乙型肝炎病毒携带者精子中HBV DNA的检测及意义

目的 检测乙型肝炎病毒携带者的精子是否携带乙肝病毒.方法 12例乙型肝炎病毒携带者的精子经辅助生育技术洗涤后制成精子涂片,应用特异性乙肝病毒DNA(HBV DNA)的探针以原位杂交法检测这些患者的精子涂片.结果 12例患者中有3例精子中检测到HBV DNA,分布于精子的核心部.结论 经辅助生育技术洗涤后的患者的精子仍存在向子代传播乙肝的可能性.     

慢性乙型肝炎肝内HBV DNA和HBsAg的双标记定位研究

应用原位杂交和免疫组化PAP法双标记技术,结合病人乙型肝炎(乙肝)病毒血清学标志物检测结果,研究了31例慢性乙肝病人肝穿刺组织中乙型肝炎病毒DNA(HBV DNA)和HBsAg的分布及意义。结果显示,肝组织内检出HBV DNA 23例,HBsAg 26例,二者同时检出者21例。从同组病人肝组织内HBV DNA和HBsAg双标检测结果与其乙肝病毒血清学标志物检测结果的比较来看,肝组织内HBV DNA和HBsAg同时阳性很可能表明HBV正处于复制及表达的活动状态。     

乙肝病毒相关肝病患者肝移植后外周血单个核细胞及肝组织中乙型肝炎病毒cccDNA的检测

背景：肝移植后受者体内绝大部分病毒负荷被清除,植入新肝后其复发性肝炎病原体从肝外进入肝内的途径及其复制规律目前尚无定论。 目的：检测肝移植前后外周血单个核细胞和肝组织中乙型肝炎病毒cccDNA及血清中乙型肝炎病毒DNA的表达。 方法：采用淋巴细胞分离液从乙肝病毒相关终末期肝病37例患者外周血中分离出单个核细胞,采用荧光定量PCR检测肝移植前后及移植后乙肝复发3个时期外周血单个核细胞和肝组织中cccDNA及血清乙型肝炎病毒DNA表达。 结果与结论：肝移植前,单个核细胞cccDNA阳性12例,肝组织cccDNA阳性6例,检出率分别为32%和16%,单个核细胞、肝组织中cccDNA拷贝范围分别为(3.028~6.508)×10⁴,(4.158~6.234)×10⁴ 拷贝/mL。肝移植后,单个核细胞cccDNA阳性1例,无血清乙型肝炎病毒DNA检测阳性病例。6例肝移植后乙肝复发病例中外周血单个核细胞cccDNA阳性4例,肝组织活检cccDNA阳性1例,6例血清乙型肝炎病毒 DNA均为阳性。提示乙肝病毒相关终末期肝病患者肝移植后乙肝复发途径可能是残留乙肝病毒在外周单个核细胞中以cccDNA为模板复制,然后再迁移到肝脏。     

早孕妇女绒毛细胞HBV感染与母婴传播关系的研究

目的探讨在携带乙型肝炎病毒的早孕孕妇中,绒毛细胞感染乙型肝炎病毒的发生情况,以及与母婴传播的关系。方法选择自愿在我院门诊行人工流产的孕妇50例,孕妇血清乙型肝炎病毒携带者为实验组（20例）,孕妇血清乙型肝炎感染标志物均阴性为对照组（30例）。分别用ELISA法检测外周血血清HBV表面标志物、荧光定量PCR法检测HBV—DNA,用免疫组织化学链霉亲和素-生物素过氧化物酶复合物（SABC）法检测孕妇的绒毛细胞。结果实验组HBV—DNA阳性16例,阴性4例。HbsAg和HbcAg均阳性5例,HbsAg和HbeAg均阳性4例,绒毛细胞出现HBV的阳性染色;对照组HBV—DNA均阴性,HbsAg和HbcAg均阴性,绒毛细胞未出现HBV的阳性染色。结论在早孕人工流产术的乙型肝炎病毒携带孕妇中,乙型肝炎病毒可感染绒毛细胞。     

144例乙型肝炎病毒相关性肾炎的临床与病理分析

目的 探讨肾活检组织内乙肝病毒标志物在乙肝病毒相关性肾炎(HBV-GN)中的表达意义及HBV-GN的临床病理特点.方法 应用免疫荧光法和免疫组化SP法,分别在石腊切片和冰冻切片中检测肾活检组织内HBV标志物.结果 2001年1月至2012年10月肾活检确诊为HBV-GN的144例患者,经免疫组织化学检测,证实HBV标志物阳性率在病例中以膜性肾病最高(117例,占81.2％),膜增殖性肾小球肾炎次之(21例,占4.5％),阳性物质沉积于毛细血管袢或系膜区.结论 鉴于我国是HBV的高感染地区,在肾活检组织中常规检测HBV标志物,对HBV-GN的早期诊断具有重要意义.     

慢性乙肝患儿HBV基因型与乙肝病毒大蛋白关系探讨

目的 探讨慢性乙型肝炎患儿HBV基因型与乙肝病毒大蛋白的关系.方法 采用实时荧光PCR法和ELISA法分别检测138例处于乙肝病毒活动期的慢性乙型肝炎患儿血清中的HBV DNA和乙肝病毒大蛋白并鉴定其基因型.结果 乙肝病毒大蛋白吸光度与HBV DNA载量存在正相关（r=0.85,P＜0.05）;HBV基因B型与HBV基因C型的ALT水平、乙肝病毒大蛋白吸光度和HBVDNA载量差异无统计学意义（P＞0.05,P＞0.05,P＞0.05）.结论 乙肝病毒大蛋白水平与HBVDNA载量具有良好的正相关性,表明乙肝病毒活动期的慢性乙肝患者体内乙肝病毒大蛋白与病毒复制程度密切相关,乙肝病毒基因型与乙肝病毒大蛋白无关.     

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.     

乙肝病毒携带产妇血浆与初乳中病毒含量的相关性研究

目的 探讨乙肝病毒携带产妇血浆及初乳中病毒含量的差异性及其临床意义.方法 选取20例乙肝病毒携带产妇作为实验组,21例乙肝表面抗原阴性的健康产妇作为对照组,采用RT-PCR法检测实验组及对照组血浆及初乳中HBV DNA病毒含量.结果 对照组产妇的初乳和血液中HBV DNA均检测阴性.实验组产妇初乳中HBV DNA含量与血浆中HBV DNA含量有一定相关性,Ct值和病毒载量相关系数r分别为0.7731和0.8053,P值均小于0.0001.实验组血浆中HBVDNA含量明显高于初乳中HBV DNA含量10～1000倍(P＜0.0001).当血浆HBV DNA国际单位(IU)的Log值下降到4.7以下时,75％的乙肝病毒携带产妇初乳中病毒DNA是低于检测限.结论 血浆和乳汁中HBV DNA载量较低的乙肝病毒携带产妇可结合其它临床检测指标部分考虑采取母乳喂养.     

乙型肝炎病毒复制期与病毒耐药基因突变相关性

目的:研究乙肝病毒不同复制期与乙型肝炎患者抗病毒用药后基因突变两者的相关性。方法:选取203例慢性乙型肝炎患者血清标本为对象。经巢式PCR扩增及磁珠分离,制备焦磷酸测序单链模板,将PCR扩增得到HBV病毒P区和BCP区产物经焦磷酸测序进行突变频率检测,并在PyroMark ID遗传分析系统上进行焦磷酸测序。结果:检测乙型肝炎病毒DNA含量,按照HBV定量结果阳性(拷贝数≥103copies/ml)和阴性(拷贝数<103copies/ml)的标本分组分析,HBV定量阳性标本具有极高的敏感性。批量突变检测,乙型肝炎病毒复制率越高,突变率越高,检测率越高。结论:选择乙肝病毒的复制阳性标本,结合焦磷酸测序进行高通量、快速、准确检测耐药基因区突变,能满足临床批量用药检测及治疗方案设计的需要。     

急性乙型肝炎患者血清中HBV-DNA定量的变化规律及其临床意义

目的 研究在临床上验证急性乙型肝炎(乙肝)时，血清中HBV—DNA被清除机制。方法 总结12例急性乙肝患者血清中HBV—DNA定量和乙肝病毒标志物的动态变化规律。结果 66．7％的患者血清HBV清除发生于患者入院之前。乙肝病毒标志物中HBsAg、HBeAg的吸光度(A值)在住院期间逐渐下降，直至阴转。结论 急性乙肝时，绝大部分患者HBV—DNA的清除可能是通过非细胞溶解机制。     

血清HBV标志物阴性肝炎肝组织中HBV DNA表达的研究

目的 :研究HBVDNA在血清HBV标志阴性肝炎肝组织中的表达。方法 :对 4 5例HBV血清标志阴性肝炎患者 ,进行肝组织HBVDNA的原位杂交检测。结果 :原位杂交表明 ,HBVDNA阳性 7例 (阳性率 15 5 6 % ) ,阳性信号主要存在于肝细胞的胞核中 ,少数位于胞浆内 ;结论 :血清HBV标志阴性肝炎肝组织中可检出HBVDNA ,有利于提高对HBV感染的诊断     

Hepatitis with isolated serum antibody to hepatitis B core antigen. A variant of non-A, non-B hepatitis?

Antibody to hepatitis B core antigen (anti-HBc) has previously been recognized to be a sensitive marker of hepatitis B virus (HBV) infection. In addition, anti-HBc has recently been suggested to be a surrogate marker for non-A, non-B hepatitis agents in donated blood. The authors studied prospectively the HBV antigen and antibody status in four patients with chronic hepatitis and persistent presence of isolated anti-HBc in their sera. The serologic and histopathologic findings of these four patients were compared with those of three groups of patients having chronic hepatitis with or without HBV markers. A low concentration of serum HBV DNA was detected in only one of the four patients with hepatitis with isolated anti-HBc and in another patient with previous HBV infection. HBV antigens and HBV DNA were not detected in the sera and liver biopsies from the remaining patients with hepatitis with isolated anti-HBc and other patients with hepatitis with or without serologic markers of previous hepatitis A or HBV infection. In contrast, all patients with chronic HBV-associated hepatitis had detectable HBV DNA, hepatitis B surface antigen (HBsAg), and hepatitis B e antigen (HBeAg) in their sera and/or liver biopsies. These findings suggest that chronic hepatitis associated with isolated anti-HBc is a heterogenous pathologic entity. The condition of some of these patients may represent a variant of non-A, non-B hepatitis, whereas the remaining patients are chronic hepatitis B carriers with low serum concentrations of HBV.     

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。     

沈阳地区乙型肝炎病毒基因型分子流行病学研究

目的 研究沈阳地区乙型肝炎病毒基因型分布和特点。方法 应用半巢式聚合酶链反应（PCR）扩增乙型肝炎病毒P基因，将PCR产物应用ABI377自动测序仪直接做核苷酸序列分析，并用DNA STAR软件进行种系发生分析及基因型鉴定。结果 HBV DNA标准株P基因片段可进行基因分型。在沈阳地区慢性HBV感染者中可检出基因型B、C和D，检出率分别为22％、50％和28％，基因型C分布与基因型B、D的差异有统计学意义，在慢性乙型肝炎患者和慢性HBV携带者间各基因型间分布比较中差异无统计学意义。结论 通过测定HBV DNAP基因序列片段可进行HBV DNA基因分型。沈阳地区乙型肝炎病毒基因型有基因型B、C和D型，其中基因型C为优势基因型。     

HBV DNA及HBV抗原在血清HBV标志物阴性肝炎肝组织中表达的研究

目的 研究HBV DNA及HBV抗原在血清HBV标志阴性的肝炎肝组织中的表达。方法 对45例HBV血清标志阴性阴性肝炎患者，进行肝组织HBV DNA的原位杂交及免疫组织化学染色检测。结果 原位杂交表明，HBV DNA阳性者7例，（阳性率15．56％），阳性信号主要存在于肝细胞的胞核中，少数位于胞浆内；免疫组化染色表明，HBsAg及HBcAg均呈阴性。结论 血清HBV标志阴性的肝炎肝组织中可检出HBV DNA，有利于提高对HBV感染的诊断。     

The absence of hepatitis B virus DNA in hepatitis B e antigen positive sera from chronic hepatitis B surface antigen carriers in China

Sera from 20 Chinese patients with chronic hepatitis B were examined for hepatitis B e antigen and hepatitis B virus (HBV) DNA. There was considerable discordance with HBV DNA not being detectable in 10 out of 13 (77%) patients who were hepatitis B e antigen positive. Further testing for anti-HBe and HBV-DNA polymerase activity confirmed the results. Possible reasons for this discordance are discussed but neither hepatitis D (delta) infection nor the acquired immunodeficiency syndrome (AIDS) could be implicated.     

Hepatitis B virus DNA in leukocytes of patients with hepatitis B virus-associated liver diseases

In order to determine the relationship between hepatitis B virus (HBV) infection of human white blood cells and different forms of HBV-associated liver diseases, we tested for HBV DNA in the sera and leukocytes of 11 healthy individuals without any serological markers of HBV infection and 91 patients with HBV infection and other gastrointestinal and urinary diseases by dot and Southern blot hybridization. HBV DNA was found in leukocytes of chronic HBV carriers, in acute and chronic hepatitis, and in patients with liver cirrhosis and hepatocellular carcinoma. Between 27 and 50% of individuals in different categories of patients examined were positive for leukocyte HBV DNA. HBV DNA was also detected in the sera of some of these patients but was absent in others. Serum HBV DNA-positive rates seemed to be highest in hepatitis B e antigen-positive asymptomatic carriers (8/10, 80%), and tended to drop to lower levels as the disease progressed to liver cirrhosis (0/8) while leukocyte HBV DNA-positive rates were highest in patients with cirrhosis (4/8, 50%). The results also show that in individuals who were serologically negative for hepatitis B surface antigen (HBsAg) and positive for antibodies to HBsAg and/or HBcAg, HBV DNA was absent in most of the sera (27/28, 96%) but it was present in leukocytes of some of these patients (7/28, 25%). In control experiments with 11 healthy individual, HBV DNA was not detected in either sera or leukocytes. In all the cases with leukocyte HBV DNA, the HBV DNA molecules were present in free forms with discrete sizes. The exceptions were a case of liver cirrhosis and a case of chronic hepatitis with possible HBV sequence integration into high molecular weight cellular DNA. Since HBV does infect human leukocytes, it may perhaps interfere with the immunological functions of the white blood cells, and thus play an important role in the pathogenesis of HBV-induced liver disease.     

Flow cytometry CD4+/CD8+ ratio of liver-derived lymphocytes correlates with viral replication in chronic hepatitis B.

T lymphocytes have been assumed to play an essential role in tissue injury in patients with chronic hepatitis B. As hepatitis B virus (HBV) is considered as a major factor controlling liver inflammation, we assessed whether a particular T lymphocyte subset could be preferentially detected in the liver in accordance with viral replication. Liver-derived lymphocytes and peripheral blood lymphocytes were analysed by flow cytometry in 21 patients with histologically confirmed chronic hepatitis B without cirrhosis. Viral replication was quantified by hybridization of serum HBV DNA. Eleven patients exhibited an active viral replication with serum HBV DNA ranging from 10 to 388 pg/ml at the time of the liver biopsy, whereas 10 patients had no detectable serum HBV DNA. In patients exhibiting viral replication, CD4+/CD8+ ratios of liver-derived lymphocytes were significantly higher (P < 0.05) than those obtained in patients without viral replication. In contrast, the percentage of T cells expressing the gamma/delta receptor and that of CD2+/CD57+ cells were similar in both groups of patients. Furthermore, in patients exhibiting viral replication, CD4+CD8+ ratios of liver-derived lymphocytes correlated with serum HBV DNA levels (P < 0.001). No relationship between CD4+/CD8+ ratio of liver-derived and peripheral blood lymphocytes was observed. Our data indicate that, in patients with chronic hepatitis B, the CD4+/CD8+ ratio of liver-derived lymphocytes correlates with viral replication. This suggests that in situ helper/inducer CD4+ T lymphocytes may positively regulate the cytotoxic T cell activity in patients with HBV-related chronic hepatitis.     

Quantitative DNA analysis of low-level hepatitis B viremia in two patients with serologically negative chronic hepatitis B.

Low-level viremia due to hepatitis B virus (HBV) was demonstrated in the sera of two patients diagnosed previously as having non-B, non-C chronic hepatitis. Both patients had a "silent" HBV infection, because they were negative for both hepatitis B surface antigen (HBsAg) and anti-hepatitis B core antibody. The TaqMan chemistry polymerase chain reaction (PCR) amplified the HBV DNA, enabling quantitation of the virus in their sera. Their serum HBV DNA concentrations were low: the amount of each HBV S or X gene amplified showed there were approximately 10(3) copies/ml and HBV DNA was detected occasionally during clinical follow-up. Positive HBsAg staining in liver tissues was demonstrated by an immunoperoxidase technique. Vertical transmission of silent HBV from one patient to her daughter was confirmed. Direct nucleotide sequencing of the amplified HBV X region revealed several mutations, suggesting reduced viral replication. One patient had a T-to-C mutation at the extreme 5'-terminus of the direct repeat 2 region and the other exhibited a coexisting X region with a 155-nucleotide deletion. These findings suggest that HBV replication is suppressed considerably in patients with silent hepatitis B.     

The detection of hepatitis B virus DNA in the blood serum of children with chronic HBsAg-positive hepatitis]

The presence of HBV DNA in the blood serum of 50 children with HBsAg-positive chronic hepatitis (36 with chronic hepatitis B, 14 with chronic hepatitis delta) was determined by molecular hybridization method. No significant differences were observed between the frequency of HBV DNA detection and clinicomorphological type of chronic HBsAg-positive hepatitis. In chronic HBV infection, HBV DNA was shown to be detected more frequently than in chronic hepatitis delta. A correlation was confirmed between the frequency of HBV DNA detection and demonstration of HBeAg in the blood. With the increase in the duration of the disease and age of the patients the HBV DNA detection rate was found to diminish.