乙型肝炎患者HBV-DNA载量及基因型与YMDD变异的相关性研究

慢性持续性乙肝病毒（mV）感染，可导致严重肝病，并可进一步发展为肝硬化和肝癌。拉米夫定具有抑制HBV复制，使肝功能正常化、肝组织病理学改善、副作用小等优点。但服用拉米夫定后，将诱发HBV-DNA聚合酶活性区酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸（YMDD）发生变异，从而导致机体产生耐药，并且这种变异与服药时间的长短有关。作者对128例经拉米夫定治疗20-96周后的乙肝患者进行回顾性分析，检测治疗前后HBV-DNA水平及基因型分析，以及治疗后YMDD变异情况，旨在探讨病毒变异与血清HBV-DNA水平及基因型的相关性，并初步探讨其临床意义。     

聚合酶链反应-单链构象多态性银染后胶回收在基因改变检测中的应用

目的:探讨聚合酶链反应-单链构象多态性银染后胶回收的方法在基因改变检测中的应用。方法:实验于2004-09/2005-10在南方医院心内科实验室完成。样本来源:于2004-09/2005-08选择南方医院心内科收治的冠状动脉粥样硬化性心脏病(简称冠心病)患者123例,均符合WHO冠心病诊断标准,并经心电图、超声心动图、CT确诊。患者均知情同意。聚合酶链反应-单链构象多态性银染方法检测冠心病患者三磷酸腺苷结合盒转运体A1基因改变,对1例怀疑存在基因改变样本的聚丙烯酰胺凝胶采用银染后单链胶回收,以胶回收后的单链聚合酶链反应产物为模板重复聚合酶链反应后进行测序。结果:在对三磷酸腺苷结合盒转运体A1基因Exon7进行单链构象多态性检测时,发现一样本泳带异常,对其样本进行银染后胶回收,结合限制性内切酶酶切,证实此样本存在碱基改变,从而验证了非变性聚丙烯酰胺凝胶银染后胶回收的准确性和可行性。结论:经聚合酶链反应-单链构象多态性银染后的非变性聚丙烯酰胺凝胶胶也可进行胶回收。聚合酶链反应-单链构象多态性银染后胶回收在基因改变检测中可以增强单链构象多态性基因突变检测的准确性,由于其无毒性、成本低及操作简单等优点,更易被实验人员所接受。     

泉州地区拉米夫定抗乙肝病毒治疗YMDD变异

目的 检测泉州地区乙肝病毒YMDD变异的类型,探讨拉米夫定治疗后及未接受拉米夫定治疗的乙型肝炎患者中乙肝病毒(HBV)发生YMDD变异情况.方法 采用聚合酶链反应(PCR)结合Taqman荧光技术对泉州地区114例慢性乙肝患者进行HBV YMDD检测,一组66例接受拉米夫定治疗及保肝治疗,另一组48例为对照组只给予保肝治疗,治疗1年对患者HBV多聚酶基因YMDD基序定性检测,观察是否有突变.结果 拉米夫定治疗组在用药1年后有24例发生YMDD突变.其中YVDD变异有15例,YIDD变异有6例,YIDD YVDD变异有3例,其中变异株中有15例,伴有HBV DNA、ALT高于正常水平;而对照组有17例发生YMDD突变.其中YVDD变异有9例,YIDD变异有4例,YIDD YVDD变异有4例.结论 泉州地区乙肝病毒变异株主要是YVDD型,在未经抗病毒治疗的慢性乙肝患者中存在YMDD变异株,临床上在应用拉米夫定对慢性乙型肝炎惠者进行抗病毒治疗前,应对患者是否存在YMDD变异加以重视.     

P21WAF1基因多态性与乳腺癌发病的关系

目的探讨P21WAF1基因多态性在乳腺癌发生、发展中的作用.方法实验在中山大学附属第三医院病理科实验室完成.收集中山大学附属第三医院2002-01/12及江门市人民医院1995-01/2000-12病理存档蜡块120块,收集同期50块乳腺良性病变标本作对照.采用聚合酶链-单链构象多态性分析法检测120例乳腺癌组织和50例乳腺良性组织P21WAF1基因的多态性.结果①聚合酶链反应扩增结果聚合酶链反应产物在琼脂糖凝胶电泳中分别显示为536 bp和300 bp.②聚合酶链反应单链构象多态性检测结果120例乳腺癌和50例良性乳腺组织P21WAF1基因多态性分别为24.2%(29/120)和6.0%(3/50),乳腺癌组织高于良性乳腺组织,两组间差异有显著性(P＜0.05).结论乳腺癌组织存在P21WAF1基因多态性,P21WAF1基因多态性与乳腺癌发生有关.     

拉米夫定治疗乙型肝炎患者发生YMDD变异早期相关临床检验指标的研究

目的:探索服用拉米夫定治疗慢性乙型肝炎患者发生酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸(YMDD)变异的相关临床检验指标,为早期预测拉米夫定发生耐药分析提供参考.方法:选择服用拉米夫定治疗的慢性乙型肝炎患者112例,在治疗前检测和治疗后6、9、12个月检测患者YMDD变异情况、HBV DNA载量、血清ALT水平、HBeAg(...     

拉米夫定耐药患者乙型肝炎病毒P区变异及其特点

【目的】观察使用拉米夫定抗病毒治疗患者乙肝病毒（HBV）P区变异及特点,为临床拉米夫定耐药的预防提供依据。【方法】对52例服用拉米夫定患者进行研究,对其HBV聚合酶逆转录酶区基因进行分析。采用荧光定量PCR试剂进行HBV基因分型。通过巢式PCR引物扩增P区,扩增产物经纯化后进行测序分析。【结果】在52例拉米夫定耐药患者中,其平均年龄（33．6±13．4）岁,共有30例发现了HBVP区耐药位点变异,检出率为57．3％。M204I为主要的突变类型。其中发现P区变异的在病毒学反弹的、治疗中应答不佳的及停药后复发的患者中分别19例,6例,5例,三者变异发生率分别为82．6％,42．9％,33．3％,其变异发生率有显著差异（P〈0．05）。疗程在1年,2年及2年以上的,P区突变检出率分别为26．3％,72．7％,81．8％,三组之间比较有显著差异。P区突变组用药时间明显长于无P区突变组,分别为（27．7±19．9）个月,（13．9±7．6）个月（P〈0．005）。【结论】拉米夫定治疗后耐药患者HBVP区变异发生率也随着时间的延长而增高。且有P区变异的平均用药时间明显长于未检测到P区变异组,故在拉米夫定长期治疗过程中,应定期观察其耐药的产生,及时调整治疗方案,以维持临床疗效的稳定性。     

拉米夫定治疗慢性乙型肝炎诱发YMDD变异相关因素分析

目的探讨拉米夫定治疗慢性乙型肝炎(简称乙肝)诱发乙肝病毒(HBV)发生YMDD(酪氨酸-蛋氨酸-天门冬氨酸-天门冬氨酸)变异的相关因素。方法采用PCR结合Taqman探针技术检测YIDD/YVDD变异,并对120例服用拉米夫定的慢性乙肝患者治疗前的丙氨酸氨基转移酶(ALT)、HBVDNA水平进行分析。结果 120例患者经拉米夫定治疗72周,有34.17%(41/120)的慢性乙肝患者血中HBV发生YMDD病毒变异。拉米夫定联合干扰素治疗组YMDD变异率低于单用拉米夫定治疗组,HBV发生YMDD病毒变异与患者的治疗方案有关,差异有统计学意义(P0.01),与HBVDNA水平呈正相关;与ALT水平呈负相关。结论服用拉米夫定治疗前严格根据患者的HBVDNA载量和ALT水平选择最佳治疗方案和治疗时机,是减少耐药、提高疗效的关键。     

影响乙型肝炎患者HBV YMDD变异的相关因素分析

目的 探讨拉米夫定治疗慢性乙型肝炎(下称乙肝)过程中乙肝病毒DNA聚合酶P区(HBV YMDD)变异的相关因素.方法 对应用拉米夫定(LAM)治疗的22例慢性乙肝患者进行肝功能、乙肝病毒血清学标志物、HBV YMDD变异检测,同时进行肝组织病理学检查.结果 丙氨酸氨基转移酶(ALT)异常率为55%,HBeAg血清转换率为13.6%,HBV YMDD变异发生率为31% ,治疗前病毒量的高低不影响其治疗效果.5例患者肝穿组织中HBsAg和HBcAg依然阳性.结论 运用拉米夫定治疗慢性乙型肝炎6个月后可出现HBV YMDD变异,随着治疗时间延长,其变异发生率越高;HBeAg血清转换率不高;肝组织内仍处于炎症及纤维化状态.     

冠状动脉粥样硬化性心脏病新致病基因MEF2A第7外显子突变的检测

目的:检测中国冠状动脉粥样硬化性心脏病患者MEF2A基因第7外显子是否存在新突变位点。方法:从1995-03/1996-08在武汉同济医院和武汉协和医院对来自湖北、黑龙江、河南、湖南等省的怀疑冠状动脉粥样硬化性心脏病的患者156例进行临床调查。利用聚合酶链反应-单链构象多态性分析结合变性高效液相色谱分析对所有对象的MEF2A基因第7外显子进行筛查,然后选取变性高效液相色谱分析图谱的峰型出现双或多峰者以及聚合酶链反应-单链构象多态性分析图谱条带数目或位置与对照有差异者的片段进行DNA直接测序,测序结果与正常序列对照确定是否存在突变。结果:参与实验的患者156例全部进入结果分析。①MEF2A基因第7外显子区域有4例患者变性高效液相色谱分析图谱呈双峰或多峰,结果疑为异常。②有7例患者的单链构象多态性分析电泳出现异常条带,即泳动比正常多出条带或移动速度与对照有差异,表明这7例可能出现MEF2A基因突变。③所有异常标本的DNA直接测序结果与正常序列对照未发现第7外显子区域基因突变。结论:冠状动脉粥样硬化性心脏病患者在MEF2A基因第7外显子未发现新的突变,变性高效液相色谱分析和聚合酶链反应-单链构象多态性结果与DNA测序结果并非平行关系,单链构象多态性分析同样存在假阳性。     

慢性乙型肝炎患者外周血单个核细胞IFN-γ、IL-6水平变化与拉米夫定疗效的关系

目的探讨慢性乙型肝炎(CHB)患者拉米夫定治疗前后外周血单个核细胞(PBMC)产生干扰素-γ(IFN-γ)、白细胞介素-6(IL-6)水平与血清乙型肝炎病毒(HBV)载量和丙氨酸氨基转移酶(ALT)的关系。方法采用荧光定量聚合酶链反应(FQ-PCR)和酶联免疫吸附试验(ELISA)分别检测拉米夫定治疗前后的CHB患者HBV DNA和PBMC经植物血凝素(PHA)刺激后分泌IFN-γ和IL-6的水平。结果CHB患者治疗前PBMC分泌IFN-γ水平显著低于正常对照组(P<0.01),分泌IL-6水平显著高于正常对照组(P<0.01);拉米夫定治疗后HBV高、中载量组分泌IFN-γ较治疗前显著升高(分别为P<0.01和P<0.05),分泌IL-6水平较治疗前显著降低(分别为P<0.01和P<0.05),治疗后中、低载量组分泌IFN-γ水平仍低于正常对照组(P<0.01),中、低载量组分泌IL-6的水平高于正常对照组(P<0.01);患者INF-γ水平变化与HBV DNA载量呈负相关(r=0.89,P<0.001),IL-6水平变化与HBV DNA载量和ALT呈正相关(分别为r=0.92,P<0.001;r=0.74,P<0.001)。结论CHB患者PBMC分泌IFNγ-、IL-6水平可作为判断拉米夫定治疗效果的参考指标。     

乙型肝炎病毒YMDD自然变异与抗病毒治疗后变异特点

目的了解未经拉米夫定抗病毒治疗的慢性乙型肝炎（CHB）患者和经拉米夫定治疗后的CHB患者乙型肝炎病毒基因组P区多聚酶YMDD变异情况，观察其特点。方法选择未接受过抗病毒治疗的CHB患者119例和经拉米夫定治疗后无明显效果的CHB患者30例，采用荧光标记杂交双探针聚合酶链反应熔解曲线法（FH—PCR—MC）对其血浆标本进行YMDD基因序列突变检测。结果119例未接受过抗病毒治疗的患者，变异检出率为19．3％（23／119），23例出现自然变异毒株的患者，其体内病毒均为变异株与野生株共生，且变异株为非优势株，变异株在总病毒量中所占的比例均在50％以下，变异类型以YVDD为主（20例）。30例经拉米夫定治疗的患者变异检出率为56．7％（17／30），17例阳性患者有11例患者体内病毒完全为变异毒株，仅6例患者为变异株与野生株共生，且变异株为优势株者占83．3％（5／6）；在变异类型上YVDD占8例，居多；YVDD与YIDD同时阳性占3例。结论未经抗病毒治疗的CHB患者存在YMDD自然变异毒株，且变异株皆与野生株共生，变异株为非优势株。经拉米夫定治疗后的变异多数为完全变异，少数为变异株与野生株共生，且变异株大多为优势株。     

Efficacy of alpha interferon therapy for lamivudine resistance in chronic hepatitis B

OBJECTIVES: The occurrence of lamivudine resistance is often associated with the clinical breakthrough, which is characterised by the reappearance of hepatitis B virus (HBV) DNA in serum and the elevation of aminotransferases. We evaluated the efficacy of alpha interferon for clinical breakthrough in patients receiving lamivudine therapy. PATIENTS: Six chronic hepatitis B patients receiving lamivudine were enrolled in the study. RESULTS: Under lamivudine therapy, clinical breakthroughs occurred in between fifteenth and thirty-fourth month of lamivudine therapy. HBV DNA reappeared, and alanine aminotransferase was elevated. Genotypic analysis showed M552V, M552I and L528M mutations. After determining the clinical breakthrough, standard alpha interferon-2b was given for 6 months. Lamivudine was also maintained. In only one patient, HBV DNA became negative by polymerase chain reaction, and serum alanine transaminase level was normal at the end of therapy. CONCLUSION: Alpha interferon added to lamivudine is generally ineffective in the treatment of lamivudine resistance.     

Cross-resistance testing of antihepadnaviral compounds using novel recombinant baculoviruses which encode drug-resistant strains of hepatitis B virus

Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in the selection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistant variants are clearly warranted but have been difficult to do because of the lack of convenient and reliable in vitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replication that relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBV genome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently been used to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation. The further development of this novel system and its application to determine the cross-resistance profiles of drug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses which encoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examine the effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir), and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526M mutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutations M550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir is active against each of these mutants. These findings are supported by the limited amount of clinical data currently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecular characterization of clinically significant HBV strains.     

HBV基因分型和耐药突变基因检测

目的探讨东营地区乙型肝炎病毒(HBV)基因型分布,乙型肝炎患者耐药情况,HBV基因型别与耐药以及突变位点关系。方法收集乙型肝炎患者血清300例,采用离心柱法提取HBV-DNA,聚合酶链反应(PCR)-反向点杂交法检测HBV分型和耐药突变。结果 300例HBV-DNA阳性患者中,检出B型、C型、B/C型及其他未检测出的基因型,未检出D型分型,检出结果中以C型为主,占81.8%;HBV患者耐药药物以拉米夫定和替比夫定的联合耐药为主,占43.6%;HBV耐药突变基因型主要为rt204I(24.35%)、rt204V(17.39%)及rt180M(17.39%);B型和C型的耐药突变率分别为30.77%和42.42%,差异有统计学意义(P0.05)。结论东营地区HBV基因C型多于B型,C型容易产生耐药,以rt204I,rt204V及rt180M基因突变型多见,拉米夫定和替比夫定的联合耐药多见,应根据基因分型和耐药突变结果对乙型肝炎患者选择适合的治疗方案。     

Hypervariable region 1 of hepatitis C virus genome and response to interferon therapy.

The relationship between the complexity of the hypervariable region 1 (HVR1) quasispecies of hepatitis C virus (HCV) and responsiveness to interferon-alpha (IFN) therapy was studied in patients with chronic hepatitis C. Twelve HCV-RNA-positive patients were treated daily with high dose IFN and ribavirin for 4 weeks, and then with IFN 3 MIU (Million International Units) TIW (three times per week) and ribavirin for 6 months. The HVR1 quasispecies complexity was analyzed by nested polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP). The baseline HCV-RNA levels in the study group ranged from 10(6) to 10(7) copies/ml. All patients exhibited HCV genotype 1 b. Initial SSCP analysis revealed four (33.3%) patients with a low complexity pattern (SSCP bands < or =4) and eight (66.6%) patients with high complexity pattern (SSCP bands >4). After 4 weeks of IFN therapy, one patient became HCV negative, and among those remaining positive, the HCV-RNA levels decreased by 2 to 3 logs and the number of SSCP decreased by 2 to 3 bands per sample. After 6 months of IFN therapy, five (41.7%) patients became HCV-RNA-negative. Seven (58.3%) patients did not respond to IFN therapy with sustained viral load from 10(3) to 10(5) copies/ml, and high complexity SSCP patterns. Our data support the HVR quasispecies complexity to be an independent predictive factor for IFN responsiveness in patients infected with HCV.     

Polymerase domain B mutation is associated with hepatitis relapse during long-term lamivudine therapy for chronic hepatitis B

Breakthrough hepatitis remains the major issue in long-term lamivudine therapy for chronic hepatitis B. However, the emergence of drug-resistant hepatitis B virus (HBV) is not always accompanied by a relapse of hepatitis. To elucidate factors predictive of breakthrough hepatitis, 53 patients with genotype C of HBV on long-term lamivudine therapy were analyzed. HBV reappeared during therapy in 19 patients with a cumulative incidence of 15% at 1 year, 34% at 2 years, and 60% at 3 years. Within this group, breakthrough hepatitis developed in 12 patients (63%). A polymerase gene domain B mutation (rt180M) emerged in 13 patients, and domain C mutations (rt204I, rt204V) were found in 19 patients. The rt180M mutation was associated with breakthrough hepatitis (p < 0.05) with a positive predictive value of 85% and a negative predictive value of 83%. Patients with the rt180M mutation had higher HBV-DNA levels during viral breakthrough compared to patients with rt180wt (p < 0.05). The mutational pattern of rt204 was not associated with breakthrough hepatitis. In conclusion, genotypic assays for the rt180M mutation after viral breakthrough may be useful in predicting the risk of breakthrough hepatitis and in deciding when to initiate alternative or additive nucleoside analogue therapy.     

Analysis of hepatitis B virus quasispecies changes during emergence and reversion of lamivudine resistance in liver transplantation

This report describes nucleotide sequence analysis of part of the polymerase gene of hepatitis B virus (HBV) during the development of lamivudine-resistant HBV in five patients who received lamivudine treatment in conjunction with liver transplantation. Samples from patients were analysed before, during and after drug treatment in conjunction with serum HBV quantification by PCR. Lamivudine resistance was found to be associated with L526M and M550V changes in two patients and M550I change in three patients. Other changes associated with lamivudine resistance in some patients were V509I, A546V, S565A and A568T. The effects on HBV surface antigen are also described. Some patients were subsequently treated with famciclovir and/or ganciclovir with variable outcomes. In two out of three patients who stopped lamivudine treatment, reversion (partial or complete) to wild-type virus was observed after about 5 months. In contrast, a complex mixture of mutant viruses emerged in a third patient who stopped lamivudine treatment.     

Hepatitis B virus antiviral drug resistance: from the laboratory to the patient

The development and application of nucleoside (and nucleotide) analogues for the treatment of chronic hepatitis B infection will transform the management of this condition. For instance, treatment with lamivudine effects a dramatic and measurable reduction of serum virus titre. This is associated with biochemical and histological improvements. Unfortunately, for the majority, replication resumes when treatment is withdrawn. Prolonged lamividine treatment may be associated with the emergence of drug-resistant species with specific polymerase mutations. Compared with the observed rate for the development of drug resistance observed during monotherapy of HIV infection, resistance is slow to emerge during treatment of hepatitis B. The rate of emergence might be dependent on the rate of infected hepatocyte turnover, which is extremely variable in chronic HBV infection (and significantly slower than infected lymphocyte turnover during HIV infection). Preliminary data suggest that pretreatment serum virus titre may be an important predictor of the development of drug resistance, an observation consistent with preexistance of the resistant virus in the hepatitis B virus quasispecies. Akin to developments in antiviral treatment of HIV infection, further progress in the treatment of chronic hepatitis B will depend on the development of drugs for use in combination therapy.     

Telbivudine: an upcoming agent for chronic hepatitis B

Telbivudine, the prototype member of β-l-2´-deoxynucleosides, has proven to be safe in in vitro animal and human studies. Telbivudine given for 4 weeks resulted in an 8-log reduction of woodchuck hepatitis virus DNA, and a 3.8-log reduction of hepatitis B virus DNA in human. After 52 weeks of telbivudine treatment there was an approximate 6-log reduction of hepatitis B virus DNA levels, hepatitis B virus DNA became undetectable by PCR assay in 61% of patients. Its antiviral efficacy is significantly better than lamivudine. The probability of tyrosine-methionine-aspartate-aspartate mutations at 52 weeks of telbivudine therapy is low, although still occurring in 4.5% of patients. After 96 weeks of therapy, the proportion of patients with undetectable hepatitis B virus DNA by PCR assay increased to 71%, but genotypic resistance also increased to 18.2%, with only 4.5% showing alanine aminotransferase flares. Telbivudine is probably one of the most potent antiviral agents for hepatitis B virus that will become available in the near future.