乙型肝炎病毒与外周血单个核细胞的相关性研究进展

由乙型肝炎病毒(HBV)所致的乙型肝炎一直是全球性公共卫生问题,但其致病机制尚未完全明确。现已证明HBV可以感染外周血单个核细胞(PBMC),并引起机体的免疫功能紊乱,但是否可在PBMC中发生复制尚无定论。研究HBV感染PBMC具有重要的理论价值及临床意义。     

乙型肝炎病毒感染外周血单个核细胞的研究现状

乙型肝炎病毒感染外周血单个核细胞并不少见,受感染的外周血单个核细胞包括粒细胞、T淋巴细胞、B细胞等。HBV可在其内复制并表达某些抗原成分。上述现象可部分解释乙型肝炎免疫功能紊乱的原因及感染途径。     

乙型肝炎病毒感染对外周血单个核细胞分泌功能的影响

目的:观察乙型肝炎病毒(HBV)感染后外周血单个核细胞(PBMC)分泌功能的动态变化,为病毒免疫实验提供参考。方法:分离健康人PBMC,分为空白对照组和病毒干预组。空白对照组PBMC正常培养,病毒干预组PBMC加入HepG 2.2.15培养上清液中浓缩的HBV共培养。分别在培养4 h、8 h、12 h、24 h、36 h、48 h后收集培养上清液进行细胞因子(CK)检测。观察细胞因子随时间变化曲线,分析病毒刺激对PBMC分泌功能的影响。结果:病毒干预后IL-2的含量随时间变化呈上升趋势,其余各因子含量随时间变化呈先上升后下降趋势,IL-4、IL-17A、IFN-γ、TNF-ɑ分泌高峰在8 h, IL-6分泌高峰在36 h, IL-10分泌高峰在24 h;空白对照组PBMC中IL-4、IFN-γ各时间点含量高于病毒干预组(P<0.05),IL-6、TNF-ɑ各时间点含量低于病毒干预组(P<0.05);IL-2、IL-10、IL-17A、含量除个别时间点均低于病毒干预组(P<0.05)。结论:病毒刺激后各细胞因子的分泌高峰不同,可根据各自分泌特点选择恰当的研究时间点;HB...     

慢性乙型肝炎患者树突状细胞功能与单个核细胞乙型肝炎病毒共价闭合环状DNA的关系

目的 探讨慢性乙型肝炎(CHB)患者外周血单个核细胞(PBMC)及树突状细胞(DC)内HBV共价闭合环状DNA(HBV cccDNA)的存在状况,DC成熟度及功能状态与DC或PBMC中HBV cccDNA载量的关系.方法 分离29例CHB患者和10例健康对照者的PBMC,用重组人粒细胞-巨噬细胞集落刺激因子(GM-CS...     

家族不良结局乙型肝炎外周血单个核细胞基因表达谱的建立

目的应用寡核苷酸基因芯片技术建立家族不良结局乙型肝炎病毒(HBV)感染者外周血单个核细胞的基因表达谱。方法在一个家族不良结局HBV感染家族中,选取患者5例,患者正常配偶4 例,提取外周血单个核细胞RNA,与涵盖2.2万个ESTs的寡核苷酸表达谱基因芯片U133A 2.0杂交,通过Affymetrix扫描仪和DNT分析软件比较患病组与对照组外周血单个核细胞基因表达谱,获得基因的相对表达比值。结果在2.2万个ESTs中初筛出55个差异表达基因,表达上调14个,表达下调41个,差异基因主要(57%)参与免疫反应、细胞信号转导、细胞周期、代谢、细胞凋亡及炎症基因。结论筛选出的55个基因,是宿主感染HBV的差异表达基因,或宿主对HBV的易感基因,为差异表达基因功能研究建立框架,为宿主HBV易感性研究提供新的靶点。     

乙肝患者外周血单个核细胞中乙肝病毒复制中间产物RNA的检测及临床意义

采用逆转我聚酶链反应技术检测乙肝患者外周血单个核细胞乙肝病毒复制中间产物ＲＮＡ。０例乙肝患者外周血单个细胞中乙肝病毒复制中间产物ＲＮＡ阳性率为８．３％（５／６０）,其中３０例慢笥肝炎患者阳性率为１３．３％（４／３０）,１５例肝炎肝硬变患者阳性率为６．７％（１／１５）,１５例急性肝炎患者中无１例阳性。认为外周血单个细胞中乙肝病毒复制中间产物与乙肝的慢性化有关,但与临床病情轻重无关。     

慢性乙型肝炎患者血清和外周血单个核细胞HBVcccDNA检测的临床意义

目的了解慢性乙型肝炎患者血清及外周血单个核细胞内是否存在HBV共价闭合环状DNA。方法以20例HBV携带者、75例慢性乙型肝炎和8例肝移植术后患者分离PBMC,应用增效PCR法检测HBVcccDNA。结果本次未在PBMC中检测到HBVcccDNA;20例HBV携带者血清HBVcccDNA阳性率为10%,75例慢性肝炎轻、中、重度患者分别为32%、52%和76%,肝移植患者为12.5%(P0.01);按血清HBVDNA载量不同分为1×105copies/ml、1×105～108copies/ml和1×108copies/ml三组,其血清HBVcccDNA阳性率分别为15.4%(2/13)、50.0%(16/32)和76.7%(23/30,P0.01)。结论慢性乙型肝炎患者肝外HBVcccDNA的检测还需要进一步研究。     

乙型肝炎患者外周血单个核细胞端粒酶活性的研究

目的 了解乙型肝炎患者外周血单个核细胞（PBMC）端粒酶活性的表达情况。方法 通过扩增端粒重复序列（TRAP）及光度酶联免疫法，分别检测健康人及各类乙型肝炎患者PBMC的端粒酶水平。结果 各组患者PBMC在植物血凝素（PHA）刺激前均有端粒酶活性的表达，以急性型肝炎组最高，重型肝炎组最低，二者差别具有显著性（P〈0．001）。PHA刺激后与刺激前比较各组端粒酶活性均有显著性升高（P〈0．001），刺激后的端粒酶水平以重型肝炎组为最低，与其他三组比较差别具有显著性（P〈0．05）。慢性重型乙型肝炎经胸腺五肽（TP5）治疗后端粒酶活性显著增高（P〈0．05）。结论 HBV急性感染期PBMC的端粒酶水平升高；慢性感染期PBMC的端粒酶水平在体内被抑制。TP5具有免疫调节作用，能使过低的端粒酶水平趋向于正常。     

慢性乙型肝炎患者外周血单个核细胞及肝组织中HBV cccDNA定量检测

目的优化HBV共价闭合环状DNA(cccDNA)特异性定量检测方法,并观察HBV cccDNA在慢性乙型肝炎患者外周血单个核细胞(PBMC)及肝组织中的分布情况。方法标本抽提核酸后,以不降解质粒的ATP依赖的DNA酶(PSAD)进行酶切,再以跨双缺口的特异性引物进行荧光PCR定量检测HBV cccDNA;用10份HBV高滴度(10~7拷贝/mL)血清和梯度稀释的HBV克隆质粒标准品,验证此检测方法的特异度和灵敏度;取慢性乙型肝炎患者PBMC和肝组织穿刺标本各50份,检测其总HBV DNA和cccDNA。结果10份血清标本均无假阳性cccDNA检测结果,灵敏度可达到10~3拷贝/mL。所有PBMC中cccDNA检测均阴性;所有肝组织中总HBV DNA和cccDNA检测均阳性,总HBV DNA含量为3．19×10~2～6．69×10~7拷贝/μg人基因组DNA,cccDNA含量为7．32×10~0～6．51×10~6拷贝/μg人基因组DNA,同一患者肝组织中总HBV DNA含量是cccDNA含量的10．7倍至9．2×10~5倍。结论本方法特异度和灵敏度高。PBMC不能支持HBV的复制。在慢性乙型肝炎患者的肝组织中均可检测到cccDNA,但其水平并非与细胞内总HBV DNA水平正相关。     

慢性乙型肝炎患者树突状细胞功能与单个核细胞乙型肝炎病毒共价闭合环状DNA的关系

Objective To investigate the relationship between the maturity and function of dendritic cells (DC) and hepatitis B virus covalently closed circular DNA (HBV cccDNA) load in the peripheral blood mononuclear cells (PBMC)/monocyte-derived DC in patients with chronic hepatitis B (CHB). Methods The peripheral blood samples were collected from 29 patients with CHB and 10healthy controls. PBMC were isolated freshly and induced with granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). A large amount of DC were harvested after seven days of culture. The expressions of CD209, CD80, CD86, human leucocyte antigen (HLA)-DR and CD1a of DC were analyzed by flow cytometry. The HBV cccDNA load in PBMC and DC were measured by real-time polymerase chain reaction (PCR). The interleukin-12 (IL-12) level in the culture supernatant of DC was determined by enzyme linked immunosorbent assay (ELISA). The effects on T lymphocyte proliferation induced by DC were tested by mixed lymphocyte reaction (MLR). The data was compared by t test and analysis of variance. Results HBV cccDNA could be detected in PBMC from 16 patients, but not in DC from all 29 patients. HBV cccDNA load was all negatively correlated with the expressions of CD209 (r= -0. 793, P＜0.01), CD80 (r= -0. 581,P＜0.05), CD86 (r=-0. 698, P＜0.01), HLA-DR (r=-0. 817, P＜0.01), CD1a (r=-0. 734, P＜0.01), IL-12 level (r=-0. 632, P＜0.05) and allogenic T lymphocyte proliferation induced by DC (r=-0. 617, P＜0.05). The expressions of CD209, CD80, CD86, CD1a and HLA-DR on DC,IL-12 level in culture supernatant of DC and the allogenic T lymphocyte proliferation induced by DC in patients with positive PBMC HBV cccDNA were all significantly lower compared to those in healthy controls, and the changes of the parameters mentioned above were greater in PBMC HBV cccDNA positive patients than those in PBMC HBV cccDNA negative patients (P ＜ 0. 05 or P ＜ 0. 01).Conclusions The function and maturity of DC are impaired in CHB patients. HBV cccDNA can be detected in PBMC from CHB patients. Moreover, the higher PBMC HBV cccDNA is, the worse DC function and maturity are, which could be one of the important mechanisms of HBV persistent infection.     

原位杂交法检测外周血单个核细胞中HCV RNA

目的比较慢性丙型肝炎患者用干扰素治疗前以及治疗后３个月ＰＢＭＣ中ＨＣＶＲＮＡ。方法应用地高辛素标记ＨＣＶＲＮＡ正链及负链探针，建立原位杂交方法检测外周血单个核细胞（ＰＭＢＣ）中的ＨＣＶＲＮＡ。结果治疗前１９例患者正链ＨＣＶＲＮＡ阳性，８例负链ＨＣＶＲＮＡ阳性，用正链探针杂交在较多的细胞中出现杂交信号，负链探针杂交仅在少数细胞中出现杂交信号，ＨＣＶＲＮＡ在ＰＢＭＣ胞浆中呈均质性分布。用干扰素治疗结束后３个月２０例患者中９例ＨＣＶＲＮＡ转阴性，近期治愈率４５％。结论原位杂交技术的敏感性及特异性较高，且重复性较好，是研究ＨＣＶＲＮＡ在组织中定位分布和病毒复制场所一种切实可行的方法     

肝移植患者外周血单个核细胞中HBV DNA检测及临床意义

目的研究乙型肝炎肝硬化终末期患者肝移植后外周血单个核细胞(PBMC)HBV DNA状态及临床意义.方法应用荧光PCR技术检测乙型肝炎肝硬化终末期肝移植术后30例患者血清及PBMC标本HBV DNA,并用细胞计数法和管家基因β-actin标定PBMC HBV DNA,观察PBMC HBV DNA与血清HBV DNA定量关系;观察患者肝移植术后不同时间PBMC HBV DNA水平.30例对照组为肝炎肝硬化失代偿期患者.结果移植后患者19份(63%)PBMC标本HBVDNA阳性,低于对照组(87%,26/30).以Ct值为定量参数,移植后患者PBMC HBV DNA水平显著低于对照组(P=0.02);肝移植术后患者PBMC HBV DNA长期维持于103～104拷贝/106细胞水平,与肝移植后时间无明显关系.移植后患者血清HBV DNA均阴性,而对照组血清阳性率为48%.结论乙型肝炎肝硬化终末期患者肝移植术后,经有效预防HBV再感染治疗后,虽然血清中不能测出HBV DNA,但PBMC中HBV DNA阳性,这可能成为肝外"病毒池",导致供肝再感染;对移植后患者监测PBMC HBV DNA,可能有助于早期诊断HBV再感染或复发.     

Dynamic changes of HBV DNA in serum and peripheral blood mononuclear cells of chronic hepatitis patients after lamivudine treatment

AIM: To study the dynamic changes of hepatits B virus (HBV) DNA in serum and peripheral blood mononuclear cells (PBMCs) of patients after lamivudine therapy. METHODS: A total of 72 patients with chronic HBV infection were included in this study. All patients were confirmed to have the following conditions: above 16 years of age, elevated serum alanine amonotransferase (ALT), positive hepatitis B e antigen (HBeAg), positive HBV DNA in serum and PBMCs, negative antibodies against HAV, HCV, HDV, HEV. Other possible causes of chronic liver damages, such as drugs, alcohol and autoimmune diseases were excluded. Seventy-two cases were randomly divided into lamivudine treatment group (n=42) and control group (n=30). HBV DNA was detected both in serum and in PBMCs by fluorescence quantitative polymerase chain reaction (PCR), during and after lamivudine treatment. RESULTS: In the treatment group, HBV DNA became negative both in serum and in PBNIC, of 38 and 25 out of 42 cases respectively during the 48 wk oflamivudine treatment, the negative rate was 90.5% and 59.5% respectively. In the control group, the negative rate was 23.3% and 16.7% respectively. It was statistically significant at 12, 24 and 48 wk as compared with the control group (P < 0.005). The average conversion period of HBV DNA was 6 wk (2-8 wk) in serum and 16 wk (8-24 wk) in PBMC. CONCLUSION: Lamivudine has remarkable inhibitory effects on HBV replication both in serum and in PBMCs. The inhibitory effect on HBV DNA in PBMCs is weaker than that in serum.     

CD150在成人乙型肝炎疫苗无应答者体外外周血单个核细胞中的表达

目的 了解信号转导淋巴细胞激活分子(SLAM)CD150在乙型肝炎(乙肝)疫苗无应答者体外外周血单个核细胞(PBMC)中的表达.方法 对2007年9月至2009年12月曾行乙肝疫苗接种、HBV标志物检测均阴性的202例患者进行标准程序再免疫,再接种后第7～12个月检测抗-HBs效价.根据抗-HBs效价判定无应答者18例(男11例,女7例);选取应答者18例(男9例,女9例)作为对照.采集无应答者与应答者静脉血18 mL,淋巴细胞分离液密度梯度离心法分离PBMC,流式细胞仪测定细胞膜表面分子CD150.采用SAS统计软件包进行t检验以及Spearman 秩相关检验.结果 乙肝疫苗免疫后,在特异性刺激剂rHBsAg作用下,PBMC中CD150的表达无应答者为(39.20±10.66)%,高于应答者的(23.73±12.41)%,差异有统计学意义(t=2.1947,P<0.05);CD3+CD4+细胞中CD150的表达无应答者为(49.64±11.94)%.亦高于应答者的(37.73±11.02)%,差异无统计学意义(t=1.7175,P>0.05).在非特异性刺激剂植物血凝素(PHA)作用下,PBMC中CD150的表达在无应答者为(39.21±7.37)%,高于应答者的(23.18±12.68)%,差异有统计学意义(t=2.2835,P<0.05).在特异性刺激剂rHBsAg作用下,CD150在PBMC及CD3+CD4+细胞中与抗体效价呈负相关(r=-0.726,P<0.05).结论 CD150可能在机体接种乙肝疫苗后的无应答者中发挥一定作用.

Abstract：

Objective To study the expression of signaling lymphocytic activation molecule (SLAM)CD150 in peripheral blood mononuclear cells(PBMCs)isolated from adult non-responders to recombined yeast gene hepatitis B vaccine.Methods A total of 202 cases were recruited.All these subjects had been immunized with recombined yeast gene hepatitis B vaccine for more than one standard scheme in two years(from Sep 2007 to Dec 2009)and remained negative for hepatitis B markers(HBsAg,anti-HBs,HBeAg,anti-HBe and anti-HBc).After recruitment,all 202 subjects received another standard scheme(0,1 and 6 month)revaccination.The blood samples were collected 7 months later after the first injection of revaccination to detect anti-HBs titer.The PBMCs were isolated from 18 adult non-responders(anti-HBs titer<10 mIU/mL)and 18 adult responders(antiHBs titer≥100 mIU/mL).CD150 expression on cell surface was analyzed by flow cytometry.SAS package was used for t test and spearman rank correlation analysis.Results After rHBsAg stimulation,the percentage of PBMCs expressed CD150 was significantly higher in non-responders (39.20%±10.66%)than responders(23.73%±12.41%)(t=2.1947,P<0.05).The same trend was also observed in rHBsAg stimulated C133+CD4+T cells,but the difference was not statistically significant(49.64%±11.94%vs 37.73%±11.02%)(t=1. 7175,P>0.05).After phytohaemagglutinin (PHA)stimulation,the percentage of CD150-positive PBMCs was also significantly higher in non-responders (39.21%±7.37%)than responders(23.18%±12.68%)(t=2.2835,P<0.05).CD150 expressions in both PBMCs and CD3+CD4+T cells were negatively correlated with anti-HBs titer after rHBsAg stimulation (r=-0.726,P<0.05).Conclusion Activation of CD150 may contribute to the non-response to hepatitis B vaccine.     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒检测及其意义

目的 探讨外周血单个核细胞（ＰＢＭＣｓ）在丙型肝炎病毒（ＨＣＶ）的感染中的作用。方法 对２２例慢性丙型肝炎患者２１例抗－ＨＣＶ（＋）血液管析患者及１２例健康献血员的ＰＢＭＣｓ分别进行ＨＣＶＲＮＡ,ＨＣＶ抗原检测及电镜观察。结果 （１）２２生丙型肝炎肝炎患者ＰＢＭＣｓ中有７７．３％（１７／２２）ＨＣＶＲＮＡ阳性,（２）感染ＨＣＶ的ＰＢＭＣｓ中电镜下发现复制的ＨＣＶ颗粒;（３）ＨＣＶ颗粒阳笥者的血清和     

慢性乙型肝炎患者树突状细胞功能与单个核细胞乙型肝炎病毒共价闭合环状DNA的关系

Objective To investigate the relationship between the maturity and function of dendritic cells (DC) and hepatitis B virus covalently closed circular DNA (HBV cccDNA) load in the peripheral blood mononuclear cells (PBMC)/monocyte-derived DC in patients with chronic hepatitis B (CHB). Methods The peripheral blood samples were collected from 29 patients with CHB and 10healthy controls. PBMC were isolated freshly and induced with granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). A large amount of DC were harvested after seven days of culture. The expressions of CD209, CD80, CD86, human leucocyte antigen (HLA)-DR and CD1a of DC were analyzed by flow cytometry. The HBV cccDNA load in PBMC and DC were measured by real-time polymerase chain reaction (PCR). The interleukin-12 (IL-12) level in the culture supernatant of DC was determined by enzyme linked immunosorbent assay (ELISA). The effects on T lymphocyte proliferation induced by DC were tested by mixed lymphocyte reaction (MLR). The data was compared by t test and analysis of variance. Results HBV cccDNA could be detected in PBMC from 16 patients, but not in DC from all 29 patients. HBV cccDNA load was all negatively correlated with the expressions of CD209 (r= -0. 793, P＜0.01), CD80 (r= -0. 581,P＜0.05), CD86 (r=-0. 698, P＜0.01), HLA-DR (r=-0. 817, P＜0.01), CD1a (r=-0. 734, P＜0.01), IL-12 level (r=-0. 632, P＜0.05) and allogenic T lymphocyte proliferation induced by DC (r=-0. 617, P＜0.05). The expressions of CD209, CD80, CD86, CD1a and HLA-DR on DC,IL-12 level in culture supernatant of DC and the allogenic T lymphocyte proliferation induced by DC in patients with positive PBMC HBV cccDNA were all significantly lower compared to those in healthy controls, and the changes of the parameters mentioned above were greater in PBMC HBV cccDNA positive patients than those in PBMC HBV cccDNA negative patients (P ＜ 0. 05 or P ＜ 0. 01).Conclusions The function and maturity of DC are impaired in CHB patients. HBV cccDNA can be detected in PBMC from CHB patients. Moreover, the higher PBMC HBV cccDNA is, the worse DC function and maturity are, which could be one of the important mechanisms of HBV persistent infection.