乙型肝炎病毒前S2区缺失突变与肝癌相关性研究

目的研究肝细胞肝癌（hepatocarcinoma,HCC）患者乙型肝炎病毒（Hepatitis B virus,HBV）前S2区缺失突变情况。方法应用PCR法扩增HBV前S区基因,应用DNAMAN软件对所测基因进行分析。结果 23.08%（6/26）的肝癌患者发现存在preS2区的缺失突变,缺失位点为nt 3223～nt 3268,缺失长度为30～45 bp。肝癌患者缺失突变发生率显著高于HBV无症状携带者。结论 HBV preS2区的缺失突变可能与肝癌的发生密切相关,该突变的发生机制仍有待进一步研究。     

乙型肝炎病毒X蛋白氨基端变异与肝癌发生的相关性分析

目的:探讨乙型肝炎病毒X蛋白(hepatitis B virus X protein, HBx)氨基端1～90位氨基酸(amino acid, AA)变异与肝细胞癌发生之间的关系.方法:采用PCR扩增产物直接测序的方法,对48例肝癌组织、159例肝癌和144例慢性肝炎患者血清中HBV X基因进行序列分析.通过病例-对照研究HBx变异与肝癌发生之间的关系.结果: 肝癌组织中HBx氨基端变异集中于1～49 AA区域,突变率为4.1%; 50～90 AA为保守区,突变率仅为0.46%. 血清检测结果显示,HBx氨基端突变在肝癌患者中的发生率显著高于肝炎患者(2.2% vs 1.8%,P<0.05); HBx第36位Ala/Thr/Pro→Ser的突变,在肝癌患者中的发生率为10.1%,显著高于肝炎患者中的2.1%[P<0.01,比数比(odds ratio,OR)=5.259, 95%可信区间为1.499～18.444)].A/T/P36S突变仅发生于C基因型病毒.结论:HBx 氨基端突变的累积可能与肝癌的发生有关.HBx A/T/P36S突变能增加C基因型HBV感染者发生肝癌的危险度.     

广西扶绥地区乙型肝炎病毒基因型分布的概况

目的研究广西扶绥地区乙型肝炎(简称乙肝)患者HBV基因型分布特点,为本地区乙肝的防治及临床抗乙肝感染的治疗提供依据。方法采用巢式PCR技术对285例(肝癌40例,乙肝病毒携带者245例)扩增HBV S区基因,经测序后与标准序列比对,得出相应的基因型,分析HBV基因型的分布概况及其特点。结果在285例患者中,B基因型59例(20.7%),C基因型223例(78.2%),未分型3例(1.1%)。在肝癌和乙肝病毒携带者中,B基因型、C基因型分别为10例(25.00%)、30例(75.00%)和49例(20.25%)、193例(79.75%)(P>0.05);男性B基因型、C基因型分别为40例、158例,女性B基因型、C基因型分别为19例、65例,HBV基因型在男性和女性间的分布差异均无统计学意义(P均>0.05);在<30岁、30~40岁、40~55岁、>55岁各年龄组间HBV基因型的分布差异均无统计学意义(P均>0.05)。结论扶绥地区HBV基因型以C基因型占主要优势,其次是B基因型,未见其他分型;HBV基因型在年龄、性别间的分布无明显差异。     

启东肝癌高发区乙肝病毒流行株全基因分析

目的：通过对江苏启东地区乙型肝炎病毒（HBV）全基因序列的分析，探讨该地区肝癌高发的分子病毒学病因。方法：以蛋白酶K消化后，酚／氯仿抽提7例肝炎和7例肝癌患者血清中DNA。应用聚合酶链反应（PCR），扩增血清中HBV基因全长，克隆至T载体后行全自动测序。以PHYLIP软件构建的系统进化树判断HBV的基因型；以Clastal W软件对序列进行突变分析。结果：14例标本中有12例为C基因型，2例为B基因型。肝癌和肝炎组中HBV基因型类别分布无差异。有5例HBV发生了PreS2的缺失突变，其中肝癌标本4例（57．1％），肝炎标本仅1例（14．3％）。HBV基因组中常见的点突变为PreS1区的nt．3116及核心启动子区的nt．1762／1764，发生率高达78．6％（11／14）。与肝炎组相比，肝癌中点突变发生率显著增高的位点为前C区nt．1899G→A的突变（P=0．01）及核心启动子区nt．1653C→T的突变（P=0．05）。结论：启东地区HBV以C基因型为主；启东HBV基因组中可能存在着与肝癌相关的点突变和缺失突变。     

广西贵港市肝癌筛查及HBV基因型分布现况调查

目的 探讨广西贵港市防癌筛查对象的乙型肝炎病毒(hepatitis B virus,HBV)感染状况及HBV基因型的分布特征。方法 分析贵港市3个城区接受肝癌早诊早治筛查的30~59岁共67.75万居民的基本情况,根据HBsAg阳性筛查肝癌高危人群(HBsAg阳性人群),从中按地区以分层抽样的方法选取1 989例高危对象,进行HBV DNA定量检测,以选取HBV DNA病毒拷贝数大于检出限(1×103)的HBsAg携带者,采用PCR-RFLP进行HBV基因分型。结果 筛查人群中HBsAg阳性率为11.99%,其中男性HBsAg阳性率高于女性(P<0.01),不同年龄组HBsAg阳性率差异有统计学意义(P<0.01),HBsAg阳性率随年龄的增长呈下降趋势。在肝癌高危人群中,HBV DNA病毒拷贝数≥1×103 755例,其中HBV A基因型6例(0.79%)。B基因型337例(44.64%)。C基因型293例(38.81%)。D基因型18例(2.39%)。UT基因型101例(13.37%),B和C基因型在男女性中的分布差异无统计学意义(P>0.05);各年龄组间HBV 基因型的分布差异亦无统计学意义(P>0.05)。结论 广西贵港市3个城区肝癌早诊早治筛查人群的HBV感染率较高,HBV基因型以B和C基因型为主,肝癌早诊率较低,需进一步完善肝癌早诊早治技术方案和管理模式,提高筛查和早诊早治效果。     

慢性HBV感染肝组织的C端截短型HBx蛋白功能研究

[目的]探讨HBx突变体在肝细胞肝癌发病机制中的作用。[方法]收集慢性HBV感染者肝穿组织标本和HBV阳性的肝细胞肝癌组织标本，提取DNA。通过3D-PCR方法扩增HBx基因并测序检测HBx基因的突变。分析两种组织标本内HBx基因的突变模式，以及突变对HBx蛋白序列的影响。进而构建所检测到的HBx突变体的真核表达质粒，体外研究其生物学活性。[结果]在所检测的7例慢性HBV感染者肝穿标本内，有3例标本内检测到因G到A突变而产生的C端截短型的HBx突变体。在所检测的2例HBV阳性的肝细胞肝癌组织内，也检测到相似的C端截短型HBx突变体。体外研究这种C端截短型HBx突变体的生物学功能发现其具有促进细胞增殖的功能。[结论]C端截短型HBx突变体在HBV相关的肝细胞肝癌发病机制中具有重要意义。     

广西HBV相关肝细胞癌及其癌旁组织中HBV S基因突变分析

目的 了解广西乙型肝炎病毒（hepatitis B virus, HBV）相关肝细胞癌 (hepatocellular carcinoma,HCC)组织及其癌旁组织中HBV S基因突变分布状况。方法 收集2015—2016 年于广西医科大学附属肿瘤医院手术切除的43例HBV相关HCC患者癌组织及其癌旁组织。采用巢式PCR扩增HBV S基因片段并测序,Chromas和DNAMAN软件分析S基因序列。结果 肝细胞癌患者HBV S基因热点突变在癌组织及癌旁组织中的分布差异无统计学意义（P>0.05）;小分子蛋白（small hepatitis B surface antigen, SHBs）终止突变只存在于癌组织中,突变位点分别为sC69*、sW74*、sL94*、sW172*和sW182*。结论 广西HBV相关HCC癌组织及其癌旁组织HBV S基因热点突变分布无特异性,SHBs终止突变可能影响HCC发生发展。     

肝癌患者乙型肝炎病毒全基因组结构分析

目的 研究肝细胞癌患者乙型肝炎病毒(hepatitis B virus,HBV)全基因组的结构及特点。方法 PCR扩增并克隆乙型肝炎病毒表面抗原(HBsAg)阳性肝癌患者血清中HBV全基因组DNA,测序并进行基因结构分析。结果 获得不同肝癌患者来源的22株HBV全基因组DNA,均属于C或B基因型和adr或adw2血清型。与标准株相比,肝癌患者来源的HBV在表面抗原、核心抗原的B／T细胞表位、X蛋白的反式激活区及增强子Ⅱ、核心启动子区发生了一些有意义的共有变异。结论 HBV的基因型与基因变异可能与HBV相关性肝癌的发生发展有关。     

反向杂交检测肝癌相关乙型肝炎病毒前C区突变方法的建立和应用

背景与目的：日益增多的研究表明,乙型肝炎病毒(hepatitis B virus, HBV)DNA前C区G1896A和G1899A突变是肝癌发生的危险因素。本研究旨在建立简单、快速、灵敏和准确的检测HBV前C区突变的反向杂交方法,并应用于检测江苏省启东地区HBV DNA前C区突变与肝癌发生的关系。方法：设计并合成HBV DNA前C区1896和1899位点的特异性探针,通过优化条件建立特异、敏感的杂交体系,并与直接测序检测结果进行比较。将该方法应用于检测启东100例肝癌和100例慢性HBV携带者(对照组),分析HBV DNA前C区突变与肝癌的关系。结果：反向杂交对血清样本的最低检测下限为HBV DNA 103 copy/mL,检测混合感染时比直接测序更占优势,混合株中10％以上的突变株均可被检测。启东地区HBV DNA前C区G1899A突变与肝癌高发具有相关性(P=0.000,OR=4.846, 95%CI：2.240～10.485),而G1896A突变未见其相关性。结论：反向杂交检测HBV DNA前C区突变方便、快速、准确,可有效监控肝癌的发生,适合临床大规模推广应用。     

Pre-S2 deletion mutants of hepatitis B virus could have an important role in hepatocarcinogenesis in Asian children

Although many studies on the risk factors and their carcinogenesis in adult hepatocellular carcinoma (HCC) have been reported, they remain poorly understood in childhood HCC. A retrospective study of 42 HCC cases in Asian children was conducted. Hepatitis B virus (HBV)-DNA in HCC tissues was detected in 36 of 42 (86%) cases tested, while no hepatitis C virus (HCV)-RNA was detectable in any of HCCs. Twenty of 36 (56%) HCC cases were accompanied by cirrhosis. Surprisingly, very high prevalence of the HBV pre-S deletion mutant was recognized in 27 of 30 (90%) HCCs examined. They occurred most frequently in pre-S2 (20/27, 74%) followed by pre-S1 (5/27, 18.5%), and both pre-S1/S2 (2/27, 7.4%). Interestingly, the pre-S2 mutant consistently appeared with deletion at nt 4-57 in all of the 20 cases with the pre-S2 mutant (100%) and within this locus in the two cases with both pre-S-1/S2 mutants. Type II ground-glass hepatocytes in non-tumorous livers were seen in 15 of the 22 HCCs with the pre-S2 deletion mutant (68%). This hotspot mutation in the pre-S2 was further confirmed by complete genomic sequence of HBV in a Japanese boy who eventually developed HCC. Our result strongly suggests that HBV is a major contributor to the development of HCC in Asian children. The HBV pre-S2 deletion mutant at nt 4-57 which has a CD8 T-cell epitope could be responsible for the emergence and aggressive outcome of childhood HCC. Determination of this hotspot mutation in the pre-S2 region could be a useful index for predicting the clinical outcome of HCC development. ( Cancer Sci 2009; 100: 2249–2254)     

Pre-S mutant surface antigens in chronic hepatitis B virus infection induce oxidative stress and DNA damage

Ground glass hepatocytes (GGHs) are the historic hallmarks for the hepatocytes in the late and non-replicative stages of hepatitis B virus (HBV) infection. We have identified type I and type II GGHs that contain two mutant types of large HBV surface antigens (HBsAg) with deletions over the pre-S1 and pre-S2 regions, respectively. These pre-S mutant HBVsAg accumulate in endoplasmic reticulum (ER), resulting in strong ER stress. Type II GGHs often appear in hepatic nodules in the late phases of HBV infection and proliferate in clusters, suggesting that these mutant pre-S1/S2 HBsAg may be involved in HBV-related hepatocarcinogenesis, associated with ER stress. In this study, we investigated the potential genomic instability imposed by pre-S mutant HBsAg. Based on the analysis of comet assays, we found that the pre-S1 and pre-S2 mutant HBsAg caused oxidative stress and DNA damage. The DNA repair gene ogg1 was greatly induced by over-expression of pre-S mutant HBsAg. Induction of the DNA repair gene ogg1 was also detected in the pre-S2 HBsAg transgenic mice, as well as the type II GGHs from patients with hepatocellular carcinoma, strongly suggesting that the pre-S mutant HBsAg contributes to the oxidative DNA damage to hepatocytes. In addition, the mutation rates in the X-linked hprt gene were enhanced in mouse hepatoma ML1-4a cells, which constitutively expressed the pre-S1/S2 HBsAg. These results indicate that pre-S1/S2 mutant HBsAg, which make up GGHs, induce oxidative DNA damage and mutations in hepatocytes in the late stages of HBV infection.     

肝细胞癌患者B和C基因型乙型肝炎病毒X蛋白的变异特点

背景与目的：X蛋白是乙型肝炎病毒(hepatitis B virus,HBV)最重要的致病因子之一,它在HBV相关性肝细胞癌(hepatocellular carcinoma,HCC)的发生发展中起着重要作用。目前已知B、C基因型HBV相关性HCC的临床及病理表现不尽相同,但目前尚不清楚这种差别是否与B、C基因型HBV X基因之间的差别有关。因此本实验拟研究B、C基因型间HBV X蛋白氨基酸差异及其在HCC患者中的变异及特点,初步探讨其与HCC发生、发展的关系。方法：PCR扩增22份乙型肝炎病毒表面抗原(HBsAg)阳性HCC患者血清HBV X基因,克隆、测序并以Vector NTI6．0软件分析其基因型。以DNAMAN软件对标准HBV及HCC来源的HBV X蛋白进行氨基酸同源分析。结果：检测的22个HBV X基因片段均属于B或C基因型。B、C基因型HBV X蛋白之间存在14个氨基酸的差异,HCC患者来源的B、C型HBV X蛋白存在4个氨基酸的共有变异,C型HBV X蛋白尚有6个型特异性变异。这些差异或变异氨基酸均位于X蛋白B、T细胞表位或反式激活区及调节区内。结论：B、C基因型HBV X蛋白之间存在氨基酸差异,且在HCC中发生基因型特异性变异,这些差异或变异氨基酸可能导致X蛋白免疫学功能及反式激活功能的不同。     

中国高发区肝细胞癌中HBVx基因的广泛存在及与p53基因249密码子高 …

目的 乙肝病毒（ＨＢＶ）是肝细胞癌（ＨＣＣ）的主要病因。ＨＢＶ中的ｘ基因被认为是它的癌基因。为确定ｘ基因在肝癌癌变中的重要性,我们主要针对ＨＢｓＡｇ阴性的ＨＣＣ,研究ＨＢＶｘ基因的存在、表达和突挛情况;同时也对ｘ基因与Ｐ５３基因２４９密码子热点突变的相关性进行了分析。方法 应用ＰＣＲ、ＲＴ－ＰＣＲ、ＰＣＲ－ＲＦＬＰ和ＤＮＡ序列分析方法,重点针对１９９１年－１９９６年在启东北京手术的的、病理确诊为Ｈ     

CYP2C19 Genotype Could be a Predictive Factor for Aggressive Manifestations of Hepatocellular Carcinoma Related with Chronic Hepatitis B Infection in Thailand

Background: Chronic hepatitis B virus (HBV) infection related hepatocellular carcinoma (HCC) is a majorhealth problem in the Asia-Pacific region including Thailand. Several factors have been proposed as contributingto hepatocarcinogenesis. This study was aimed to investigate the impact of CYP2C19 genotypic polymorphismin HCC related to chronic HBV infection in Thailand. Materials and Methods: A cross-sectional study wasperformed between April 2014 and January 2015. Chronic HBV patients with HCC (n=50) and without HCC(n=50) were included. Clinical information and blood samples of all patients were collected. The CYP2C19genotype was determined by polymerase chain reaction-restriction fragment length polymorphism method, andwas classified as rapid metabolizer (RM), intermediate metabolizer (IM) or poor metabolizer (PM). Results:The CYP2C19 genotype frequencies of RM, IM and PM in HBV patients were found to be 19/50 (38%), 25/50(50%) and 6/50 (12%), respectively. The CYP2C19 genotype frequencies of RM, IM and PM in HBV with HCCpatients were 21/50 (42%), 25/50 (50%) and 4/50 (8%), respectively. The distribution of CYP2C19 genotype wasnot different between patients with and without HCC. Interestingly, among HBV with HCC patients, the RMgenotype of CYP2C19 tended to increase risk of aggressive manifestation (OR=2.89, 95%CI=0.76-11.25, P-value= 0.07), compared with non RM genotype carriers. Conclusions: CYP2C19 genotype IM was the most commongenotype in Thai patients with chronic HBV infection. In addition, genotype RM could be an associated factorfor aggressive presentation in HCC related to chronic HBV infection.     

Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis and hepatocellular carcinoma

Hepatitis B virus (HBV) genotype C and the basic core promoter (BCP) mutations were reported to be associated with the development of hepatocellular carcinoma (HCC). In this study the full sequences of HBV genomes were analyzed in order to find the other predictors of HCC development. We determined the full sequences of HBV genomes in 24 genotype C carriers who developed HCC (HCC group) at the beginning of follow-up and at the time of HCC diagnosis, and 20 patients who did not develop HCC (non-HCC group) served as a control. The number of nucleotide and amino acid substitutions in most regions was higher in the HCC group than in the non-HCC group, and the following substitutions and deletions were found more frequently in the HCC group than in the non-HCC group: G1317A and T1341C/A/G in the X promoter region were detected in 13 and six of the HCC cases, four and none of the non-HCC cases, respectively; and pre-S2 deletion was detected in eight HCC and none of the non-HCC cases. Compared with the wild type X promoter, the mutant type X promoters, M1 (G1317A), M2 (T1341C), and M4 (T1341G) showed increases in activity of 2.3, 3.8, and 1.4 times, respectively, in HepG2 cells. Substitutions and deletion of nucleotides of the HBV genome, especially the pre-S2 deletion and G1317A and T1341C/A/G mutations may be useful markers for predicting the development of HCC. ( Cancer Sci 2007; 98: 1921–1929)