HBV阳性血清诱导人外周血单个核细胞凋亡的检测及意义

目的研究乙型肝炎病毒(HBV)对外周血单个核淋细胞(PBMC)凋亡的作用.方法利用10名健康献血员外周血分离PBMC,分为加HBV阳性血清组和加健康人血清对照组,经培养72h后,采用PI染色法在流式细胞仪上检测细胞的凋亡情况.结果加HBV阳性血清组培养细胞的凋亡率为(39.56±7.03)%,明显高于加健康人血清组培养细胞的凋亡率(27.57±7.78)%,两组间具有非常显著的差异(P＜0.02).结论HBV可能具有诱导PBMC凋亡的能力,这可能是形成HBV慢性感染免疫耐受的一个重要机制.     

慢性乙型肝炎患者外周血单个核细胞基因表达谱的研究

目的 分析慢性乙型肝炎患者外周血单个核细胞差异表达基因，探索慢性乙型肝炎形成的分子机制。方法 应用含14000条人体cDNA的微阵列芯片和来自外周血单个核细胞的标记cDNA，分析了10例慢性乙型肝炎患者和10例健康人基因表达谱。通过应用GenePix 4000B扫描仪和ImaGene3．0分析软件比较cy5标记的慢性乙型肝炎来源cDNA与Cy3标记的健康人来源cDNA的杂交结果，获得个体基因的相对表达比值。结果 在分析的14000条基因中，差异表达的基因有92条，占0．66％。其中51条基因表达水平显著上调，41条基因表达水平显著下调。这些差异表达的基因主要为细胞信号转导，细胞周期和代谢，凋亡及炎症相关类基因。结论 在乙型肝炎病毒致慢性乙型肝炎过程中，涉及到了众多基因的差异表达，为进一步阐明慢性乙型肝炎形成的分子机制提供基础。     

外周血单个核细胞中乙型肝炎病毒感染的研究进展

乙型肝炎病毒（ＨＢＶ）感染乙肝（ＨＢ）患者外周血单个核细胞（ＰＢＭＣｓ），ＨＢＶ－ＤＮＡ以游离和整合型两种形式存在于ＰＢＭＣｓ内。在ＰＢＭＣｓ内复制与表达。研究ＰＢＭＣｓ内ＨＢＶ感染对于探讨ＨＢ的临床进程、治愈和指导治疗等均具有重要的意义。最近提出了一种与上述相反的观点，ＰＢＭＣｓ内有关的ＨＢＶ－ＤＮＡ和ＲＮＡ是吸附的结果，而非病毒的复制。     

慢性乙型肝炎患者外周血单个核细胞Toll样受体3的表达降低

目的 探讨慢性乙型肝炎患者外周血单个核细胞Toll样受体3(TLR3)的表达及其临床意义.方法 分别采集慢性乙型肝炎患者和健康志愿者外周血,荧光定量PCR法检测血清HBV DNA复制水平;使用RT-PCR、流式细胞术以及免疫印迹技术分别检测外周血单个核细胞TLR3的mRNA、蛋白的表达;使用ELISA法检测血清中肿瘤坏死因子α(TNF-α)和干扰素β(IFN-p)水平.结果 慢性乙型肝炎患者外周血单个核细胞中的TLR3表达显著低于健康志愿者,且降低水平与血清HBV DNA复制水平相关;慢性乙型肝炎患者外周血TNF-α、IFN-β浓度显著低于健康志愿者,且降低的水平与血清HBV DNA复制水平相关.结论 慢性乙型肝炎患者外周血单个核细胞TLR3的表达与乙肝病毒的复制水平相关.     

慢性乙型肝炎患者外周血单个核细胞INF-α和IL-8表达的检测及临床意义

目的检测慢性乙肝患者外周血单个核细胞α干扰素（IFN—α）的诱导表达及白细胞介素8（IL-8）表达,评价慢性乙肝患者外周血单个核细胞（PBMC）细胞免疫功能。方法PolyIC体外刺激正常对照组和慢性乙型肝炎病毒（Hepatitis Bvirus,HBV）感染组病人PBMC,取培养上清液利用病毒保护试验测定IFN—α2b治疗前后病人PBMC表达的干扰素抗病毒生物学活性。同时在IFN—α2b治疗前后,RT-PCR检测慢性乙肝患者不同干扰素应答组病人PBMCIL-8表达的变化。结果治疗前应答组病人（n=13）和无应答组病人（n=27）PBMC分泌的干扰素生物学活性显著低于正常对照组（n=20）（P〈0．01;P〈0．01）。应答组病人PBMC分泌的干扰素活性随治疗时间延长而增加,1个月时已显著高于无应答组病人（P〈0．01）;无应答组病人PBMC分泌的干扰素活性始终处于低下水平。治疗前,应答组病人和无应答组病人PBMCIL-8mRNA水平都显著高于正常对照组（均为P〈0．01）;治疗后,应答组病人IL-8水平随治疗时间延长而降低,1个月时已显著低于无应答组病人（P〈0．01）,无应答组病人IL8水平始终处于较高水平。结论慢性HBV感染病人的细胞免疫功能受损,不能正常表达IFN-α但应答组病人经干扰素治疗后IFN—α表达能力逐渐恢复。慢性乙肝患者PBMCIL-8表达与肝脏炎症活动有关。慢性乙肝患者PBMCIFN—α活性检测结果也许可以作为干扰素治疗预后的判断指标。     

慢性乙型肝炎患者外周血单个核细胞中IL—18表达水平

目的探讨白细胞介素 18(IL- 18)在乙型肝炎病毒感染中的作用。方法应用流式细胞免疫学方法 ,对 30例慢性乙型肝炎活动期、缓解期 ,15例 HBS Ag阳性无症状携带者 ,10例正常对照外周血单个核细胞 (PBMC) IL- 18的表达进行检测。结果无症状携带者表达最低 ,慢性乙型肝炎缓解期低于正常对照 (P<0 .0 1) ,活动期与正常对照无显著差异 (P=0 .2 5 )。活动期 IL- 18的表达与肝组织炎症活动度有关 (P<0 .0 1) ,与血清 AL T水平呈正相关 (r=0 .6 3,P<0 .0 1)。结论 IL- 18与慢性乙型肝炎病情的活动相关 ,与肝组织炎症程度相关     

外周血单个核细胞中乙型肝炎病毒核酸与细胞因子含量变化

目的：探讨慢性乙型肝炎患者外周血单个核细胞中乙型肝炎病毒核酸与血清细胞因子变化关系。方法：采用荧光实时PCR技术定量检测76例慢性乙型肝炎患者和15例健康者外周血单个核细胞中HBVDNA含量，用ELISA法定量检测血浆细胞因子(IFN-γ、TNF—α、IL-4、IL-6、TGF-β1、sIL-2R)水平。结果：①慢性乙型病毒性肝炎中细胞因子(IFN-γ、TNF-α、IL-4、IL-6、TGF-β1、sIL-2R)水平明显高于健康对照组；②单个核细胞HBVDNA阳性组IFN-γ、TNF-α含量明显低于阴性组，而sIL-2R含量则高于阴性组但无统计学差异，IL-4、IL-6、TGF-β1含量无差异；③随着PBMCs中HBVDNA含量升高，TNF—α含量逐渐降低，IL-4和sIL-2R含量逐渐升高，而IFN-γ，IL-6、TGF-β1含量无显著性变化。结论：外周血单个核细胞HBVDNA持续存在及含量变化与细胞因子相对异常有关，进而导致肝细胞损伤。     

乳癌患者外周血及肿瘤局部单个核细胞亚群分析

对13例女性乳癌患者外周血MNC(半个核细胞)与15名正常女性比较时发现,乳癌患者血中Leul~+细胞及DR~+细胞较低(P<0.05,P<0.01),Leu2a~+细胞较高(P<0.01),Leu3a/Leu2a比值下降(P<0.001),从数量上反映患者机体细胞免疫功能紊乱,并发现可能存在的Leu3a~+、Leu2a~+双标记细胞增多的现象,也可能与免疫抑制有关。DR~+细胞的减少由何引起有待于进一步研究。配对资料分析表明:癌周间质MNC亚群与血中MNC各亚群水平相近(Leu7、Leullb细胞除外),而癌间间质则显示较低的MNC水平。     

哮喘患者外周血单个核细胞IL-5、IL-3 mRNA表达的研究

嗜酸细胞（ＥＯＳ）是哮喘慢性炎症中的关键效应细胞。ＩＬ５、ＩＬ３能促进ＥＯＳ在骨髓的分化、成熟,使机体的ＥＯＳ产生增多,并促使ＥＯＳ在支气管肺组织的聚集及活化。哮喘患者外周血、支气管粘膜、支气管肺泡灌洗液（ＢＡＬＦ）中ＩＬ５明显升高［１］。ＩＬ３对ＥＯＳ的作用较ＩＬ５弱,它在哮喘发病机理中的作用仍有争议。本文通过对哮喘患者ＰＢＭＣＩＬ５ｍＲＮＡ、ＩＬ３ｍＲＮＡ表达水平的研究,探讨其在哮喘发病中的作用。１　材料与方法１．１　研究对象　哮喘组：哮喘患者１３例,男８例,女５例,平均年龄３…     

Anti-HBV effects of CpG oligodeoxynucleotide-activated peripheral blood mononuclear cells from patients with chronic hepatitis B

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) are known as a potent Th1-like immune enhancer in vertebrates. Chronic hepatitis B is the immunocompromising condition. We therefore investigated the effects of CpG ODN on cultured cells from chronic hepatitis B patients and healthy controls. The inhibitory effects of CpG ODN on hepatitis B virus (HBV) were also studied. The secretion of IFN-alpha by CpG ODN-activated peripheral blood mononuclear cells (PBMCs) from chronic hepatitis B patients and healthy controls was significantly increased when compared with PBMCs alone or GpC ODN-stimulated PBMCs. After activation with CpG ODN, the IFN-alpha secretion by chronically HBV-infected patient PBMCs is less than that by healthy control PBMCs. Treatment of HepG2 2.2.15 cells with culture supernatants of PBMCs activated by CpG ODN can significantly suppress the secretion of HBsAg, HBeAg and HBV DNA as compared with that of PBMCs without CpG ODN activation under the same conditions. No inhibitory effect on the replication of HBV was found for CpG ODN treatment alone. Our results indicated that CpG ODN could efficiently enhance the immune response of chronic hepatitis B patients. Moreover, the CpG ODN-activated PBMCs from chronic hepatitis B patients were able to significantly inhibit HBV replication in vitro, suggesting that CpG ODN may be a potential immunoregulator against HBV infection in the future.     

Detection of hepatitis B surface antigen (HBsAg) in peripheral blood mononuclear cells of patients with acute and chronic active hepatitis B

Seventy five patients with acute and chronic active hepatitis (CAH) were studied by indirect immunofluorescence with monoclonal antibodies for the presence of hepatitis B surface antigen (HBsAg) on peripheral blood mononuclear cells (PBMC). The viral surface antigen was detected in the PBMC of all the patients with hepatitis B virus (HBV)-induced CAH and in acute patients with more than 2 months of evolution. No HBsAg was detected in the samples obtained from 12 normal controls or from 14 non-A, non-B CAH patients. Analysis of PBMC subsets revealed that HBsAg was present in non-T cells; dual fluorescence studies showed HBsAg on surface Ig-positive lymphocytes. The binding of anti-HBs monoclonal antibodies was higher than that of a goat anti-HBs serum, and the highest reactivity was observed with an antibody against the pre-S(2)-region sequence. Both HBsAg and hepatitis B core antigen (HBcAg) were also detected in lysates of PBMC by dot blot analysis.     

沙利度胺对慢性乙型肝炎患者外周血单个核细胞的影响

目的探讨沙利度胺（Thalidomide,Thal）对慢性乙型肝炎（Chronic hepatitis B,CHB）外周血单个核细胞（Peripheral blood mononuelear cells,PBMC）增殖、克隆形成、分泌TNFα和IFNγ的影响。方法选择慢性乙型肝炎患者23例,常规方法分离PBMC,体外扩增,用细胞计数法观察Thal对PBMC增殖的影响,胎盘蓝染色法观察其对细胞存活的影响,琼脂糖凝胶法观察Thal对PBMC克隆形成的影响,酶联免疫吸附法（ELISA）观察Thal对CHB PBMC分泌TNFα和IFNγ的影响。结果Thal对PBMC的增殖具有促进作用,而无毒性影响（P〈0．05）;抑制PBMC克隆的形成（P〈0．05）;抑制TNFα的产生而促进IFNγ的产生（P〈0．05）。结论Thal对CHB PBMC的免疫功能具有调节作用,可望用于临床上慢性乙型肝炎的治疗。     

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．     

慢性乙型肝炎患者树突状细胞表型的研究

目的探讨慢性乙型肝炎患者外周血来源树突状细胞（Dendritic cell，DC）数量及表型的改变，并对其与肝功能、乙肝病毒复制水平的关系进行研究。方法检测37例慢性乙肝患者和21例健康人肝功能及血清HBV DNA水平，并提取外周血单个核细胞（Peripheral blood mononuclear cell，PBMC）进行体外诱导培养，促使其发育成DC，计数其数量并检测膜表面分子的变化。分析DC数量及表型与肝功能、乙肝病毒复制水平的关系。结果与正常对照组比较，慢性乙肝患者的树突状细胞数量明显减少（P〈0．05），且其膜表面分子CD83、CD86的表达均明显降低（P〈0．05）。在慢性乙肝患者中，DC数量、DC膜表面分子CD83和CD86与血清HBV DNA之间呈负相关关系，而与肝功能之间无明显相关关系。结论慢性乙肝患者体内存在DC数量减少及成熟障碍，这种改变与肝内炎症反应程度不相关，但与乙肝病毒（Hepatitis Bvirus，HBV）的复制水平呈负相关，提示DC参与慢性乙肝患者体内HBV的清除。     

Correlation of AIM2 expression in peripheral blood mononuclear cells from humans with acute and chronic hepatitis B

The AIM2 (absent in melanoma 2) protein promotes host defenses against invading viruses and pathogenic bacteria through corresponding adapter molecules leading to the initiation of innate immune responses. We investigated the expression of AIM2 in peripheral blood mononuclear cells (PBMCs) from patients with acute hepatitis B (AHB) and chronic hepatitis B (CHB) during different clinical phases, and analyzed the correlation between AIM2 and clinical profiles in these groups. This study indicated that there is higher expression of AIM2, IL-1β, and IL-18 in AHB compared with expression in CHB. The expression of AIM2 mRNA was significantly negatively correlated with serum hepatitis B virus (HBV) load, HBeAg, and significantly positively correlated with IL-1β and IL-18 in AHB patients and CHB patients with immune clearance, which suggests that AIM2 expression is correlated with the immune clearance of HBV in the host. We summarized that there is a higher immune status in AHB, and a lower immune response in CHB. This suggests that the down-regulation of AIM2 may be associated with the chronic development of HB.     

Hepatitis B virus infection of peripheral blood mononuclear cells is common in acute and chronic hepatitis

Hepatitis B virus (HBV) infection of peripheral blood mononuclear cells (PBMCs) has been observed in all stages of liver disease. Thus far all information about the physical state of HBV in mononuclear blood cells comes from Southern blot analysis and in situ hybridization. In this study we focused our attention on the presence of HBV DNA sequences in PBMCs of 30 patients with acute type B hepatitis and 6 patients with chronic active hepatitis by utilizing both Southern blot analysis and the polymerase chain reaction (PCR). Southern blot analysis showed no HBV DNA sequences in PBMCs of the acute hepatitis patients, although the sensitivity of our method enabled us to detect as little as 1 pg of cloned HBV insert. As far as the chronic hepatitis patients are concerned Southern blot analysis revealed the presence of HBV DNA sequences in 5 out of 6 patients but intermittently at successive follow-up times. On the other hand we were able to demonstrate the presence of HBV related sequences in 14 out of 30 acute hepatitis patients (5 HBeAg positive, 9 antiHBe positive) and in all 6 chronic hepatitis patients by PCR. Our results indicate that the involvement of PBMCs with HBV during acute HBV infection occurs at a very low level, often below the detection limit of the Southern blot technique.     

慢性乙肝患者外周血单个核细胞TRAIL受体表达及其临床意义

目的探索慢性乙肝患者外周血单个核细胞(PBMC)肿瘤细胞坏死因子相关的凋亡诱导配体(TRAIL)受体在PBMC凋亡中的作用,及其与机体肝脏损伤的相关性。方法用RT-PCR和流式细胞术检测55例慢性乙肝患者(包括轻度、中度、重度)外周血单个核细胞TRAIL各受体(DR4、DR5、DcR1和DcR2)表达水平,同时检测肝功能相关指标,并进行相关性分析。以30例正常人作为对照。结果诱骗受体DcR1在慢性乙肝患者中的表达显著低于对照组(P<0.05)。DcR1的表达随慢性乙肝病情的加重而逐渐降低。慢性乙肝患者的DcR1表达与转氨酶(ALT)呈显著负相关,与血清白蛋白成显著正相关。结论慢性乙肝患者外周血单个核细胞DcR1表达下调可能是其细胞凋亡增加的机制之一,且DcR1表达情况可从一定程度上反映肝脏的损伤程度。     

慢性乙型肝炎患者外周血细胞因子诱导的杀伤细胞的动态观察

目的:动态观察慢性乙型肝炎患者细胞因子诱导的杀伤(CIK)细胞的增殖及杀伤活性。方法:抽取10例健康人及20例慢性乙型肝炎患者的外周血,常规分离单个核细胞(PB-MC),加IFN-γ、IL-2及抗人CD3单克隆抗体(mAb)后培养,诱导CIK细胞形成。于培养后3、6、12、24及30 d,取培养的 细胞,用流式细胞仪检测CIK细胞表面CD3、CD4和CD8以及CD4、CD25、CD3、CD56和CD95、CD28分子的表达水平、增殖及杀伤活性。结果:慢性乙型肝炎患者CIK细胞的增殖及杀伤活性均低于正常对照组。培养不同时间乙肝患者CIK细胞的增殖倍数、杀伤活性和上述表面标志的表达水平,均较培养前明显增高,于培养后12 d达高峰。结论:慢性乙型肝炎患者CIK细胞的增殖倍数、杀伤活性均低于正常人,但明显高于培养前;这可能是导致HBV感染持续发展的原因之一。