortality of rats and prolonged their survival time. Meanwhile, insulin significantly reduced the levels of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and high mobility group box 1 (HMGB1) in bronchoalveolar lavage fluid (BALF). Besides, insulin markedly inhibited the expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB). Taken together, these data provided information that insulin attenuated LPS-induced ALI may attribute partly to the inhibition of the production of cytokines, and the expression of TLR2, TLR4 and NF-κB.