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

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...     

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     

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.     

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。     

慢性乙型肝炎患者外周血单个核细胞内HBV DNA存在的意义

目的探讨外周血单个核细胞（PBMC）内HBVDNA与HBV标志物的关系及在慢性肝病发展过程中的作用．方法应用RCR技术结合斑点杂交技术测定血清HBVDNA及PBMC内HBVDNA血清学标志物采用ELISA法结果在HBsAg＋，HBeAg十组48例中，血清与PBMC内HBVDNA阳性检出率分别为93．8％和83．3％；在HBsAg＋，抗-HBe 组41例中，其阳性检出率分别为48．8％和58．5％.两组间存在显著性差异（P＜0．01）在其他各组中血清及PBMC内HBVDNA检出率均较抵有6例呈单纯PBMC内HBVDNA阳性.在不同类型慢性乙肝患者中，CSH和CMoH组PBMC-HBVDNA检出率分别为78．6％和61．8％，与CMiH组（25．0％）、ASC组（14．3％）比较，存在非常显著性差异（P＜0．01），呈随病情加重而检出率增高的趋势结论PBMC清除HBVDNA较血液缓慢，在病毒持续感染及造成肝细胞损伤过程中可能起一定作用．     

慢性丙型肝炎患者外周血单个核细胞中HCV RNA和NS5抗原的检测

目的 研究丙型肝炎病毒（ＨＣＶ）感染外周血个核细胞（ＰＢＭＣ）的情况及其意义。方法 运用非核素原位杂交法（ＮＩＳＨ）和抗生蛋白链菌素－生物素法（ＳＡＢＣ）分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ ＲＮＡ和ＮＳ５抗原。结果 ２０例患者中有８例（４０％）,ＰＢＭＣ中ＨＣＶ ＲＮＡ呈阳性,其中６例（３０％）ＰＢＭＣ中ＮＳ５抗原同时呈阳性。ＨＣＶＲＮＡ主要分布于胞浆中,而ＮＳ５抗原还可以出现在胞膜     

外周血单个核细胞介导乙型肝炎病毒感染的transwell 小室体外模型研究

目的：观察感染 HBV 的 PBMC 经人绒毛膜癌滋养层细胞（Bewo 细胞）构建的胎盘屏障迁移情况,探讨 PBMC 作为载体转运 HBV 的生物学作用。方法培养并用细胞计数试剂盒-8检测培养 Bewo 细胞和 PBMC 的增殖和活性。用 HBV DNA 含量为5×106拷贝／mL 的乙型肝炎患者血清100μL 感染 PBMC 后,用羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）荧光染料标记感染的细胞, transwell 小室建立 Bewo 细胞和感染 HBV 的 PBMC 共培养模型。用流式细胞仪检测感染 HBV 的PBMC 迁移情况,荧光定量 PCR 法检测 transwell 下室中 PBMC HBV DNA 含量。结果 PBMC、Bewo细胞均在接种24 h 左右开始增殖,120 h 左右开始进入生长停滞期。transwell 小室共培养0、12、24和48 h,下室中绿色荧光标记的 PBMC 量分别为（0．445±0．021）％、（21．180±4．653）％、（34．830±7．156）％和（64．185±3．161）％,随着培养时间的延长,下室中检测到的标有绿色荧光的 PBMC 量越多（F ＝68．983,P ＝0．001）。共培养24、48 h 时,下室 PBMC HBV DNA 分别为（1．925±0．431）×103、（2．565±0．361）×103拷贝／mL,即下室中的 PBMC 发生感染。结论 PBMC可以作为 HBV 肝外感染的靶细胞,利用 transwell 小室构建胎盘滋养层屏障可以为体外研究 HBV 跨胎盘传播提供新思路。     

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.     

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     

Intracellular levels of hepatitis B virus DNA and pregenomic RNA in peripheral blood mononuclear cells of chronically infected patients

Summary.  It remains uncertain whether hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and pregenomic RNA (pgRNA) can be detected in the serum or peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B (CHB) infection. We examined HBV cccDNA and pgRNA in the serum and PBMC, and investigated the effect of lamivudine therapy on the viral loads in the PBMC of CHB patients. Paired serum and PBMC samples from 50 treatment-naïve CHB patients [25 hepatitis B e antigen (HBeAg) positive and 25 HBeAg negative] were quantified for total HBV DNA, cccDNA and pgRNA by real time polymerase chain reaction. HBV cccDNA and pgRNA were below the lower detection limit in all serum samples, and in 84% of PBMC. HBV DNA ( r  = 0.889, P  <   0.001) and pgRNA ( r  = 0.696, P  <   0.001) in PBMC correlated with the HBV DNA in serum. In the longitudinal study, 30 patients treated with lamivudine therapy for a median duration of 34 weeks (range 12–48 weeks) were examined. The median HBV DNA reduction in PBMC before and after treatment was 1.318 (range −0.471 to 3.846) log units, which was significantly lower than serum HBV DNA reduction [3.371 (range −0.883 to 9.454) log units, P  <   0.05]. HBV cccDNA and pgRNA were undetectable in the serum of CHB patients. HBV viral loads in PBMC correlated with serum HBV DNA. Lamivudine therapy had less effect on the HBV viral loads in PBMC compared with the serum viral loads.     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒感染研究

目的 以分子生物学、免疫学有电镜等技术对慢性丙型肝炎患者ＰＢＭＣｓ进行综合检测,以证实其受ＨＣＶ感染,并试图在电镜下发现和证实感染细胞内ＨＣＶ颗粒。方法 对逆转录聚合酶链反应（ＲＴ－ＰＣＲ）和免疫组织化学等技术分别检测患者ＰＢＭＣｓ内的ＨＣＶ ＲＮＡ和ＨＣＶＡｇ,以常规和免疫电镜技术观察研究感染细胞内ＨＣＶ的形态、定位和超微结构。结果 ＨＣＶ ＲＮＡ阳性检出率为７７．２％（１７／２２）,ＨＣＶＡｇ     

早期病毒学应答是评估拉米夫定治疗慢性乙型肝炎疗效的重要指标

目的观察拉米夫定治疗HBeAg阳性慢性乙型肝炎病人的疗效及影响因素。方法336例HBeAg阳性慢性乙型肝炎病人予以拉米夫定100mg每日一次口服治疗9月～24月。疗效评估包括血清学、病毒学、血生化学和综合应答。结果血生化学应答率为86.6%(291/336),治疗前ALT水平与血清HBeAg/抗-HBe血清转换呈正相关,从高至低依次为治疗前ALT≥5倍组>ALT2～5倍组>ALT≤2倍组。HBV DNA应答率与疗程有关,治疗6～9月时完全应答率最高为75.0%～74.7%,后随疗程延长转阴率下降,依次9月时为74.7%>12月时69.8%>18月时61.7%>24月时61.2%。治疗前HBV DNA水平≤1×106copies/ml者血清HBeAg/抗-HBe血清转换率高于HBV DNA>1×106copies/ml者,治疗24月时两组比较有显著差异(P<0.05)。性别、年龄、病程与HBV DNA转阴无显著相关(P>0.05)。早期治疗3月无应答者16例延长疗程至6月时2例发生完全应答占12.5%、9月时3例完全应答18.8%,延长疗程至12月、18月时无新增完全应答病例。YMDD变异发生率随疗程延长而增加。结论基线ALT水平显著升高、HBV DNA水平较低是HBeAg/抗-HBe血清转换的重要预测指标。治疗6～9月的疗效在一定程度上可看作是判断远期疗效的标准。治疗6～9月无应答者应考虑改用或联合其它药物治疗。     

阿德福韦酯长期治疗对慢性乙型肝炎患者PBMCs中线粒体DNA的影响

目的探讨长期服用阿德福韦酯对CHB患者外周血单个核细胞（PBMCs）线粒体的可能损伤。方法本研究包括18例服用阿德福韦酯2年、25例服用3年和22例未服用阿德福韦酯的CHB患者;采用实时定量PCR法检测各组患者PBMCs中线粒体DNA（mtDNA）含量（mtDNA／nDMA）;采用ELISA法检测血浆丙二醛（MDA）、F2一isoprostanes含量;采用分光光度法检测血浆总抗氧化能力（TAOC）。结果2年组患者mtDNA含量为0．6±0．4copies／cell,3年组为0．8±0．5copies／cell,均显著低于对照组（1．4±1．2copies／cell,P均〈0．05）;2年组F2一iso—prostanes含量为1．5±0．8ng／ml,3年组为2．2士1．3ng／ml,显著低于对照组（3．7±2．7ng／ml,P均〈0．05）;2年组TAOC含量为3．0±1．1单位／毫升,3年组为2．3±1．4单位／毫升,显著低于对照组（4．3±1．8单位／毫升,P均〈0．05）。3组问MDA含量无统计学差异（F=2．404,P〉0．05）。结论长期应用阿德福韦酯治疗慢性乙型肝炎,对PBMCs中mtDNA水平有一定的影响,其意义还有待探讨。     

拉米夫定治疗慢性乙型肝炎病人的长期疗效

通过多中心,承机,双盲,安慰剂对照的临床试验,研究拉米夫定（ｌａｍｉｖｕｄｉｎｅ）对慢性乙型肝炎（乙肝）病人的疗效和安全性。方法随机选择３２２例慢性乙肝病拉米夫定治疗（１００ｍｇ／ｄ）,１０７例病人服用安慰剂作对照,共治疗１２周,在１２周治疗结束后,拉米夫定组和安慰剂组病人均继续服用拉米夫定１００ｍｇ治疗至５２周。疗效评估包括临床症状和体征,肝功能和ＨＢＶ复制指标。结果治疗１２周,拉米夫定组ＨＢＶ     

荧光定量检测乙型肝炎患者外周血单个核细胞中HBV DNA的含量及其意义

探讨乙型肝炎患者外周血单个核细胞（PBMC）HBV DNA和血清HBV DNA的感染情况及其临床意义。采用荧光定量PCR技术对216例HBV感染者和20例健康人进行了PBMC中及血清中的HBV DNA检测。患者PBMC内HBV DNA的总检出率为51．3％（111／216）,有随着病情加重而逐渐增高的趋势,而血清中HBV DNA阳性率无此趋势;从定量结果上看各组血清和PBMC定量HBV DNA含量对数值间均呈正相关。所以PBMCHBV DNA较血清HBV DNA检测能更好反映肝细胞内HBV DNA的存在情况,从而对慢性乙型肝炎患者HBV的复制状态有更准确的评价。     

改良PCR法检测外周血单个核细胞中的HBV DNA与HBVcccDNA

目的检测慢性HBV感染者外周血单个核细胞(PBMCs)中HBV DNA的存在状况。方法采用改良PCR法检测慢性HBV感染者PBMCs中HBV DNA、共价闭合环状DNA(cccDNA)。结果120例慢性HBV感染者PBMCs中HBV DNA、cccDNA阳性检出率分别为62.50%、45.83%,HBV DNA阳性检出率高于cccDNA(P<0.01);其中HBeAg阳性组PBMCs中HBV DNA(80.00%)和cccDNA阳性检出率(45.00%)分别明显高于HBeAg阴性组(61.67%)(30.00%)(P均<0.01);PBMCs中HBVDNA检测阳性者血清HBVDNA水平明显高于HBVDNA检测阴性者(P<0.01)。结论改良PCR法检测表明HBV DNA不仅可感染PBMCs,且部分参与复制;PBMCs中HBVDNA存在和复制能力与HBeAg阳性相关;PBMCs中HBV DNA阳性检出率与血清HBV DNA水平有一致性。