HBV前C区基因变异与中医证候的相关性研究

研究慢性乙型肝炎患者HBV前C区基因变异的变化规律与中医证候的相关机制。基因芯片检测慢性乙型肝炎不同证候的HBV前C区基因变异。不同位点变异率(%)与证候相关,肝肾阴虚1764(38%),1896(68%),1899(38%);瘀血阻络1762(93%),1764(90%),1899(50%)。不同证候变异株信号强度差异显著,1762、1764;瘀血阻络>肝肾阴虚>肝郁脾虚;1862:肝肾阴虚>肝郁脾虚;>1896:肝肾阴虚>瘀血阻络>湿热中阻>肝郁脾虚;1899:瘀血阻络>肝肾阴虚>湿热中阻>肝郁脾虚。慢性乙型肝炎HBV前C区基因变异与中医证候相关,为分子证候辨证提供了一定实验依据和研究基础。     

清热利湿法对慢性乙型肝炎HBV基因突变株复制的影响

目的:研究清热利湿法对慢性乙型肝炎(CHB)患者HBV前C区基因突变株复制的影响。方法:采用基因芯片技术定量检测CHB肝胆湿热/湿热中阻证患者治疗(清热利湿法)前后的HBV前C基因变异。结果:治疗前的HBV前C区1762、1764、1896位点变异率明显高于1899位点变异率,差异有显著性意义(P<0.01);治疗后各位点变异率下降,其中治疗组1764、1896位点变异率显著下降(P<0.05)。治疗前不同位点变异株信号强度1896>1764>1762>1899,差异具有显著性意义(P<0.05);治疗后1762、1764、1896位点变异株信号强度下降,其中1896位点信号强度显著下降(P<0.05)。结论:清热利湿法在改善CHB肝胆湿热/湿热中阻证患者临床证候的同时,可在一定程度上抑制HBV前C区突变株的复制。     

慢性重型乙型肝炎前C区及BCP区基因突变的临床意义

应用基因芯片杂交技术检测慢性重型乙型肝炎（慢重肝）及非重型肝炎（包括慢乙肝及肝硬化）患者血清中HBV前C区A1896、A1899及BCP区T1762/A1764联合突变的发生率,比较慢重肝好转或治愈患者及恶化或死亡患者前C区及BCP区基因突变情况。结果慢重肝前C区A1896、A1899,BCP区T1762/A1764联合突变率及两区多位点突变率与慢乙肝或肝硬化患者比较均有显著统计学差异（P均〈0.01）;慢重肝好转或治愈患者A1896、A1899及多位点变异率与恶化或死亡患者比较均有统计学差异（P均〈0.05）。提示HBV前C区及BCP区基因变异为慢重肝的重要发病机制之一。     

慢性乙型肝炎患者HBV前C区G1896A、核心启动子1762/1764变异与乙型肝炎自然史、血清HBsAg定量及疾病程度的相关性

目的研究HBV前C区G1896A、核心启动子1762/1764变异与HBV自然史、血清HBsAg水平间的相关性及对疾病严重程度的影响。方法分别各选取40例HBV野生株、前C区G1896A或核心启动子1762/1764变异以及两者联合变异的慢性乙型肝炎感染者为研究对象。采用PCR反向点杂交技术检测HBV前C区G1896A位点、核心启动子1762/1764变异及HBV基因分型。同时检测HBsAg定量、HBeAg、HBV DNA定量、肝脏生化指标等。结果(1)HBV前C区G1896A、核心启动子1762/1764变异患者的年龄、ALT高于野生株感染者(P0.001),Alb低于野生株感染者(P0.001)。(2)HBV自然史免疫耐受期以野生株感染为主(P0.001),免疫清除期、低(非)复制期野生株及PC G1896A或/和BCP1762/1764变异株差异无统计学意义(P0.05),再活动期以PC G1896A或/和BCP1762/1764变异株为主(P0.001)。(3)PC G1896A或/和BCP1762/1764变异株的HBsAg水平(lgIu/mL)、HBeAg阳性率较野生株组降低(P0.001),基因C型、HBV DNA水平(≥6 lg拷贝/mL)较野生株组差异无统计学意义(P0.05)。(4)HBV PC G1896A或/和BCP1762/1764变异中肝硬化患者多于野生株(P0.05)。结论慢性HBV感染者前C区G1896A、核心启动子1762/1764变异与乙型肝炎自然史、HBsAg水平、HBeAg的状态及疾病严重程度相关。     

HBeAg阴性慢性乙型肝炎患者的基因变异测定及意义

应用PCR方法检测45例HBeAg阴性慢性乙型肝炎(慢乙肝)患者(HBeAg阴性组)及15例慢乙肝并肝硬化患者(肝硬化组)的基因突变情况.结果 HBeAg阴性组1762位点变异3例,前(pre)-1896位点变异12例,1762 1764位点联合变异9例,1762 1764 pre-1896位点联合变异21例;肝硬化组分别为9例、3例、3例.认为HBeAg慢乙肝患者基因突变以1762 1764 pre-1896位点联合变异为主,其中pre-1896位点变异可能在肝硬化的发生中有重要意义.     

234例慢性乙型肝炎患者HBV前C/BCP区突变分析

目的调查慢性乙型肝炎患者血清HBV前C/基本核心启动子（BCP）区的突变情况,分析各种突变对HBeAg表达的影响。方法抽提患者血清DNA,采用改进的巢式PCR技术,扩增HBV前C/BCP区基因,对PCR产物进行DNA测序。用NTI软件比对结果,根据文献报道,选取1753、1762、1764、1862、1896和1899共6个位点进行突变分析,并重点分析不同突变对患者血清HBeAg阳性率和病情的影响。结果在234例中6个位点均无突变者为74例（31.6%）,其血清HBeAg阳性率为74.3%（55/74）。在234例中6个位点检出至少1种突变的病例为160例（68.4%）,突变形式包括4种单一位点突变和21种组合形式的突变;检出G1896A突变73例（31.2%）,其中36例检出有共存未突变序列,37例仅检出突变序列,后者血清HBeAg的阳性率为18.9%（7/37）。检出G1896A以外其他形式突变的有87例,HBeAg阳性率为63.2%（55/87）;其中以A1762T＋G1764A最为常见,HBeAg阳性率为69.4%（34/49）。在1753、1862位点上检出4种特殊碱基突变,前C区有基因片段插入或缺失的有2例。结论多数慢性乙型肝炎患者在HBV前C/BCP区可检出突变,突变形式多样,其中G1896A突变样本血清HBeAg阳性率显著下降,而其他突变对其影响较小。应用DNA序列测定法分析HBV前C/BCP区基因突变,获得的信息全面,对临床评估病情进展和实施抗病毒治疗有参考价值。     

家族聚集性慢性乙型肝炎的病毒前C区和基本核心启动子变异与临床病理的关系

目的: 分析家族聚集性慢性乙型肝炎的基因变异特征和肝组织病理学改变的关系.方法: 选择家族聚集性慢性乙型肝炎112例作实验组和非家族聚集性慢性乙型肝炎110例作对照组;用基因芯片法检验前C1896和BCP1762、1764双突变;并肝组织活检进行肝组织炎症活动度计分和肝纤维化计分.结果: 实验组BCP T1762/A1764双突变率显著高于对照组(X2=4.5626,P=0.0044);实验组炎症评分显著高于对照组(u=6.3397,P=0.0000);家族聚集性慢性乙型肝炎者中单独BCP T1762/A1764双突变者炎症计分显著高于与无此类位点变异者和单独前C区1896变异者(t1=8.0111,7.958,t1=2.1651,2.059,均P<0.05).结论: 家族聚集慢性乙型肝炎肝组织炎症计分和纤维化计分较高,与BCP1762和1764的变异率较高有关.     

家系感染乙型肝炎病毒基因变异与HLA-Ⅱ类等位基因的关系

目的研究家系内慢性乙型肝炎病毒基因变异特征及与HLA-II类分子基因多态性的关系。方法采用PCR扩增和产物测序检测HBV前C区1896位及BCP区1762/1764位变异,分析家系内慢性HBV感染者121例（实验组）病毒变异特征及与非家系慢性乙肝患者83例（对照组）病毒变异频率差异;用PCR/SSP对家系内慢性HBV感染者的HLA-DQA1和DQB1等位基因多态性进行测定;分析HLA-II类分子基因多态性与家系内慢性乙型肝炎病毒基因变异的关系。结果实验组前C区1896位变异、BCP区1762/1764位双变异及联合变异率均高于对照组;HLA-DQA1＊0501和DQB1＊0301等位基因频率和家系慢性HBV感染者前C区1896位、BCP区1762/1764位及联合变异有关;母亲为第一代的感染家系中,子女的前C区1896位及BCP区1762/1764位变异与母亲是否有该位点变异差异无显著性。结论家系感染HBV患者中,HBV前C区1896位、BCP区1762/1764位双突变及联合变异频率明显高于无家系感染的慢性乙型肝炎患者;家系内感染HBV前C区1896位、BCP区1762和1764位变异并非都是由上一代遗传或传播而来,而主要是与个体的免疫状况有关。     

乙型肝炎病毒前C区和核心启动子区突变与慢性乙型肝炎病情的关系

乙型肝炎病毒（HBV）感染能导致急、慢性肝病，其中全球慢性乙型肝炎感染者近4亿。每年由于HBV慢性感染引起的肝硬化，肝细胞癌死亡患者超过47万人。而由于HBV病毒复制过程缺乏校对机制，在慢性感染的过程中发生基因突变的概率很高，相关研究最多的HBV突变株主要有：发生在翻译水平的前C区突变（G1896A）；发生在核心启动子（BCP）区，即A1762T和G1764A的联合突变；发生在HBV DNA聚合酶区的变异包括P区528、552位点的突变。研究表明，HBV前C区1896位突变以及BCP区突变普遍存在于CHB患者中，     

慢性乙型肝炎患者HBV C基因启动子变异的临床意义

目的:探讨慢性乙型肝炎中C基因启动子(BCP)变异的临床意义。方法:采用错配PCR与限制性长度片段多态性分析(RFLP)相结合,检测35例慢性乙型肝炎患者BCP区核苷酸(nt)1762碱基A→T和1764G→A联合突变及前C区nt1896G→A终止变异。结果:在35例慢性乙型肝炎中检出BCP区T1762A1764变异8例(23％),其中6例血清HBeAg( ),2例抗HBe( ),而7例前C区A1896变异中HBeAg( )2例,抗HBe( )5例,未见T1762A1764变异和A1896变异同时出现者。结论:提示HBV毒株BCP区T1762A1764变异可能与前C区A1896变异不同,它的出现不足以导致HBeAg(—)型的慢性肝炎。     

乙型肝炎病毒前C区突变株感染的临床和病理

探讨乙型肝炎病毒（ＨＢＶ）前Ｃ区突变株（１８９６位点Ｇ→Ａ点突变）慢性感染患者的临床特点、病理特征及与干扰素疗效的关系。方法采用突变特异性ＰＣＲ检测１０８份慢性ＨＢＶ感染患者血清和／或肝组织中ＨＢＶ前Ｃ区突变株，按Ｋｎｏｄｅｌｌ方法评价肝组织病理损伤。结累ＨＢＶ前Ｃ区突变株在不同ｅ系统均存在，但ＨＢｅＡｂ（＋）组中单纯突变株感染或突变株感染占优势的混台感染（１４．２９％，４６．２３％）显著地高于ＨＢｅＡｇ（＋）组（０％，６．４５％）；突变株感染与肝脏疾病严重程度相关，慢性肝炎重度组检出率（１３．６４％，４０．９１％）极显著地高于慢性无症状携带者（０％，１０．２６％）。５例前Ｃ区突变株感染者应用干扰素治疗可产生应答和无应答２种结果。临床症状较轻的ＨＢＶ前Ｃ区突变株感染者的肝脏病理损伤程度无明显加重。结论前Ｃ区突变株感染普遍存在，但更常见于ＨＢｅＡｂ阳性者。【关键词】##4乙型肝炎病毒;;突变;;病理学;;治疗     

A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular retention and impaired secretion of HBe-antigen

Aim: Hepatitis B virus (HBV) e antigen (HBeAg) is translated from precore mRNA as a precore/core protein, which is post-translationally modified to give rise to the protein that is secreted into the serum. The G1862T mutation in HBV occurs in the bulge of the encapsidation signal within the pregenomic RNA. When the precore mRNA is translated, this mutation results in a valine to phenylalanine substitution at the -3 position to the signal peptide cleavage site at the amino end of the precursor protein. The aim of this study was to determine whether this mutation could affect HBV replication and/or HBeAg expression. Methods: Following transfection of Huh 7 cells, HBV replication was followed using real time polymerase reaction (PCR) and expression of HBeAg expression was monitored using confocal microscopy. Results: HBV replication was reduced when this mutation was introduced into genotype D but not into genotype A replication-competent constructs. Using mutant HBeAg-expressing plasmids, we demonstrated a 54% reduction in HBeAg secretion relative to the wild type. Confocal microscopy demonstrated that the mutant HBeAg accumulated in the endoplasmic reticulum, endoplasmic reticulum intermediate compartment and Golgi. These aggregates of mutant protein increased in size following treatment of the cells with a proteasome inhibitor, MG132, and had the hallmark features of aggresomes. They attracted ubiquitin, heat shock proteins and proteasomes and were isolated from the cytosol by the intermediate filaments, vimentin and cytokeratin. Conclusion: The formation of aggresomes, as a result of the G1862T mutation, may play a contributory role in HBV-induced liver disease.     

Cytoplasmic expression of hepatitis B core antigen in chronic hepatitis B virus infection: role of precore stop mutants.

AIMS/BACKGROUND: The cytoplasmic expression of HBcAg is a possible target of immune hepatocytolysis and it is important for the pathogenesis of hepatic injury in chronic hepatitis B virus (HBV) infection. Cytoplasmic HBcAg expression has been suggested to be related to the precore sequence of HBV, HBV DNA level or cell cycle of hepatocytes and the aim of this study was to understand its mechanism. MATERIAL/METHODS: We studied the expression pattern of HBcAg and its relationship to serum HBV DNA levels, cell proliferation activity of hepatocytes and precore mutation using 66 patients' sera and biopsied liver specimens of chronic hepatitis B. RESULTS: The expression patterns of HBcAg were cytoplasmic predominant (CP) in 17 cases, nuclear and cytoplasmic (NC) in 10 cases and nuclear predominant (NP) in 9 cases and negative in 30 cases. CP expression cases showed a higher grade of hepatitis activity than NP expression cases. Serum HBV DNA levels showed a wide range and there was no significant difference according to the expression pattern of HBcAg. Cell proliferation activity of hepatocytes, measured by Ki-67 (MIB-1) labelling index was higher in CP expression cases than in NP expression cases and it was also significantly higher in cases of high grade than in low grade hepatitis activity. The precore region was evaluated by primer extension assay in 51 cases and there were 16 cases of 1896 precore mutant, 23 cases of wild type, 12 cases of mixed infection of 1896 precore mutant type and wild type. CP expression of HBcAg was significantly more frequent in 1896 precore mutant cases (86%) than in wild type cases (26%). CONCLUSION: CP expression of HBcAg is the major pattern of 1896 precore mutant cases and it might be involved in the severe liver injury of precore mutants. One of the mechanisms regulating CP HBcAg expression is suggested to be precore mutation of HBV as well as cell cycle of hepatocyte.     

Cytoplasmic expression of hepatitis B core antigen in chronic hepatitis B virus infection: role of precore stop mutants

Abstract: Aims/Background: The cytoplasmic expression of HBcAg is a possible target of immune hepatocytolysis and it is important for the pathogenesis of hepatic injury in chronic hepatitis B virus (HBV) infection. Cytoplasmic HBcAg expression has been suggested to be related to the precore sequence of HBV, HBV DNA level or cell cycle of hepatocytes and the aim of this study was to understand its mechanism. Material/Methods: We studied the expression pattern of HBcAg and its relationship to serum HBV DNA levels, cell proliferation activity of hepatocytes and precore mutation using 66 patients' sera and biopsied liver specimens of chronic hepatitis B. Results: The expression patterns of HBcAg were cytoplasmic predominant (CP) in 17 cases, nuclear and cytoplasmic (NC) in 10 cases and nuclear predominant (NP) in 9 cases and negative in 30 cases. CP expression cases showed a higher grade of hepatitis activity than NP expression cases. Serum HBV DNA levels showed a wide range and there was no significant difference according to the expression pattern of HBcAg. Cell proliferation activity of hepatocytes, measured by Ki-67 (MIB-1) labelling index was higher in CP expression cases than in NP expression cases and it was also significantly higher in cases of high grade than in low grade hepatitis activity. The precore region was evaluated by primer extension assay in 51 cases and there were 16 cases of 1896 precore mutant, 23 cases of wild type, 12 cases of mixed infection of 1896 precore mutant type and wild type. CP expression of HBcAg was significantly more frequent in 1896 precore mutant cases (86%) than in wild type cases (26%). Conclusion: CP expression of HBcAg is the major pattern of 1896 precore mutant cases and it might be involved in the severe liver injury of precore mutants. One of the mechanisms regulating CP HBcAg expression is suggested to be precore mutation of HBV as well as cell cycle of hepatocyte.     

Prevalence of Hepatitis B Virus Genotype D in Precore Mutants Among Chronic Liver Disease Patients from New Delhi,India

Hepatitis B is one of the most important causes of chronic viral hepatitis world wide. Mutations in the precore region of the hepatitis B virus (HBV) genome are frequently found in hepatitis B envelope antigen-negative cases. Data from India on the HBV genotype-associated distribution of precore mutations are limited. Our objective in this study was to genotype and detect the precore mutant with a point mutation from G to A at nucleotide 1896 using ligase chain reaction (LCR) and direct sequencing. A total of 115 cases of chronic liver disease were screened. The cases were evaluated on the basis of history, clinical examination, liver function profile, and serological test for HBV infection, which includes HBsAg, anti HBcIgG, HBeAg using commercially available ELISA kits. The cases, which were HBeAg+, HBeAg-, and HBV DNA+, were subjected to LCR and confirmed by direct sequencing. Of 115 chronic liver disease cases, 50 (43.5%) cases were HBV DNA positive. All cases were subjected to LCR; 11 (22%) cases confirmed the presence of precore mutants, while the remaining 39 (78%) were classified as the wild form of the virus. HBV genotyping by direct sequencing revealed that genotype D was predominant in both wild and mutant forms of the virus. We conclude that the HBV genotype distribution was not significantly different between precore mutants and the wild form of the virus (P>0.05). North Indian patients with genotype D were more likely to have persistent HBV infection with precore mutants. HBV genotypes correlate with the clinical outcome of chronic HBV infection.     

慢性乙型肝炎HBV前C区基因突变与中医虚实证的关系

目的:探讨慢性乙型肝炎HBV前C区基因突变与中医虚证和实证的关系。方法:采用聚合酶链反应-单链构象多态分析(PCR-SSCP)银染技术,对91例慢性乙型肝炎患者HBV前C区基因突变进行了研究。结果:53例慢性乙型肝炎实证患者,HBV DNA阳性36例,其中前C区基因突变26例,突变率为72.2％;38例虚证患者,HBV DNA阳性16例,其中前C区基因突变6例,突变率为37.5％。两组比较有显著性差异,P<0.01。实证突变组肝功能ALT、AST和SB水平显著高于虚证突变组及未突变组,P<0.01。结论:HBV前C区基因突变与中医虚证和实证有一定的关系,实证突变率高于虚证,且实证突变组的炎症活动较虚证组剧烈。     

Response to interferon-alpha in chronic hepatitis B with and without precore mutant strain detected by mutation site-specific assay

GOALS: We investigated whether the presence of precore mutant (stop codon mutation at codon 28) affects the response to interferon-alpha in patients with chronic hepatitis B. BACKGROUND: Mutations of hepatitis B virus (HBV) may influence the response to treatment. The association of precore mutant with the response to interferon is controversial. STUDY: Thirty-one Japanese patients with hepatitis B e antigen-positive chronic hepatitis were treated with natural interferon-alpha. HBV DNA with the precore mutation was assayed in serum using a mutation site-specific assay before and after treatment. RESULTS: Before treatment, precore mutant was detected in 22 cases (group A) and not detected in 9 cases (group B). Serum HBV DNA level before treatment was not different between the 2 groups. At the end of treatment, serum HBV DNA was decreased to undetectable levels in 13% (4 of 31). Six months after treatment, the percentage of cases with loss of hepatitis B e antigen and a decrease in the transaminase level to within the normal range was significantly higher in group B than in group A (67%, 18%, P = 0.015). CONCLUSIONS: Chronic hepatitis without precore mutant strain before treatment is more responsive to IFN-alpha.     

慢性HBV感染前C区变异与病毒复制水平关系

探讨HBV前C基因变异与病毒复制水平的关系在慢性HBV感染者中的意义。应用错配聚合酶链反应（PCR）－限制性片段长度多态性（RFLP）分析与荧光定量聚合酶链反应检测HBVDNA相结合,对30例HBsAg（＋）、抗－HBe( )及抗－HBc( )慢性HBV感染者,其中无症状携带者（AsC)9例、慢性乙型肝炎（CHB）12例及慢性重症肝炎（CHF）9例进行前C区基因变异与HBVDNA水平关系进行分析。AsC组3例（33．33％）,CHB组9例（75％）及CHF组8例（88．89％）有A83(nt1896)变异。荧光定量PCR结果表明HBVDNA含量在CHF组中最高。HBV前C变异与HBV不同感染状态中都可见,其病毒复制水平与肝病活动相关。     

重型肝炎血清HBV前C区A83变异株比率的动态观察

目的 了解ＨＢＶ前区Ｃ区Ａ８３变异与重型肝炎的关系。方法 用套式错配ＰＣＲ限制片段长度多态性分析和凝胶光密度图象分析仪及定量ＰＣＲ,对９例重型肝炎患者血清ＨＢＶ变异株的比率和ＨＢＶ ＤＮＡ含量进行测定和动态观察。结果 Ａ８３变异株在６例阳性率中均与野生株呈混合感染,其比率〉５０．０％的仅２例,存活死亡各１例,〈３１．０％的４例均死亡,变异株的出现及其比率变化与ＨＢＣＶ ＤＮＡ含量的消长一致,但无统