Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

Interleukin-10 promoter polymorphisms in patients with hepatitis B virus infection or hepatocellular carcinoma in Chinese Han ethnic population

BACKGROUND: Since single nucleotide polymorphisms (SNPs) can serve as gene markers, polymorphism profiles may help scientists to identify the full collection of genes that contribute to the development of complex diseases such as cancer. The distribution of interleukin-10 (IL-10) promoter polymorphisms in Chinese Han ethnic patients with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) was investigated in this study. METHODS: The polymorphisms of IL-10 promoter region were detected by pulymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. Sixty-six health controls, 42 patients with HBV infection, 30 HCC patients, and cell line SMMC-7721 were examined this way. RESULTS: Polyrnorphisms of T/C or T/N on-872 site occurred frequently in Han ethnic population. Pulyrnorphisms were detected in HBV and HCC patients and cell line SMMC-7721. The hotspot among the pulymorphisms was inserting base A between-1058 and-1057. CONCLUSION: Polymorphisms of IL-10 promoter in HBV and HCC patients may be associated with HBV infection and HCC development.

Hepatitis C and B Virus Infection in Chinese Patients with Extrahepatic Bile Duct Carcinoma

Objective In China, the incidence of extrahepatic bile duct carcinoma (EBDC) tends to increase over the past decades. The etiology of the noted increase in EBDC is not identified. Approximately, in a half of the overall Chinese patients with EBDC, the causative factors in the development of EBDC have not been demonstrated.There is a high prevalence of hepatitis C virus (HCV) or hepatitis B virus (HBV) in China, both of which can induce malignant transformation of infected cells and strongly associated with hepatocellular carcinoma (HCC). In this study, EBDC tissues from Chinese patients were examined for the presence of HCV and HBV infection to investigate further the potential causes of EBDC.Methods HCV NS5 protein and HBsAg were detected by labeled streptavidin biotin (LSAB) method; HCV RNA and HBV DNA were detected by in situ polymerase chain reaction (IS-PCR) in formalin fixed, paraffin embedded specimens from 51 Chinese patients with EBDC. HCV RNA and HBV DNA were detected by IS-PCR in 34 Chinese patients with specimens of benign lesions of hepatobiliary tract (control group) .Results In 51 case tissue sections of EBDC, NS5 protein was detected in 14 (27.5%), and HBsAg in 5 (9.8%), HCV RNA in 18 (35.4%) and HBV DNA in 8 (15.9%) .respectively, of which HCV and HBV co-infection was detected in 2 (3.9%). In 34 case tissue sections of the control group, HCV RNA was detected in 2 (5.9 % ), and HBV DNA in 3 (8.8%).Conclusion In this study using standard histochemical and PCR techniques, HCV and HBV genomes and their encoding proteins were detected in the tissues of EBDC. The data show that there is a higher than expected incidence of HCV and HBV presence in EBDC tissues than would be expected on serologic grounds. The detectable rate of HCV RNA in EBDC tissues was significantly higher than in control group (x2 = 9.808, P = 0.002). As a result, this study indicates that there is a correlation between the presence of HCV infection and EBDC, and HCV infection has possible etiologic significance in the development of EBDC in China. While HBV DNA was detected in EBDC tissues with the difference in the detectable rate of HBV, DNA being not significance between EBDC tissues and the control group (x2 = 0.853, P = 0.356) . Further research is necessary to determine the presence of a causal relationship between HCV/HBV infection and the development of EBDC.

Investigation on Hepatitis C and B Virus Infection in Carcinoma of the extrahepatic bile duct in CHINA

Objective: The incidence of carcinoma of extrahepatic bile duct tends to increase during recent decade in China, but its cause is unclear. This study is to investigate the hepatitis C virus (HCV) and Hepatitis B virus (HBV) infection in the tissues of carcinoma of extrahepatic bile duct and study their correlation. Methods: HCV RNA and HBV DNA was detected by in situ polymerase chain reaction (IS-PCR) in sections of 51 cases of carcinoma of extrahepatic bile duct and 34 cases of control group. Results: Of 51 carcinoma of extrahepatic bile duct, HCV RNA was detected in 18 (35.4%), HBV DNA in 8 (15.9%). In 34 cases of control group, HCV RNA was detected in 2 (5.9%), and HBV DNA in 3 (8.8%). Conclusion:The prevalence of hepatitis C and B viralinfection in the tissues of carcinoma of extrahepatic bile duct was significantly higher than in control group. The findings suggest a correlation between HCV, HBV infection and carcinoma of extrahepatic bile duct, inferring HCV and HBV might be involved in the development of carcinoma of extrahepatic bile duct.

The Paper Symposium on PLC VIRAL HEPATITIS（HBV AND HCV）BACKGROUND IN CHINESE PATIENTS WITH HEPATOCELLULAR CARCINOMA

Antibody to hepatitis C virus (AntiHCV) was detected using antiHCV EIA (Abbott Kit) in the sera of Chinese patients with hepatocellular carcinoma (HCC), acute hepatitis, chronic hepatitis and blood donors, the positive rates being 10.4% (61/586),11. 8% (10/85),19.2% (44/229), ana 1. 9% (3/160) respectively. HBV DNA was detected by polymerase chain reaction (PCR) in sera from 61 HCC patients with positive antiHCV, the positive rate for HBV DNA being 55.7% (34/61),which was lower than those with negative antiHCV (78.7%) , 413/525). These results indicate that in Chuia the role of HBV infection in the causation of HCC seems to be more important than that of HCV infection.

聚合酶链反应检测血清中HBV DNA的临床意义

我们开发了一种敏感又快速的方法,以检测低水平乙型肝炎病毒(HBV)感染。通过聚合酶链反应(PCR)检测了12例慢性肝炎和52例肝癌病人血清中的HBV DNA。将PCR分析结果与常规血清标志物相比较,并评价检测血清中HBV DNA的临床意义。

肝细胞肿瘤患者癌组织中HBx基因的检测

目的通过检测肝细胞肿瘤（ＨＣＣ）患者癌组织中ＨＢｘ基因探讨乙型肝炎病毒（ＨＢＶ）在 ＨＣＣ中的重要性。方法根据ＨＢｘ基因ＤＮＡ序列设计引物，采用特异性聚合酶链式反应（ＰＣＲ） 对３１例病理确诊并经手术切除后的ＨＣＣ患者癌组织进行了检测，同时采用ＰＣＲ方法检测了同一 患者血清ＨＢＶ ＤＮＡ。结果ＰＣＲ显示３１例ＨＣＣ患者癌组织中ＨＢｘ基因和血清中ＨＢＶ ＤＮＡ 的阳性率分别为７７．４％（２４／３１）和３２．３％（１０／３１）。结论ＨＢｘ基因片段广泛存在于ＨＣＣ患者癌 组织中并且在癌细胞中可能存在整合。

应用原位PCR法检测肝癌组织中的HBV DNA

目的探讨乙型肝炎病毒（ＨＢＶ）与肝细胞癌（ＨＣＣ）的关系。方法采用直接原位聚合酶链反应（ｉｎｓｉｔｕＰＣＲ，ＩＳＰＣＲ），对６４例福尔马林固定石蜡包埋的ＨＣＣ组织（其中６１例附癌周组织）切片中的ＨＢＶＤＮＡ进行检测，并与传统的免疫组织化学原位杂交和组织抽提核酸ＰＣＲ法进行了比较。结果ＩＳＰＣＲ对石蜡包埋的ＨＣＣ组织中的ＨＢＶＤＮＡ检测高度敏感，检出率为７１．９％（４６／６４），而相应组织应用免疫组织化学法（ＨＢｓＡｇ）、原位杂交和组织抽提核酸ＰＣＲ法的结果分别为１７．２％（１１／６４）、５４．７％（３５／６４）和５７．８％（３７／６４）。癌周组织中的ＨＢＶＤＮＡ的检出率明显高于癌组织者，为７８．７％（４８／６１）。ＩＳＰＣＲ检测还显示，ＨＢＶＤＮＡ在肝细胞与癌细胞中主要分布于胞浆，较少见于胞核。在癌组织中，核阳性占４８．４％（３１／６４），癌周组织中则为２４．６％（１５／６１）。结论ＨＢＶＤＮＡ在癌周及癌组织存在情况的复杂性提示ＨＢＶ的感染、整合在ＨＣＣ发生中的作用远非清楚。

PCR技术用于检测肝细胞癌石蜡包埋肝组织中的HBV DNA

用多聚酶链反应(PCR)技术检测了16例肝细胞性肝癌(HCC)患者癌灶和癌旁双份组织石蜡包埋标本中的乙型肝炎病毒脱氧核糖核酸(HBV-DNA)。以C基因引物扩增HBV-DNA，32份标本中检出率71．87％。阻新鲜冷冻肝标本作对照检出率81.25％，二者相比无显著性差别(X^2=0.57，P>0．05)。以质粒提取HBV-DNA作灵敏度测定，PCR-EB可达10fg，PCR-SBH可达1ag。应用非放射性的地高辛素标记探针作杂交灵敏度达^31P标记探针的水平。

肝癌患者HBV血清学标志和HBV-DNA的关系

目的 为了探讨肝癌患者血清中 HBV标志物与HBV- DNA之间的相关性 .方法 运用酶联免疫吸附试验和聚合酶链式反应同时检测血清中 HBV标志物和 HBV- DNA.结果 在 42 7例肝癌患者的血清中 ,HBV- M(+)为 74.0 % ,HBV- DNA(+)为 44 .0 % ,与正常对照组差异非常显著 .

HBV相关肝细胞癌患者HBV前S/S基因变异的分析

目的:研究肝细胞癌(Hepatocellularcarcinoma,HCC)患者体内乙型肝炎病毒(HepatitisBVirus,HBV)前S/S基因变异特点。方法:分别对9例乙型肝炎病毒表面抗原(HBsAg)阳性肝癌患者及其家族中11例HBsAg阳性者血清乙肝病毒前S/S基因PCR扩增﹑克隆﹑测序并进行结构分析,找出基因变异活跃位点,再对两组资料作统计学分析。结果:除对照组中1例未能扩增出前S/S基因,仅能扩增出S基因,其它样本均扩增出前S/S基因片段。所有HBV株均为B或C基因型和adrq+或adw2血清型。肝癌患者来源的HBV在包膜蛋白抗体结合区点突变发生率较高,但与对照组相比,没有统计学差异(P>0.05)。肝癌组4例发生HBV前S区删除变异,具有特异性。与对照组(0/10)相比,有统计学差异(P<0.05)。结论:HBV前S/S区点突变可能与肝癌发生没有相关性,而前S区删除变异可能与肝癌发生有密切联系,应当对HBV感染者进行前S区基因变异监测。

原发性肝癌与HCV、HBV感染关系的探讨

在ELISA的基础上,本文进一步应用PCR技术对广东地区80例PHC、40例非肝癌恶性肿瘤病人(对照组1)、80例正常人(对照组2)进行病例对照研究。结果发现,PHC组中,HBsAg、抗-HBe、HaeAg、HBV-DNA的阳性率分别为77.5％、55.0％、17.5％和40.0％,高于两对照组,抗-HCV及HCV-RNA的阳性率分别为12.5％、13.8％,与对照组1相比无显著的差别,但高于对照组2。HBV感染率(81.3％)显著高于HCV的感染(20.0％)。PHC病人中,若按HBV与HCV感染情况进行年龄分层,发现在HBV与HCV混合感染的PHC病人中,发病年龄有提早发生的倾向。表明,广东地区HCV感染在PHC发生的病因上具有一定的作用,但HBV仍是主要的因素。

原发性肝细胞癌HBx基因缺失突变及意义

作者应用聚合酶链反应技术分析了原发性肝细胞癌45例及其癌旁肝组织中HBc及HBx基因的存在形式.结果84.4％的癌及癌旁组织出现HBc扩增带,其中野生型分别占73.7%和81.6％,突变型分别占26.3％和18.4%.当同时扩增HBc加HBx基因时,癌及癌旁组织阳性率分别为71.1%和68.9%,其中野生型分别为25.0％和29.0％,突变型分别为75.0％和71.0％.表明HBx基因的缺失突变是一频发事件,其突变型和野生型的出现频率与单一扩增HBc基因比较,具有显著差异(P<0.01)、HBx基因的缺失突变,可能更有利于发挥反式激活作用,对于HBV的复制及原发性肝细胞癌的发生具有重要意义.

肝细胞癌病理切片中HBVs基因分析

1目的 探讨乙型肝炎病毒 (HBV) S区在肝细胞癌 (HCC)发病机制中的重要性。 2方法 根据 HBs基因 DNA序列设计引物 ,采用聚合酶链式反应 (PCR)对 14例病理确诊 HCC病理切片中的 DNA进行检测。 3结果 PCR显示 ,14例 HCC病理切片中HBs基因阳性率为 5 7.1% (8/14 )。 4结论 HBVs基因片段在 HCC中广泛存在 ,并可能在

Analysis of point mutation in site 1896 of HBV precore and its detection in the tissues and serum of HCC patients

INTRODUCTION Hepatitis B is one of the common infectious diseases,which severely impairs the health of the people in our country and has close relationship

HBV和HCV感染与原发性肝细胞癌发生的相关性研究

应用聚合酶链反应（ＰＣＲ）技术，检测了２０例肝细胞癌患者肝组织ＨＢＶＤＮＡ和ＨＣＶＲＮＡ。检测发现，８０％的ＨＣＣ癌组织中可检出ＨＢＶ－ＤＮＡ的存在，２５％的病例可以查到ＨＣＶ－ＲＮＡ；所有ＨＢＶ和ＨＣＶ阳性病例均为原发性肝细胞癌，而继发性肝细胞癌病例未能检出二种病毒核酸序列。本研究提示我国引起ＨＣＣ发生的肝炎病毒以ＨＢＶ为主，ＨＣＶ也可能起到一定作用。

ANALYSIS OF POINT MUTATION IN SITE 1896 OF HBV PRECORE AND ITS DETECTION IN THE TISSUES AND SERUM OF HCC PATIENTS

Aim The 3’-base specific polymerase chain reaction (3’- BS- PCR) method was es-tablished to investigate the relationship between the mutation of precore region of Hepatitis B virus(HBV) and the liver damage to the patients caused by HBV and the possibility of HBV precore gene in-tegration in liver cells。 Mdthods According to the DNA sequence of precore region of HBV,themethod of 3’- BS- PCR is applied to analyze the point mutation site 1896 of HBV precore in 126 clini-cal serum specimens and 23 hepatoeellular carcinoma (HCC) patients’ tissues and serum whose trmorshave been surgically excised and pathologically diagnosed.Rdsults The point mutation in site 1896 ofHBV precore has been successfully rates of preore gene of HBV in the 23 patients’ tissues and serum are52.2 % (12/23) and 30.4 % (7/23) respectively.Conclusion The established method for HBV ore-core mutation analysis is simple and results can well repeated.It has provided a new approach to clinicalHBV research and its relationship to liver damage.The results obtained suggested that HBV precoremutation exists in a wide range among serum and tissue of the patients infected by HBV and HCC pa-tients,and the pre-c gene of HBV can not be detected in the serum of 21.8% of the HCC patients(tissue HBV precore gene positive).We may deduce that there may be the integration of HBV precoregenee in the genome of liver cells,which may play an important role in the carcinogenesis of HCC.

血清乙型肝炎病毒RNA定量分析系统的建立

目的 :建立针对血清乙型肝炎病毒 (hepatitisBvirus,HBV)转录体的定量分析系统 .方法 :从血清中提取病毒DNA和RNA ,采用竞争性PCR技术、Southern blotting及分子克隆等技术 ,建立循环病毒DNA和RNA(转录体 )的定量分析系统 ,并应用其对抗病毒药物Lamivudine干预治疗后HBV感染患者血清中HBVDNA和RNA结构和数量的变化进行分析 .结果 :建立了两个针对X区RNA的定量分析系统 ,可检测带有全长型和顿挫型 3′端结构的转录体 .我们还建立了HBVX ,Core和X PreCore区段的DNA/RNA定量分析系统 ,三者分别与HBV基因复制的早、中、晚期对应 .此研究初步阐明了Lamivudine治疗期间患者血清中HBVDNA和转录体结构和数量的变化 .治疗 8wk以后 ,在Core和X PreCore区DNA拷贝数从 10 9·mL-1下降为 10 5·mL-1,下降幅度明显高于X区(下降至 10 7·mL-1) ,其比率更准确地反映了Lamivudine的作用效率 ,可用于疗效的评估 .转录体的拷贝数仅有小幅下降 ,采用锚定的Oligo(dT)引物检测的两种全长和顿挫型Ploy(A)RNA (10 5·mL-1)明显低于X区 (10 7·mL-1)RNA的拷贝数 ,提示在基因复制过程中前基因组RNA的Ploy(A)尾被去除 .结论 :这一分析系统可显示血清中HBV转录体含量及定量不同 3′端结构 。

肝癌组织内乙型肝炎病毒基因组前C区终止密码的发现

用聚合酶链反应(PCR)在3例乙型肝炎病毒(HBV)C基因阳性的原发性肝癌(HCC)组织中扩增和克隆了HBV C基因片段(部分前C区和全部C区),获得了重组质粒pHBC_1,pHBC_2,pHBC_3。将重组质粒中HBV C基因亚克隆至M_(13)DNA序列测定系统,用双脱氧法测定了HBV C基因片段的核苷酸序列。在2例HCC中,发现前C区第1898位核苷酸点突变,结果形成了终止密码。在3例HCC中,C区也发现有核苷酸点突变,其中部分可致编码氨基酸改变。