肝细胞癌复发或转移与HBV DNA水平及其基本核心启动子区1762/1764双突变的关系

目的 探讨肝细胞癌(HCC)复发或转移与HBV DNA水平及其基本核心启动子(BCP)区1762/1764双突变的关系. 方法 选择163例HCC患者进行120周的随访,收集患者一般资料、生物化学指标、瘤体资料、HBV病毒学指标等,用实时定量PCR检测HBV DNA水平,基因测序法检测BCP区1762/1764双突变情况,并用logistic回归分析HCC复发或转移的发生率与这些资料的关系.两均数比较采用两独立样本t检验;多个样本均数比较采用方差分析;计数资料采用x2检验;筛选影响因素采用二分类logistic回归分析. 结果 共有157例患者进入最终的有效追踪.110例在手术后或肝动脉栓塞化疗术(TACE)后2年内出现肿瘤的复发或转移,其发生率随着时间的推移而逐渐上升,在12、24、48、72、96周和120周分别为2.55％(4例)、8.92％ (14例)、17.83％ (28例)、40.76％ (64例)、58.60％ (92例)和70.06％(110例).对基线资料的单因素分析显示血清高水平Y-谷氨酰转移酶、无包膜瘤体、门静脉栓塞、较大肝癌、BCP变异、HBV DNA水平与HCC的高复发或转移率有关.多因素分析显示HBV DNA水平和BCP区1762/1764双突变与HBV相关性HCC复发和转移高度相关.复发或转移率随着HBV DNA水平的上升而增加,在基线HBV DNA水平＜3log10拷贝/ml的HCC患者中,肿瘤的复发或转移率为42.1％ (8/19);在基线HBV DNA水平≥5log10拷贝/ml的HCC患者中,肿瘤的复发或转移率升为87.0％ (67/77,x 2=22.308,P＜0.01).在157例HCC患者中,BCP区1762/1764双突变阴性43例,有22例(51.2％)发生肿瘤的复发或转移;BCP区1762/1764双突变阳性114例中有88例(77.2％)发生肿瘤的复发或转移(x2=6.022,P＜0.05).通过logistic回归分析,显示BCP区1762/1764双突变阳性患者较双突变阴性患者有更高的HCC复发或转移率(OR=5.264,95％可信区间为1.436～ 12.574).结论 HBV BCP区1762/1764双突变和基线HBV DNA水平与HBV相关性HCC复发和转移相关.     

肝细胞癌组织中HBV基因型的检测及分析

目的 了解肝细胞癌组织中HBV基因型分布状况及特点.方法 应用型特异性引物分型法和基因测序与生物信息学相结合的分型方法分析肝细胞癌组织中HBV基因型,并与HBV携带者血清中HBV基因型进行比较.结果 在63份肝细胞癌组织标本中,B、C、混合型B C和混合型B D基因型分别占20份(31.8%)、37份(58.7%)、4份(6.3%)和2份(3.2%).与HBV携带者血清相比,两者基因型构成比无显著性差异(P＞0.05).结论 中国南部地区的肝细胞癌组织中存在HBV基因型B、C、混合型B C和混合型B D,以C基因型为主,其次为B基因型.肝细胞癌组织中HBV基因型分布与当地HBV携带者基因型密切相关.     

HBV基因型和HBV核心启动子双点突变与肝损伤的关系探讨

目的 了解HBV基因型在荆州地区的流行病学状况；探讨HBV基因型和HBV核心启动子双点突变与肝损伤的相关性。方法 采用PCR微板核酸杂交-ELISA技术，对临床诊断为不同程度的乙型肝炎患者血清中的HBV DNA进行基因分型和核心启动子双点突变检测。结果本地区基因型以B型和D型为主，分别为31．4％和24．4％。其它为：A型3．5％，C型18．6％，B、C混合型11．6％，B、D混合型10．5％。结论 HBV基因型在本地区分布有其独特之处，HBV核心启动子双点突变在C型发生频率高于B型，有显著性差异，但与D型比较无显著性差异。HBV基因型及HBV核心启动子双点突变与肝损伤彼此间无明显相关性。肝损作可能主要与机体免疫等相关，HBV变异仅起次要作用。     

广西慢性乙型肝炎患者HBV核心基因启动子突变的研究

目的 了解HBV核心基因启动子突变与肝损害程度或HBeAg状态的关系。方法 用套式PCR扩增59例慢性乙型肝炎患者血清HBV核心基因启动子,阳性者用直接测序法检测。结果35例HBV DNA阳性,阳性率为59.3％。无正常序列标本,最常见的突变类型是nt1762、1764发生双突(A→T、G→A),占57.1％;其次为nt1799位点突变,由C→G,占54.4%,为无义突变;nt1752位点突变,由A→G,使该密码由异亮氨酸变为缬氨酸,占37.1％;nt1753T→C,占20.0%。T_(1762)A_(1764)突变株在HBeAg阳性、阴性患者组中的分布分别为31.3％、79.0％,两者差异有显著性,x~2 8.068 8,P<0.05。结论 HBV核心基因启动子突变在广两慢性乙型肝炎患者较常见,T_(1762)A_(1764)突变株与HBeAg阴性及慢性肝炎有关。     

乙型肝炎病毒基本核心启动子和前C区突变与肝硬化肝细胞癌和非肝硬化肝细胞癌发生的关系

目的 研究慢性HBV感染者抗病毒治疗前HBV基本核心启动子(BCP)和前C区突变与肝细胞癌发生的关系.方法 收集2003年1月至2010年12月浙江省上虞市人民医院感染疾病科门诊和住院收治的慢性HBV感染患者,其中慢性乙型肝炎166例(对照),肝硬化肝细胞癌患者158例和非肝硬化肝细胞癌患者57例,采用PCR扩增后直接测序法检测HBV BCP和前C区突变,同时确定基因型.数据采用x2检验及Logistic回归分析.结果 患者以HBV基因B型为主,其中慢性乙型肝炎有124例,肝硬化肝细胞癌有126例,非肝硬化肝细胞癌有50例.以慢性乙型肝炎患者作为对照组,在单变量分析中BCP V1753突变(x2=7.927,P=0.005)、BCP T1762/A1764双突变(x2=12.796,P＜0.01)、前C区A1896突变(x2=6.890,P=0.009)和前C区A1899突变(x2=11.850,P=0.001)与肝硬化肝细胞癌的发生有关;前C区A1896突变(x2 =27.310,P＜0.01)和前C区A1899突变(x2=7.575,P=0.006)与非肝硬化肝细胞癌的发生有关.多因素Logistic回归分析发现,在HBeAg阳性患者中,BCP T1762/A1764双突变(wald=6.180,P=0.016,OR=8.883)和前C区A1899突变(wald=10.279,P=0.001,OR=7.475)是肝硬化肝细胞癌发生的危险因素;前C区A1896突变(wald=4.324,P=0.038,OR=4.439)和前C区A1899突变(wald=4.850,P=0.028,OR=6.010)是非肝硬化肝细胞癌发生的危险因素.在HBeAg阴性患者中,仅前C区A1896突变(wald=15.448,P＜0.01,OR=12.128)是非肝硬化肝细胞癌发生的危险因素.结论 BCP T1762/A1764双突变与HBeAg阳性患者的肝硬化肝细胞癌发生有关,前C区A1896突变与HBeAg阳性和阴性患者的非肝硬化肝细胞癌发生有关,前C区A1899突变与HBeAg阳性患者的肝硬化肝细胞癌和非肝硬化肝细胞癌发生有关.     

HBV基因型F1b核心突变和阿拉斯加原住民肝细胞癌的最新关联

正【据《Hepatology》2018年6月报道】题:HBV基因型F1b核心突变和阿拉斯加原住民肝细胞癌的最新关联(作者Hayashi S等)HBV基因型F1b感染与年轻阿拉斯加原住民(AN)的肝细胞癌(HCC)密切相关。然而,基因型F1b引起HCC的机制尚不清楚。该研究分析了来自20例HBV感染的HCC患者的长期系列样品中基因型F1b的临床和     

肝细胞癌组织中HBV、HCV和HGV感染的研究

为了从组织学角度探讨乙型肝炎病毒 (ＨＢＶ)、丙型肝炎病毒 (ＨＣＶ)及庚型肝炎病毒 (ＨＧＶ)与肝细胞癌 (ＨＣＣ)之间的相互关系 ,我们采用免疫组织化学法 ,以抗 ＨＢｓ,抗 ＨＣＶＮＳ5及抗 ＨＧＶＮＳ5 3种单克隆抗体 (单抗 ) ,对 2 6例ＨＣＣ组织中的ＨＢＶ、ＨＣＶ及ＨＧＶ的表达和分布情况进行了研究。材料和方法一、组织来源ＨＣＣ组织均来源于中山医科大学附属肿瘤医院 1998～ 1999年 2 6例原发性肝癌患者手术切除后的石蜡存档标本。其中男 2 4例 ,女 2例 ,年龄 2 5～ 77岁 ,平均年龄 47岁 ,所有标本均经病理确诊。二、免疫组织…     

原发性肝细胞癌相关HBV G588C突变的功能研究

目的探索乙型肝炎病毒(hepatitis B virus,HBV)G588C突变在原发性肝细胞癌(HCC)组织HBV cccDNA中的存在情况及其对HBV复制的影响。方法通过滚环扩增(rolling cycle amplification,RCA)和PCR扩增测序,寻找G588C突变在18对HCC患者癌与癌旁组织中的存在情况。在HBV 1.2×质粒(HBV C基因型)的基础上构建G588C突变位点,将G588C突变质粒与野生型质粒分别转染HepG2和Huh7肝癌细胞系细胞,检测上清和细胞中HBsAg含量及上清中HBV DNA水平。结果在18对癌与癌旁组织中G588C突变只存在于3例癌组织中;G588C突变组上清中HBsAg 3、4d含量分别为HepG2细胞5.605±1.182,8.270±2.241,Huh细胞2.714±0.371,10.148±2.793,细胞内HBsAg 3、4d含量分别为HepG2细胞4.852±1.024,7.736±1.762,Huh细胞18.349±3.040,34.110±2.129;野生型组上清中HBsAg 3、4d含量分别为HepG2细胞8.083±1.428,13.170±0.938,Huh细胞6.231±0.373,23.971±1.573,细胞内HBsAg 3、4d含量分别为HepG2细胞2.937±0.876,4.270±1.659,Huh细胞13.498±3.06,21.010±3.488。G588C突变组上清中HBsAg含量显著低于野生型组(F=44.88,P0.01),G588C突变组细胞内HBsAg含量显著高于野生型组(F=34.65,P0.01);G588C突变组上清和细胞中HBsAg总量与HBV 1.2×野生型组比较差异无统计学意义(F=7.69,P0.05);G588C突变组上清中HBsAg与细胞内HBsAg的比值显著低于HBV 1.2×野生型组(F=23.59,P0.05),HBV DNA水平与两组间差异无统计学意义(F=6.23,P0.05)。结论 G588C突变不影响HBV的复制,但影响HBsAg由细胞内向细胞外的分泌,这可能与HCC的发生发展有关。     

在感染乙肝病毒亚基因型C2的患者中X和核心启动子突变对肝细胞癌的影响

在HBV基因组中HBV基因型／亚基因型及其相关突变已有报道与肝细胞癌（HCC）有关。为了确定HBV基因组中完全X、核心启动子、前核心／核心区与HCC相关的突变，本项研究对感染过HBV C2且患有HCC的80例日本患者与无HCC且在年龄、性别和乙肝e抗原（HBeAg）状态均匹配的80例患者中进行了交叉层面上的对照性比较。HBeAg阳性组（HCC占31例；无HCC占29例）和HBeAg阴性组（HCC占49例；无HCC占51例）在年龄和性别上也是匹配的。     

HBV相关肝细胞癌防治进展

HBV相关肝细胞癌(HCC)占目前中国大陆HCC总数的63.9%,HBV通过直接和间接致癌作用2个途径导致HCC产生或复发,理论上抗病毒治疗可以减少或推迟HCC的发生。核苷(酸)类似物(NAs)具有明确的循证医学证据可以减少HCC发生,最新研究提出聚乙二醇干扰素(PEG-IFN)的二级预防作用优于NAs;数据显示NAs可以有效降低治愈性手术后HCC的累积复发率,而IFN的应用可以延长患者的总生存期。HBV相关HCC的二级/三级预防应重视以下2点:重视IFN/PEG-IFN在慢性乙型肝炎患者中的合理应用,将HBV DNA20 IU/ml和HBs Ag1000 IU/ml作为治疗的长期控制目标。在抗病毒时代应个体化应用2类抗病毒药物,把HCC的发生率和复发率降到最低。     

Optimization of competitively differentiated polymerase chain reaction in detection of HBV basal core promoter mutation

AIM: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation. METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control. To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CDPCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to 10μmol/L and mutual primer to about 100 nmol/L. Optimized CD-PCR could detect both mutant and wild strain independent of the amount of templates and the number of PCR cycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.     

Optimization of competitively differentiated poiymerase chain reaction in detection of HBV basal core promoter mutation

AIM: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation. METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control. To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CD-PCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to 10μmol/L and mutual primer to about 100μmol/L Optimized CD-PCR could detect both mutant and wild strain independent of the amount of templates and the number of PCR cycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.     

Double point mutation in the core promoter region of hepatitis B virus (HBV) genotype C may be related to liver deterioration in patients with chronic HBV infection

BACKGROUND AND AIM: Hepatitis B virus (HBV) genotype C has a more severe pathogenesis than genotype B in Japan. We retrospectively investigated the relationship between HBV genotype and the core promoter (CP) (nt 1762 and 1764) and precore (PreC) (nt 1896) mutations of the HBV genome. METHODS: A total of 129 Japanese patients (42 genotype B and 87 genotype C) with chronic HBV infection, living in two different geographical areas in Japan, were evaluated (mean follow-up period 10.1 +/- 3.8 years). In 2000, CP and PreC HBV mutations were analyzed by direct sequencing from sera. Hepatitis B e antigen (HBeAg), HBV DNA and serial alanine aminotransferase (ALT) changes were followed and determined using serological methods. RESULTS: Genotype C patients had significantly higher rates of HBeAg (40.2%vs 2.4%), HBV DNA positivity (75.9%vs 7.1%) and ALT abnormality (71.3%vs 11.9%) than genotype B patients (all P < 0.05). Among genotype B patients, CP wild type (92.9%) was predominant and PreC mutation (88.1%) was predominant. However, among genotype C patients, CP mutation (75.9%) was predominant and PreC mutation (66.7%) was predominant. The CP mutation was found significantly more in genotype C than in genotype B (P < 0.05). Of the 67 patients with ALT abnormality, five (7.5%) genotype B and 62 (92.5%) genotype C patients (31 HBeAg positive and 31 negative) were found. Among the 31 genotype C patients who were HBeAg positive, the combination of CP mutation and PreC wild (54.8%) was predominant, while among the remaining 31 genotype C patients who were HBeAg negative, the combination of CP mutation and PreC mutant (71.0%) was predominant. CONCLUSION: Genotype C might be one of the worse prognostic markers in patients with chronic HBV infection, possibly because of mutation in the CP region.     

Virological analysis, genotypes and mutational patterns of the HBV precore/core gene in HBV/HCV-related hepatocellular carcinoma

We investigated the replicative profile of hepatitis B (HBV) and hepatitis C (HCV) viruses and the mutational pattern of the HBV precore/core (pre-C/C) domain in hepatocellular carcinoma (HCC). Thirty-eight consecutive patients with HCC were included in the study - 18 of them with HBV/HCV co-infection and 20 with HBV single infection. Twenty-three additional patients with co-infection, without HCC were recruited as the control group. Replication activity was evaluated by detecting and quantitating both HBV and HCV genomes. The HBV pre-C/C region, encompassing the pregenome encapsidation signal involved in viral replication, was analysed by direct sequencing. HBV viraemia levels were significantly lower (P = 0.04) in patients with co-infection in comparison with single-infected HCC, whereas two different HBV viraemia profiles were detected in co-infection with or without circulating HCV. HBV genotype D was prevalent in the three groups and HCV genotype 1b was found to be the infecting strain in all patients. Lower variability in the pre-C/C region was found in co-infection in comparison with HBV single infection (P = 0.0004). A synonymous T1936C mutation was found in all co-infected HCC cases not related to the presence or absence of circulating HCV, and a hypermutated pre-C strain, characterized by the same mutational pattern, was identified in three HCC cases. The mutational pattern of the pre-C/C region was closely related to HBV replication efficiency, and specific HBV mutations selectively associated with HCV co-infection could be linked with accelerated HBV/HCV-related disease progression.     

HBV C基因基本核心启动子变异对乙型肝炎患者血清病毒复制和细胞因子水平的影响

目的 探讨乙型肝炎病毒（HBV）DNAC基因基本核心启动子（BCP）变异对机体免疫状态及乙型肝炎病毒复制的影响。方法采用PCR微板核酸杂交结合ELISA技术检测HBVDNABCP变异；采用双抗体夹心ELISA法检测患者血清IL-2、IFN-γ、IL-4和IL-10水平；采用荧光定量PCR法测定HBVDNA水平。结果HBVDNABCP变异病人和非变异病人血清IL-2水平分别为61．4±24．7ng／L和65．1±25．3ng／L，IFN-γ为82．0±50．1ng／L和71．8±67．0ng／L，IL-4为62．3±46．0ng／L和59．4±51．0ng／L．IL-10为74．0±88．2ng／L和81．4±67．0ng／L（P均〉0．1）；HBVDNABCP变异病人的HBVDNA水平为1×10^5.82±2.01 copies／ml，明显高于非变异病人的1×10^4.71±1.78 copies／ml（P〈0．01）。结论HBVDNABCP变异对机体血清细胞因子水平无明显影响，但对HBVDNA的复制具有一定的促进作用。     

慢性重型乙型肝炎患者HBVDNA前C／BCP区突变基因分析

目的分析慢性重型乙型肝炎（慢重乙肝）患者HBVDNA前C区和基本核心启动子（前C／BCP）区突变特点与意义。方法收集87例慢重乙肝和196例慢性乙型肝炎（慢乙肝）患者血清，提取HBVDNA，用巢式PCR扩增HBVDNA前C／BCP区基因，PCR产物进行DNA测序，用NBI软件比对结果，重点分析G1896、G1862、G1899、A1762、G1764、T17536个位点突变。结果慢重乙肝组和慢乙肝组6个位点突变全阴率分别为3．4％和28．1％（P〈0．01）；慢重乙肝组在其中5个位点上的突变检出率显著高于慢乙肝组。此外，慢重乙肝组和慢乙肝组≥三联突变检出率分别为56．3％和35．2％（P〈0．01），≥四联突变检出率分别为25.3％和8．7％（P〈0．01），插入／缺失突变检出率分别为10．3％和1．0％（P〈0．01）。结论HBVDNA前c／BcP区基因突变发生频率的增加与慢乙肝发生重症化相关，结合临床资料分析突变的意义将有助于认识慢乙肝重症化的发生机制。     

乙型肝炎病毒adr型C区突变调节人类白细胞抗原-Ⅰ的表达

目的研究HBV adr型C区突变对机体HLA-I表达的调节机制。方法构建HBV C区突变株L97、G87、V60的真核表达载体,转染HepG2细胞,检测转染细胞中HLA-I的表达以及抗原提呈相关基因LMP2、TAP1、tapasin的mRNA表达情况。结果各转染细胞株均能有效表达HBeAg,但表达量不同,野生株高于C区突变株;各转染细胞株均有HLA-I的表达,其中以L97突变株表达量最高;TAP1 mRNA表达上调,LMP2 mRNA及tapasin mRNA均未见明显表达。结论HBV C区突变株可降低HBeAg的表达,且均能维持甚至上调HLA-I的表达,TAP1 mRNA的表达上调可能是HLA-I表达增强的原因之一。