TRAIL及TRAIL受体在慢性乙型肝炎患者外周血淋巴细胞的表达

目的　探讨TRAIL(TNF relatedapoptosis inducingligand)及TRAIL受体 (TRAIL R)mRNA在慢性乙型肝炎患者外周血淋巴细胞 (peripheralbloodlymphocytes,PBL)中的表达。方法　分离 2 0例正常人、2 4例慢性乙型肝炎患者及 2 4例慢性重型乙型肝炎患者之PBL ,体外单独或与植物血凝素(PHA)共同培养 4 8h ,荧光素吖啶橙和溴乙啶染色PBL ,观察其凋亡细胞比例 ,采用逆转录 聚合酶链反应 (RT PCR)方法检测TRAIL、TRAIL RmRNA在PBL中的表达。结果　与正常对照组相比 ,活化诱导的淋巴细胞凋亡在慢性乙型肝炎患者明显增高 ,而在慢性重型肝炎患者则降低 ,差异有显著性 (P<0 .0 1)。PHA活化前 ,TRAILmRNA在上述 3组研究对象外周血淋巴细胞均不表达 ;PHA活化后 ,其表达明显上调 ,且慢性乙型肝炎组高于正常对照组 ,慢性重症肝炎组低于正常对照组 ,差异有显著性(P <0 .0 1)。 4个TRAIL R的mRNA在 3组研究对象外周血淋巴细胞均有表达 ,两两比较差异无显著性 (P >0 .0 5 )。经PHA活化后 ,TRAIL R2mRNA表达明显上调 (P <0 .0 1) ,TRAIL R3mRNA表达完全消失 ,TRAIL R1与 R4的mRNA表达无明显改变 (P >0 .0 5 )。结论　慢性乙型肝炎患者外周血淋巴细胞存在凋亡紊乱现象 ,TRAIL参与了乙型肝炎患者外周血淋巴细胞活化诱导细胞死亡 (     

慢性乙型肝炎PBMC凋亡及淋巴细胞亚群的检测

目的了解慢性/慢性重型乙型肝炎外周血淋巴细胞激活诱导细胞死亡(AICD)现象的存在情况、各免疫细胞的状况、AICD与淋巴细胞状况的关系,以探讨乙型肝炎慢性化和重型化的机制.方法利用慢性、慢性重型乙型肝炎病人和健康献血员外周血单个核细胞(PBMC)在PHA-P刺激下培养72h,通过流式细胞仪检测PBMC的凋亡情况;采用流式细胞仪结合全自动血液分析仪对慢性、慢性重型乙型肝炎病人及正常对照组外周血各淋巴细胞亚群进行检测.结果慢性乙型肝炎组PBMC凋亡率高于慢性重型乙型肝炎组(P＜0.01),高于正常对照组(P＜0.01).慢性乙型肝炎组总淋巴细胞百分率高于慢性重型乙型肝炎组(P＜0.01),慢性重型乙型肝炎组淋巴细胞数低于正常对照组(P＜0.01);CD3+、CD3+CD4+细胞数低于正常对照组(P＜0.01),低于慢性乙型肝炎组(P＜0.05);CD3+CD8+细胞数低于正常对照组(P＜0.05),低于慢性乙型肝炎组(P＜0.05).单核细胞百分率高于正常对照组(P＜0.01),高于慢性乙型肝炎组(P＜0.05).结论慢性乙型肝炎患者外周血淋巴细胞活化与凋亡共存,慢性重型乙型肝炎患者外周血淋巴细胞消耗严重,AICD参与乙型肝炎慢性化、重型化的发生机理.     

单核巨噬细胞极化在慢性活动性乙型肝炎中的表达及意义

目的:研究慢性活动性乙型肝炎患者单核巨噬细胞的数量与功能变化。方法:随机选择51 例慢性乙型肝炎患者(其中轻中度20 例,重度31 例)以及正常对照13 例。采用Percoll 分层液分离PBMCs,以CD14 标记单核细胞,流式细胞仪检测PBMCs 表面分子CD80、CD86、HLA-DR、CD163 的表达;ELISA 检测血清细胞因子IL-10、IL-12 与IL-23 的水平;免疫组织化学染色检测CD68 在肝脏的分布。结果:轻中度慢性乙型肝炎组、重度慢性乙型肝炎组CD80 表达水平低于对照组。慢性乙型肝炎患者外周血单核细胞CD86 的表达低于对照组,其中重度组与对照组比较有统计学差异(P<0.01)。慢性乙型肝炎患者HLA-DR 的表达低于对照组,其中轻中度组与重度慢性乙型肝炎组间HLA-DR 的表达水平差异有统计学意义(P<0.01)。此外,慢性乙型肝炎患者外周血单核细胞CD163 的表达明显高于对照组(P<0.01)。慢性乙型肝炎患者肝脏汇管区CD68 阳性细胞浸润增加,汇管区增大(P<0.05)。轻中度慢性乙型肝炎组、重度慢性乙型肝炎组与对照组血清IL-10 表达水平之间两两比较差异显著,均具有统计学意义(P<0.01)。结论:慢性乙型肝炎患者巨噬细胞参与肝脏病变的发生,外周血中单核细胞存在M1 型/ M2 型平衡失调,向M2 型极化的现象,可能参与其慢性化发展。     

类风湿关节炎CD4+CD28-T细胞与淋巴细胞凋亡的相关性研究

目的: 研究类风湿关节炎(RA)患者外周血CD4⁺CD28^-T细胞比例与淋巴细胞凋亡异常的相关性。方法: 采用流式细胞术三色分析法检测50例患者和50例健康志愿者的外周血淋巴细胞中CD4⁺CD28^-T细胞比例;通过加入PHA孵育检测RA病人外周淋巴细胞和正常对照的淋巴细胞对激活诱导细胞死亡(AICD)易感性差异;分析CD4⁺CD28^-T细胞比例与外周血淋巴细胞凋亡率的相关性。结果: RA组CD4⁺CD28^-T细胞比例的均数明显高于健康对照组(7.79%±3.52% vs 1.89%±1.78%,P<0.05)。RA组病人外周血淋巴细胞的AICD凋亡率低于健康对照组(11.38%±5.73% vs 19.46%±6.32%,P<0.05)。Spearman相关分析结果显示CD4⁺CD28^-T细胞比例与外周血淋巴细胞AICD凋亡率负相关(r=-0.433,P<0.01)。结论: RA患者外周血中CD4⁺CD28^-T细胞比例增多,活化淋巴细胞生存期延长,这可能参与RA的发病机制。     

早期自然流产患者淋巴细胞FAS/FASL的表达

目的探讨外周血和蜕膜淋巴细胞凋亡在早期自然流产中的作用.方法采用流式细胞技术检测外周血淋巴细胞FAS/FASL、Annexin-Ⅴ及蜕膜组织淋巴细胞FAS/FASL的表达.结果正常妊娠组Annexin-Ⅴ,FAS和FASL分别是(1.19±1.39)%,(56.64±11.19)%,(4.37±2.65)%;自然流产组分别是(1.96±2.43)%,(57.47±10.41)%,(4.45±2.10)%,两组差异均无统计学意义,P值分别为 0.262;0.710;0.596.正常妊娠组蜕膜组织淋巴细胞表达FAS,FASL分别为(30.88±16.13)%,(2.24±0.62)%,自然流产组分别是(32.63±13.45)%,(3.91±1.90)%,两组FAS表达差异无统计学意义,P值为0.515;两组FASL表达差异有统计学意义,P值为0.001.结论蜕膜淋巴细胞FASL表达上调可能是早期自然流产发生的原因之一.     

肺结核病患者外周血T淋巴细胞上PD-1的表达及意义

目的研究程序性死亡因子-1(PD-1)在肺结核病患者外周血T淋巴细胞上的表达,初步探讨其在结核慢性发生发展中的意义。方法选择肺结核患者(30例)为研究对象。对照组40例,分别为20例确诊为肺部感染的患者及20例正常健康体检者。分别用流式细胞术及RT-PCR检测PD-1蛋白及mRNA在T淋巴细胞上的表达情况。采用ELISA法检测各组血清中肿瘤坏死因子α(TNF-α)、γ-干扰素(IFN-γ)细胞因子浓度。结果与两对照组相比,肺结核患者外周血T淋巴细胞上PD-1蛋白及mRNA的表达均明显升高(P<0.01),外周血TNF-α和IFN-γ水平明显降低(P<0.05)。肺部感染与健康体检者相比,各项指标均无统计学差异。结论 PD-1在肺结核病患者T淋巴细胞上的表达明显升高,这可能在肺结核病的发生、发展及慢性化过程中起重要作用。     

泛素调控蛋白A20对慢性乙肝患者单核细胞活性的影响北大核心CSCD

目的:观察泛素调控蛋白A20在慢性乙肝患者外周血中的变化及其对CD14^(+)单核细胞功能的影响。方法:选取慢性乙肝患者47例和健康对照者26例,纯化外周血CD14^(+)单核细胞和CD4^(+)T细胞,流式细胞术检测CD14^(+)单核细胞中A20水平,实时定量PCR检测CD14^(+)单核细胞中A20 mRNA相对表达量。A20 siRNA或对照siRNA转染慢性乙肝患者纯化的CD14^(+)单核细胞,ELISA检测上清中炎症细胞因子水平,实时定量PCR检测FasL和TRAIL mRNA相对表达量。CD14^(+)单核细胞与HepG2.2.15细胞共培养,检测CD14^(+)单核细胞诱导靶细胞死亡的比例。CD14^(+)单核细胞与CD4^(+)T细胞共培养,流式细胞术检测CD4^(+)T细胞分泌IFN-γ和IL-17的比例。结果:慢性乙肝患者外周血CD14^(+)单核细胞中A20^(+)细胞比例高于健康对照者(P=0.0004),慢性乙肝患者外周血CD14^(+)单核细胞中A20平均荧光强度和A20 mRNA相对表达量亦高于健康对照者(P<0.001)。A20 siRNA转染慢性乙型肝炎患者纯化的CD14^(+)单核细胞后,分泌炎症细胞因子水平高于未转染细胞和对照siRNA转染细胞(P<0.05),但FasL和TRAIL mRNA未见明显变化(P>0.05)。A20 siRNA转染的CD14^(+)单核细胞诱导靶细胞死亡的比例高于未转染细胞和对照siRNA转染细胞(P<0.05),诱导CD4^(+)T细胞分泌IFN-γ和IL-17的比例亦高于未转染细胞和对照siRNA转染细胞(P<0.05)。结论:慢性乙肝患者CD14^(+)单核细胞中高表达的A20有抑制细胞毒性及CD4^(+)T细胞活化的作用,可能与导致乙型肝炎病毒感染慢性化相关。     

职业性三氯乙烯药疹样皮炎CXCR2和CXCR3基因的表达

目的: 建立荧光定量PCR检测三氯乙烯药疹样皮炎(DMLT)患者外周血淋巴细胞CXCR2和CXCR3 mRNA表达的方法.方法: 利用SYBR Green荧光定量PCR分别检测24例DMLT患者、 26例正常人外周血淋巴细胞CXCR2和CXCR3基因mRNA表达情况,以β2微球蛋白基因作为内参,根据相对定量公式 (2-△△CT)计算DMLT患者与正常人CXCR2和CXCR3基因表达差异倍数.结果: 24例患者外周血淋巴细胞荧光定量PCR均检出CXCR2和CXCR3 mRNA表达,其中CXCR2表达情况有两种: 表达上升14例(58.3%)和表达下降10例(41.7%),与正常人CXCR2 mRNA相比表达倍数分别为16.76±7.01、 0.54±0.30;CXCR3表达显著升高(△CT=6.3±2.8,11.4±1.9;P<0.01),与正常人CXCR3 mRNA相比表达倍数为33.37±31.61.结论: 成功建立了DMLT患者外周血淋巴细胞CXCR2和CXCR3mRNA表达的荧光定量PCR方法.     

慢性乙型肝炎患者iNKT细胞PD-1表达的变化

目的检测慢性乙型病毒性肝炎患者外周血中iNKT细胞比例、细胞因子分泌能力以及其表面程序性死亡因子1(programmed death 1,PD-1)的表达情况。方法采集慢性乙型肝炎患者外周血,利用CD3、TCR Vα24、TCR Vβ11、CD279单克隆抗体标记后经流式细胞术直接检测iNKT细胞比例及其PD-1表达比例;采用PMA+Ionomycin体外活化iNKT细胞后流式细胞术检测其胞内IFN-γ分泌情况。结果慢性乙型肝炎患者外周血iNKT细胞比例[(0.09±0.04)%]与健康对照者[(0.11±0.07)%]相比无明显差异,但体外活化后胞内IFN-γ分泌量减少[(24.64±7.71)%vs(42.35±11.60)%],且iNKT细胞PD-1表达升高[(36.7±9.7)%vs(16.2±5.4)%]。结论慢性乙型肝炎患者外周血iNKT细胞功能的下降可能与其表面负性刺激分子PD-1表达上调密切相关。     

慢性乙型肝炎患者外周血淋巴细胞CD44v6的表达及意义

目的：研究慢性乙型肝炎及乙型肝炎后肝硬化患者外周血淋巴细胞（PBL）CD44v6的表达情况及意义。方法：用流式细胞术分别检测50名健康体检者、30例乙型肝炎病毒（HBV）携带者、60例慢性乙型肝炎患者及60例乙型肝炎后肝硬化患者PBLCD44v6的表达水平。结果：慢性乙型肝炎患者PBLCD44v6表达明显高于正常对照组和HBV携带组（P〈0.01）;肝炎后肝硬化组明显高于慢性乙型肝炎组（P〈0.01）;HBV携带组与正常对照组之间的差异也有统计学意义（P〈0.01）。结论：CD44v6在慢性乙型肝炎HBV致肝细胞损伤过程中可能起着重要作用。     

Telomerase activity of peripheral blood lymphocytes in patients with chronic hepatitis B

Chronic hepatitis B is the immunocompromising condition. The decrease of lymphocyte telomerase is linked to immunosenescence in hosts. To know whether telomerase activity of lymphocytes is involved in immunopathogenesis in patients with chronic hepatitis B, telomerase activity of peripheral lymphocytes was determined in such patients. The results showed that telomerase activity in resting peripheral lymphocytes of healthy subjects was detectable at low level, and obviously increased (P<0.001) after stimulation in vitro with phytohaemagglutinin (PHA). Telomerase activity of lymphocytes decreased with age in both groups with or without PHA stimulation. Telomerase activity of resting lymphocytes in patients with chronic hepatitis B was also observed at detectable level and markedly upregulated after PHA stimulation. The decreased telomerase activity of resting lymphocytes was found in patients with chronic hepatitis B (n=14, 0.32+/-0.27) compared to that in healthy subjects (n=17, 0. 52+/-0.28; P<0.05). However, there was no difference present between these two groups in telomerase activity of activated lymphocytes with PHA. In addition, no effect of recombinant human interleukin-12 (rhIL-12) on telomerase expression was observed in either the patient group or the healthy group. We concluded that the decreased telomerase activity of lymphocytes in chronic hepatitis B patients is present, which may be partly responsible for immunosuppressive condition in such patients.     

Quantitative gene expression of cytokines in peripheral blood leukocytes stimulated in vitro: modulation by the anti-tumor nerosis factor-alpha antibody infliximab and comparison with the mucosal cytokine expression in patients with ulcerative colitis

Emerging data indicate that alterations in cytokine synthesis play a role in inflammatory bowel disease (IBD) pathogenesis. In this study, we quantified mRNA expression of the main acute-phase cytokines and T-cell cytokines in biopsies from patients with established ulcerative colitis (UC) and compared it with that obtained in biopsies from normal controls. Quantification of cytokine gene expression was also evaluated in in vitro phytohemagglutinin (PHA)-treated peripheral blood leukocytes (PBLs) at the RNA and protein levels. The in vitro influence of the anti-tumor necrosis factor-alpha (TNF-alpha) antibody infliximab (INFL) on PHA-treated PBLs was also evaluated. Analyzing inflamed specimens from UC patients compared with control samples, interleukin (IL)-6 was sharply the most induced cytokine. Interestingly, similar results were found in activated PBLs, where acute-phase cytokines were more abundantly expressed compared with T-cell cytokines. IL-6 was confirmed to be the most induced with a maximum increase of 1110-fold after 4 h of PHA stimulation, followed by TNF-alpha and IL-1beta as well as interferon-gamma (IFN-gamma). Surprisingly, analyzing cytokine-mRNA expression from activated PBLs, the time kinetics and quantity of IFN-gamma was more similar to that of the acute-phase proteins than to that of the T-cell cytokines, which were upregulated after 1 h. The upregulation of cytokine-mRNA was translated into protein as demonstrated by enzyme-linked immunosorbent assay. IFN-gamma was also strongly expressed in the RNA from UC biopsies. TNF-alpha protein was not detectable at all in INFL-treated cultures. INFL did not induce a reduction of TNF-alpha-mRNA nor of IL-1beta-mRNA, but it reduced IFN-gamma- mRNA and, to a lesser extent, IL-6-mRNA; it also reduced the T-cell-derived cytokine IL-2. The in vitro model of PHA-stimulated PBLs may mimic inflammation processes observed in vivo. INFL may reduce inflammation in vivo through inhibition of both monocyte and T-cell activation.     

Cell-mediated immunity to HBcAg in chronic HBV infection

The role of hepatitis B core antigen (HBcAg) as a possible target of cell-mediated immune response in chronic hepatitis B virus (HBV) infection has been recently emphasized. Peripheral blood leukocytes (PBLs) from 35 chronic carriers of hepatitis B surface antigen (HBsAg) were studied in vitro for their immune response to a purified preparation of HBcAg isolated from circulating Dane particles. PBLs from all the studied HBsAg-positive patients yielded a stimulation index above 3, with values ranging from 3.1 to 38.1. None of the healthy seronegative subjects, taken as control group, had a stimulation index above 2, with a mean value +/- SD of 1.28 +/- 0.35. Levels of PBL stimulation correlated with the histologic activity of liver disease, and the differences reached statistical significance. These results indicate that lymphocyte response to HBcAg may be relevant in determining liver cell damage.     

CpG-ODN对慢性乙型肝炎患者外周血单个核细胞T-bet表达的影响

目的 探讨T-bet在乙型肝炎病毒(HBV)感染中的作用以CpD-ODN对其表达的调节作用。方法应用半定量RT-PCR，对22例慢性乙型肝炎活动期患者、10例HBsAg阳性无症状携带者和12例正常对照外周血单个核细胞(PBMC)T-bet的表达进行检测；同时用CpD-ODN体外刺激以上3组实验对象的PBMC，RT-PCR观察T-bet的表达情况。结果无症状携带者T-bet表达最低，慢性乙型肝炎活动期患者T-bet表达最高；经cpG-ODN刺激后，无症状携带者和正常对照的T-bet表达明显上调，结论T-bet在HBV感染的不同状态中具有差异表达，提示T-bet可能参与了机体抗HBV的免疫应答，具体的机制有待进一步研究。CpG-ODN作为一种新型的免疫调节剂，可在一定程度上恢复HBV感染者特别是无症状携带者的Th1型细胞免疫应答．     

Abnormal T-cell activation in chronic hepatitis B viral infection: a consequence of monocyte dysfunction?

The process of T-cell activation in chronic hepatitis B virus (HBV) carriers has been investigated by measurement of membrane expression of lymphocyte-activation markers in response to a variety of mitogenic stimuli in order to delineate further the abnormality of T-cell-mediated immunity present in such patients. A substantial proportion of unstimulated T cells from the peripheral blood of patients but not controls expressed HLA-DR; in contrast the IL-2 and transferrin receptors were rarely expressed spontaneously in either group and there was no difference in spontaneous lymphocyte transformation. After stimulation with monocyte-dependent T-cell mitogens, phytohaemagglutinin (PHA) or anti-T3, patients had significantly reduced expression of the IL-2 and transferrin receptors and of HLA-DR in association with impaired lymphocyte transformations compared to controls. In contrast, lymphocyte activation was normal in response to the monocyte-independent T-cell mitogen phorbol-myristate-acetate (PMA). These data confirm that the process of T-cell activation is abnormal in chronic HBV carriers but suggest that the T cell is intrinsically normal. In allogeneic co-cultures, monocytes from patients inhibited the transformation of normal and patients' lymphocytes in response to PHA, suggesting that defects of T-cell-mediated immunity in chronic HBV carriers may be a consequence of monocyte dysfunction.     

乙型肝炎患者外周血单个核细胞凋亡的检测及意义

目的研究激活诱导细胞死亡 (AICD)现象在乙型肝炎慢性化和重型化机制中的意义。方法分离 2 0例慢性 /慢性重型乙型肝炎病人与 10例健康献血员外周血单个核细胞 (PBMC) ,在 PHA的刺激下培养 72 h后收集细胞 ,经流式细胞仪检测凋亡。结果 PBMC凋亡率乙型肝炎组明显高于正常对照组 [(2 5 .48± 14.0 7) % vs(11.45± 5 .2 7) % ,P<0 .0 1];慢性乙型肝炎组 [(30 .5 7± 13.43) % ]明显高于正常对照组 [(11.45± 5 .2 7) % ,P<0 .0 1]和慢性重型乙型肝炎组 [(13.5 9± 6 .44 ) % ,P<0 .0 1];PBMC凋亡率乙型肝炎 HBe Ag(+ )组明显高于正常对照组 [(2 9.5 0± 12 .5 4) % vs(11.45± 5 .2 7) % ,P<0 .0 1]。结论 AICD可能是形成 HBV慢性感染免疫耐受的一个重要机制。     

Interleukin 2 activity in chronic liver disease and the effect of in vitro alpha-interferon.

To investigate mitogen induced helper interleukin 2 (IL-2) production in patients with chronic liver disease (CLD), IL-2 activity was assessed by an IL-2 bioassay using phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMNC). IL-2 activity was significantly reduced in patients with autoimmune chronic active hepatitis, primary biliary cirrhosis and alcoholic hepatitis with or without cirrhosis (P less than 0.01), and was comparable to controls in those with alcoholic cirrhosis alone. In vitro preincubation of PBMNC with lymphoblastoid alpha-interferon (alpha-IFN) before stimulation with PHA, led to a significant increase in IL-2 activity in all subjects (P less than 0.01), except those with alcoholic hepatitis, but in none of the groups did the levels of IL-2 activity reach those seen in normal subjects. The decrease in IL-2 activity in patients with CLD may be due to low IL-2 production or presence of an IL-2 antagonist(s). Such an abnormality may occur, not only as a result of liver damage, but may also be important in determining immunological disturbances involved in the pathogenesis of the liver disease.     

SLE患者外周血IFN-γ及IL-4细胞因子mRNA的表达研究

为利用实时监测定量PCR技术检测细胞因子mRNA的表达 ,从mRNA水平探讨IFN γ、IL 4mRNA的变化。对初诊SLE患者外周血IFN γ、IL 4的mRNA表达进行检测 ,并同时使用LPS及PHA作刺激剂研究其对SLE患者细胞因子mRNA表达的影响。结果是PHA刺激后 ,正常组IFN γmRNA的表达明显高于SLE组 (P =0 0 2 4 ) ;LPS对正常对照的IFN γ和IL 4的mRNA表达均能明显上调 ,但SLE患者仅表现为IFN γ升高 ,IL 4升高不明显 ;未刺激组SLE患者与正常对照的比较结果显示IFN γ的表达及IFN γ/IL 4有明显降低 (P =0 0 14 ,P =0 0 33)。故SLE患者与正常对照相比 ,IFN γmRNA的表达下降 ,同时SLE患者的IFN γ、IL 4mRNA表达对LPS及PHA的反应下降。由于不同的患者受累系统不同 ,临床表现不一 ,可能T细胞的异常并不相同 ,其免疫系统紊乱的机制也不一致