树鼩、熊猴感染人乙型肝炎病毒血清指征的研究

目的：验证树Qu、熊猴感染人乙型肝炎（乙肝）病毒（HHBV）血清感染指征。方法：用含HBV的人血清接种给233只树Qu，28只熊猕，每周定期采血，用不同公司生产的ELISA试剂检测接种后的动物血清感染指标。结果：树Qu感染HHBV后，90．0％呈急性感染，44．4％为可持续1年以上的慢性感染，33．3％持续2年2个月。熊猴感染HBV后，血清HBV标志物呈一过性。结论：熊猴对HHBV并不敏感，而树Qu对HHBV高度敏感，有望成为HHBV的动物模型。     

人类乙肝病毒体外感染树鼩肝原代细胞的研究

目的 为进一步研究人类乙肝病毒(HBV)提供接近自然感染状态的理想细胞模型.方法 体外二步灌流法分离树鼩原代肝细胞,纯化后的乙肝患者血清感染上述肝细胞,Southern blot和Noahem blot检测感染后细胞内的DNA和RNA,ELISA方法 检测细胞上清的HBsAg,免疫组化检测细胞内HBsAg的表达.结果 可检测出肝细胞内cccDNA(共价闭合环状DNA)、pgRNA(前基因组RNA)和sgRNA(亚基因组mRNA),感染后第7天,信号开始增强,持续到实验结束的第14天.细胞上清中HBsAg自第1天到第5天S/CO值逐渐下降,随后S/CO值逐渐升高.结论 HBV可在原代树鼩肝细胞中复制和表达.     

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。     

慢性乙型肝炎病毒感染树鼩Kupffer细胞TLR2和TLR4的表达及其意义

目的探索慢性乙型肝炎病毒(HBV)感染树鼩Kupffer细胞Toll样受体(TLR)家族中的TLR2和TLR4在mRNA水平的表达情况及其对Kupffer细胞功能的影响。方法树鼩分为确定慢性感染HBV的树鼩、疑似慢性感染HBV的树鼩和未接种HBV的正常对照树鼩。全部动物定期抽血和进行肝活检手术,采用实时荧光定量PCR(qRT-PCR)分析血清和肝组织的HBV DNA水平;对手术切取的树鼩肝组织进行Kupffer细胞的分离、纯化和原代培养,采用qRT-PCR检测TLR2、TLR4以及TNF-α的mRNA表达水平;采用迁移实验及溶酶体荧光探针等方法分析TLR2和TLR4对Kupffer细胞迁移能力及溶酶体数量的影响。结果确定慢性感染HBV的树鼩TLR2 mRNA和TLR4 mRNA表达水平均低于疑似慢性感染HBV的树鼩和未接种HBV的正常对照树鼩(P0.05),表达水平均与动物肝组织的HBV DNA拷贝数呈负相关(P0.05),与Kupffer细胞的细胞迁移数、溶酶体密度及TNF-αmRNA表达水平呈正相关(P0.05)。结论 Kupffer细胞中的TLR2和TLR4可能通过影响Kupffer细胞功能而参与树鼩HBV感染后肝脏病变的慢性化发展过程。     

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。     

血清HBV标的阴性肝炎肝组织中HBV DNA表达的研究

目的：研究HBV DNA在血清HBV标志阴性肝炎肝组织中的表达。方法：对45例HBV血清标志阴性肝炎患者，进行肝组织HBV DNA的原位杂交检测。结果：原位杂交表明，HBV DNA阳性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；结论：血清HBV标志阴性肝炎肝组织中可检出HBV DNA，有利于提高对HBV感染的诊断。     

慢性活动性肝炎HBV DNA原位分子杂交研究

对50例慢性活动性乙型肝炎肝组织进行了原位杂交及免疫组化研究；部分作了上述两法的双标记。发现HBV DNA阳性物质在肝细胞内的分布方式可分为全浆型、膜下型、核型和膜间型4种。后者提示HBV DNA有可能通过肝细胞膜直接传播至邻近的肝细胞。双标记显示，大多数HBV DNA强阳性肝细胞并不同时含有HBcAg或HBsAg；反之亦然。此外，少数病例见胆小管上皮和窦内皮细胞胞浆内亦呈HBV DNA阳性。     

病毒接种剂量对HBV感染成年树鼩的影响

目的探讨不同病毒接种剂量对乙肝病毒(hepatitis B virus,HBV)感染树鼩进程的影响。方法分别给4组树鼩接种含101、102、104和108GE(genome equivalents)乙肝病毒的感染者血清,于不同时间点采集树鼩血清标本,应用实时荧光定量PCR(real-time fluorescence,PCR)检测血清HBV DNA浓度,用电化学发光免疫测定法(electrochemiluminescence immunoassay,ELICA)检测血清中HBV感染标志物,用临床生化分析仪测定谷丙转氨酶(alanine transaminase,ALT)水平。结果接种108GE HBV后树鼩体内HBV感染持续3周,接种104GE HBV后树鼩体内HBV感染延长至15周,接种101和102GE HBV后树鼩体内HBV感染可以存在9周。结论病毒接种剂量对HBV感染成年树鼩的进程有显著影响,中低剂量(101、102和104GE)接种利于树鼩体内HBV感染的维持。     

慢性乙型肝炎患者血清前S1抗原检测的临床应用价值探讨

乙型肝炎病毒(HBV)表面抗原(HBsAg)编码区由S、前S1和前S2基因组成，分别编码S、前S1和前S2三种主要蛋白，从而共同构成HBV外壳蛋白。前S1蛋白含有肝细胞膜受体，在HBV感染肝细胞和机体免疫应答反应中起重要作用。作为一项新的HBV血清学检测指标，乙型肝炎病毒前S1抗原(HBV pre S1-Ag)的检测已被普遍应用于乙型肝炎的实验室诊断和疗效观察。检测慢性乙型肝炎患者乙型肝炎血清标志物(HBV—M)、乙型肝炎前S1抗原(HBV pre S1-Ag)、乙型肝炎病毒DNA定量(HBV-DNA)，     

CAS基因在原发性肝癌组织中的表达及其与HBV感染的关系

目的 探讨细胞凋亡易感基因(CAS)可否作为肝癌的病理诊断标志物,以及肝细胞癌中CAS蛋白的表达与HBV感染之间的关系.方法 应用免疫组化法检测肝癌、癌旁组织及未发生肿瘤的肝硬化、肝炎组织中CAS蛋白的表达情况,同时应用免疫组化法、核酸原位杂交法检测HBV感染的肝细胞癌组织、癌旁组织中HBsAg、HBcAg以及HBV DNA的表达情况,分析肝细胞癌中CAS蛋白的表达与HBV感染之间的关系.结果 CAS蛋白在肝癌组织中的表达较癌旁组织明显升高(P＜0.01),而癌旁组织中CAS蛋白表达较未发生肿瘤的肝硬化、肝炎组织显著增强(P＜0.01).低分化型肿瘤细胞的CAS蛋白表达明显高于中分化型和高分化型(P＜0.01).CAS蛋白在HBV感染的肝细胞癌组织中的表达明显高于非HBV感染的癌组织(P＜0.01),其中HBV DNA阳性的肝细胞癌组织中CAS蛋白表达水平显著高于HBV DNA阴性的肝细胞癌组织(P＜0.05).结论 CAS蛋白在肝细胞癌组织中呈高表达,肝细胞癌分化愈低其表达愈强,表明CAS蛋白可作为肝细胞癌的病理诊断与分化程度的评价标志物.并推测HBV DNA可能通过上调CAS的表达,在HBV感染相关性肝癌的发生发展过程中发挥重要的作用.     

Serial pathologic changes in livers of tree shrews and macaca assamensises infected with human hepatitis B virus]

BACKGROUND: To serially observe the pathologic changes in livers of tree shrews and macaca assamensises infected with HHBV. METHODS: 10 adult tree shrews and 28 macaca assamensises were inoculated with HBV rich human sera. The liver of the animals were regularly biopsied. The liver samples were examined histopathologically by HE staining. Some samples were stained for HBsAg by immunohistochemistry (IH), and HBV DNA by in situ hybridization (ISH). RESULTS: HBsAg in 80% of tree shrews infected with HHBV can be detected by IH, HBV DNA in 50% of those can be found by ISH.The positive rates of HBsAg in macaca assamensises' livers were 25% by IH, none HBV DNA was detected. CONCLUSION: The tree shrew model seems to be applicable for the research of human hepatitis B.     

A study on primary liver cancer in tree shrews induced by human hepatitis B virus and aflatoxin B1

Among 41 tree shrews exposed to aflatoxin B1(AFB1), 17 were experimental infected by human hepatitis B virus (HHBV) and 24 were uninfected. After 158 weeks, 9 cases (52.94%) of primary liver cancer (PLC) were found out of the 17 tree shrews infected by HHBV and only 3 cases (12.5%) developed PLC in the 24 uninfected animals. Significant difference of PLC incidence was seen between the HHBV-infected and uninfected groups (P less than 0.05). Moreover, 1/9 of the tree shrews that had been infected by HHBV but without exposure to AFB1 developed PLC at the 83rd week. No PLC was found in 6 tree shrews that had neither been infected with HHBV nor been exposed to AFB1. These results demonstrate the possible etiologic relationship between HHBV infection and PLC, and the synergistic effects of HHBV and AFB1 during PLC development.     

原位杂交法检测肝组织中丁型和乙型肝炎病毒核酸

利用国外引进的重组质粒获得纯化基因片段，分别以随机引物法和ＰＣＲ法制备地高辛素标记的ＨＢＶＤＮＡ探针和ＨＤＶｃＤＮＡ探针。用原位杂交法检测了石蜡包埋的肝组织切片ＢＶＤＮＡ和ＨＤＶＲＮＡ。４９例感染肝组织分为两组：丁肝组２３例；单纯乙肝组２６例，ＨＢＶＤＮＡ的检出率丁肝组（７８．２６％）与乙肝组（７６．９２％）无统计学差异；而ＨＤＶＮＡ的检出率丁肝组（６０．８７％）明显高于乙肝组（１５．３８％）。ＨＢＶＤＮＡ可见于受染肝细胞的胞核或胞浆内，而ＨＤＶＲＮＡ绝大部分见于肝细胞胞核。两种病毒核酸阳性细胞在肝组织中的分布特点大致相同：弥漫或散在地分布于肝小叶或假小叶内，或局灶性分布于小叶周边。ＨＤＶＲＮＡ阳性的肝组织都或多或少地同时存在ＨＢＶＤＮＡ。同一例肝组织中，ＨＢＶＤＮＡ阳性细胞从数量和颗粒密度上似略高于ＨＤＶＲＮＡ。将乙肝组和丁肝组两组病人肝内ＨＢ－ｓＡｇ、ＨＢｃＡｇ和ＨＢＶＤＮＡ及血清ＨＢｅＡｇ作了比较，各指标阳性率虽有差异，但均无统计学意义。因此，未发现ＨＤＶ感染对ＨＢＶ的复制有明显抑制作用。此结果对以往用血清学或免疫组化方法对ＨＤＶ的研究有所补充和深入，亦可为研究其它类型病毒性肝炎之间的重叠感染所借鉴。     

Sequencing of human-viral DNA junctions in hepatocellular carcinoma from patients with HCV and occult HBV infection

DNA of free hepatitis B viruses (HBV) has been detected in the liver of patients infected with hepatitis C virus (HCV). It is unknown whether HBV DNA is integrated into such livers; if so, it may affect hepatocarcinogenesis. Hepatocellular carcinomas (HCCs) from 34 patients without HBV surface antigen (HBsAg) and with anti-HCV, and from 7 patients with HBsAg and without anti-HCV as controls, were examined, using the cassette-ligation-mediated polymerase chain reaction and primers based on HBV DNA sequence. In the controls, HBV DNA had been integrated into human DNA of all HCCs. On the basis of HBV DNA in tumor tissue, 23 of the 34 patients with anti-HCV had occult infection. Junctions between human DNA and HBV DNA were detected in 10 of the 34 patients without HBsAg and with anti-HCV. HBV DNA was integrated into chromosome 11q in 4 of the 10 HCCs with junctions. The DNA to either side of the human-viral junctions was sequenced. Clinically, the mean tumor size of these 10 HCCs was 39 mm; that of the 24 HCCs without integrated HBV was 25 mm. The surrounding tissue was cirrhotic in 2 of the 10 former HCCs and in 16 of the latter 24 HCCs. In conclusion, integrated HBV was detected in some patients with HCV infection; in these patients, the integrated DNA was associated with accelerated hepatocarcinogenesis.     

In situ hybridization using a biotinylated DNA probe on formalin-fixed liver biopsies with hepatitis B virus infections: in situ hybridization superior to immunochemistry

In situ hybridization (ISH) using a biotinylated hepatitis B virus (HBV) DNA probe was compared with immunohistochemistry (IHC) using antibody to HBsAg in liver biopsies of 98 patients with HBV-related hepatitis. The positive reaction for ISH and/or IHC was demonstrated in 83 cases from 98 cases studied. In 60 cases, the findings of ISH and IHC were concordant, and in 20 cases, ISH showed evidence of HBV infection by detecting HBV DNA, but IHC did not. The study here confirms the advantages of ISH detecting HBV-DNA over IHC detecting HBsAg. Since ISH using biotinylated probe is simple and rapid, it may be used as a routine diagnostic procedure.     

HBV体外感染卵巢颗粒细胞

目的建立HBV体外感染颗粒细胞模型,研究HBV在颗粒细胞中的复制情况,为深入研究HBV经卵细胞母婴垂直传播提供研究平台。方法原代颗粒细胞体外培养后用HBV阳性血清感染。收集培养上清,在不同时点检测HBsAg、HBeAg定量,实时定量PCR检测HBVDNA。免疫组化检测培养细胞中的HBsAg和HBcAg。巢式PCR检测细胞中的HBVDNA及HBV-mRNA。原位杂交检测细胞内的HBVDNA。结果成功建立了HBV体外感染颗粒细胞模型,在培养上清中可以持续96h检测到HBsAg和HBV DNA,在细胞内检测到HBsAg和HBcAg的阳性信号,PCR扩增显示细胞内有HBVD-NA及HBV-mRNA的存在,原位杂交证实细胞内HBVDNA阳性。结论 HBV能够在体外感染颗粒细胞,并在其内复制,该结果为深入研究HBV经卵细胞传播机制提供了很好的研究平台。     

成年树鼩实验感染丁型肝炎病毒的初步研究

在证实成年树可感染人乙型肝炎病毒（ＨＢＶ）的基础上，进行了丁型肝炎病毒－乙型肝炎病毒（ＨＤＶ／ＨＢＶ）实验感染的探索。ＨＤＶ／ＨＢＶ阳性人血清经同时和重叠感染方式接种于成年树后，定期留取感染树血清及肝组织，检测血清中ＨＢｓＡｇ、ＨＤＡｇ、抗－ＨＤ、ＨＢＶＤＮＡ及ＨＤＶＲＮＡ，初步探讨成年树实验感染ＨＤＶ的可能性。研究发现：①成年树对人ＨＢＶ易感，ＨＢｓＡｇ阳性率为７５％，其血清学反应及肝组织病理改变与文献报道一致；②ＨＤＶ可通过同时和重叠两种方式感染成年树，感染树血清中可出现ＨＤＡｇ及抗－ＨＤ，经分子杂交证实其血清及肝内均有ＨＤＶＲＮＡ和ＨＢＶＤＮＡ存在，且可导致明显的肝细胞损伤。结果提示：成年树既可感染人ＨＢＶ也可感染ＨＤＶ，可作为研究ＨＤＶ人工感染的实验动物；成年树感染ＨＤＶ后的特点与人及黑猩猩感染时十分相似，能较好地反映人丁型肝炎的真实情况。     

肝细胞异型增生的产生及其与肝细胞癌发生关系的实验研究

82只实验树(鼠句)分为4组:A组26只,HBV阳性并加喂黄曲霉毒素B_1(AFB_1);B组34只,HBV阴性并加喂AFB_1;C组13只,为HBV感染者;D组9只,为HBV阴性。实验158周。A组发生LCD16例(61.53%),其中9例(56.25%)发生肝细胞癌(HCC)。B组出现LCD 18例(52.94%),其中3例发生HCC(16.66%)。C组出现LCD 6例(46.15%);D组无LCD产生。82只树(鼠句)有40只出现LCD,其中12只(30%)发生HCC;而无LCD的42只,只有1例(2.83%)产生HCC。它们之间发生HCC的危险性相差12.6倍。血清HBV阳性的树(鼠句),其LCD发生率显著高于HBV阴性者(6/13对0/9.P<0.05);在HBV阴性的动物中,摄入AFB_1的其LCD发生率也同样比未摄入者高(18/34对0/9,P<0.05)。表明HBV和AFB_1都能诱发LCD;LCD可能是HCC的癌前病变。     

The significance of 'anti-HBc only' in the clinical virology laboratory.

BACKGROUND: Isolated detection of hepatitis B core antibody (anti-HBc) in the absence of surface antigen (HBsAg) or antibody (anti-HBs) has been reported, particularly among individuals infected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The significance of this phenomenon is unknown and it is unclear whether all individuals with such serological pattern need further molecular investigations. OBJECTIVES: To determine the prevalence of 'anti-HBc only' in samples referred to a clinical virology laboratory and to evaluate its significance and possible mechanisms. STUDY DESIGN: Samples identified as anti-HBc positive (389/4359, 8.9%) during an 11-month period were investigated for HBsAg, anti-HBs, anti-HCV and anti-HIV. 'Anti-HBc only' samples were tested for HBV DNA using a nested qualitative PCR. Viral loads were measured in samples with detectable HBV DNA and the DNA sequences were analysed. RESULTS: Of 379 samples with detectable anti-HBc, 155 (40.9%) were 'anti-HBc only'. HBV DNA was detected in 6/151 (4%), all of which had a viral load <400 copies per ml. Anti-HIV was found in 50/151 (33.1%) and anti-HCV in 14/151 (9.3%). Of these, only one of the HIV infected patients had detectable HBV DNA. Phylogenetic analysis of the HBV surface gene from three patients showed a variety of genotypes (A, E and G). One sequence had a mutation in codon 144, which has previously been reported to give false negative HBsAg results. CONCLUSIONS: 'Anti-HBc only' is a common phenomenon in the clinical virology laboratory but only a small proportion of samples had detectable HBV DNA. The presence of HBsAg mutants with possible false negative HBsAg test result is of concern. Samples with 'anti-HBc only' could be used to monitor the emergence of these mutants.