HBV核心启动子A1762T/G1764A双突变与慢加急性肝衰竭关系的研究

目的 分析HBV BCP A1762T/G1764A双突变与慢加急性肝衰竭（Acute on chronic liver failure,ACLF）之间的相关性.方法 对166名HBV慢性感染后处于疾病不同阶段的患者进行HBV前BCP A1762T/G1764A双突变检测,比较不同患者之间突变率的差异.结果 慢性肝炎（CHB）45人,肝硬化（LC）45人,ACLF 49人,肝细胞癌（HCC）27人,各组A1762T/G1764A双突变率分别为40.0%（18/45）、84.4%（38/45）、73.5%（36/49）、92.6%（25/27）.但是,以CHB为基础的ACLF患者和以LC为基础的ACLF患者A1762T/G1764A双突变率差异无统计学意义[（81.3%）vs.（69.7%）,P=0.502].HBeAg阳性者和HBeAg阴性者BCP双突变率差异无统计学意义（P=0.735）.A1762T/G1764A双突变者HBV DNA水平（10g）为5.68±1.36,阴性者HBV DNA水平（log）为6.14±1.81,差异无统计学意义（P=0.075）.结论 A1762T/G1764A双突变与HBV感染后疾病进展相关,不具备ACLF特异性;A1762T/G1764A联合突变者HBV DNA水平及HBeAg状态也与非联合突变者差异无统计学意义.     

HBV核心启动子A1762T/G1764A双突变与慢加急性肝衰竭关系的研究

Objective To analysis the relationship between HBV BCP A1762T/G1764A double mutation with acute on chronic liver failure(ACLF).Methods HBV BCP A1762T/G1764A double mutation was detected in 166 HBV chronic infection patients by nested PCR and direct DNA sequencing.The mutation rate was compared among the patients with different disease course.Results Among 166 patients,45 patients,45 patients,49 patients and 27 patients were diagnosed as chronic hepatitis B(CHB),liver cirrhosis(LC),ACLF and hepatocellular carcinoma(HCC),respectively.A1762T/G1764A double mutation rate was 40.0%(18/45),84.4%(38/45),73.5%(36/49)and 92.6%(25/27) respectively in different groups.However,A1762T/G1764A double mutation rate has no difference between ACLF based on CHB and LC(P=0.502)and between patients with HBeAg positive and negative(P=0.735).HBV DNA level (log) of patients with A1762T/G1764A double mutation was 5.68±1.36,lower than but having no significant statistic difference compared to patients without the double mutation(6.14±1.81,P=0.075).Conclusion A1762T/G1764A double mutation has a close relationship with the progress of HBV-infection diseases,but is not specific to patients with ACLF.And patients with BCP double mutation have similar HBV DNA levels and HBeAg status with patients without the double mutation.     

慢加急性肝衰竭中IL-10的表达及其启动子甲基化状态的研究

目的 探讨慢加急性肝衰竭患者血清IL-10的表达及其甲基化状态的意义.方法 慢加急性肝衰竭组25例,慢乙肝组25例,正常对照组10例,ELISA法测定血清IL-10的水平,MSP方法检测IL-10启动子甲基化状态,三组间比较.结果 肝衰竭组、慢乙肝组较正常对照组血清IL-10水平明显升高,有统计学意义(P<0.05);肝衰竭组较慢乙肝组升高,无统计学意义(P>0.05).肝衰竭组IL-10水平与TBIL、MELD明显正相关(分别为r=0.566,r=0.443,P<0.05),与PTA呈明显负相关(r=-0.581,P<0.05),与ALT、HBV-DNA无明显相关性(分别为r=-0.022,r=0.033,P>0.05).肝衰竭组甲基化分布状态较慢乙肝组和正常对照组有统计学差异(P<0.05).结论 肝衰竭、慢乙肝患者IL-10水平明显升高.IL-10水平随肝衰竭的严重程度升高.IL-10启动子区甲基化可能是基因失活的重要机制.     

慢加急性肝衰竭中IL-10的表达及其启动子甲基化状态的研究

目的 探讨慢加急性肝衰竭患者血清IL-10的表达及其甲基化状态的意义.方法 慢加急性肝衰竭组25例,慢乙肝组25例,正常对照组10例,ELISA法测定血清IL-10的水平,MSP方法检测IL-10启动子甲基化状态,三组间比较.结果 肝衰竭组、慢乙肝组较正常对照组血清IL-10水平明显升高,有统计学意义(P<0.05);肝衰竭组较慢乙肝组升高,无统计学意义(P>0.05).肝衰竭组IL-10水平与TBIL、MELD明显正相关(分别为r=0.566,r=0.443,P<0.05),与PTA呈明显负相关(r=-0.581,P<0.05),与ALT、HBV-DNA无明显相关性(分别为r=-0.022,r=0.033,P>0.05).肝衰竭组甲基化分布状态较慢乙肝组和正常对照组有统计学差异(P<0.05).结论 肝衰竭、慢乙肝患者IL-10水平明显升高.IL-10水平随肝衰竭的严重程度升高.IL-10启动子区甲基化可能是基因失活的重要机制.     

HBV核心启动子突变与临床表现及病毒复制的关系

目的探讨HBV核心启动子nt 1 762～1 764突变与肝病严重性的关系以及对e系统状态和病毒复制的影响.方法采用半套式突变特异PCR(msPCR)技术检测97例各型肝病患者血清nt 1 762～1 764突变株的感染状况.结果经测序和扩增产物电泳证实,采用我们设计的引物建立的msPCR方法检测HBV感染者血清nt 1 762～1 764突变株和野生株的特异性强,方法可靠.急性肝炎、慢性肝炎轻度、中度、重度及肝硬化患者nt 1 762～1 764突变株感染比例分别为2/5、7/43、10/31、1/3和7/15,肝硬化患者nt 1 762～1 764突变株感染率显著高于慢性肝炎轻度患者(P＜0.025).在92例慢性HBV感染者中,野生株(25/92)、突变株(42/92)和混合感染者(25/92)血清HBeAg阳性率分别为80.0%、56.0%和64.3%;HBV DNA含量分别为(4.4±8.5)×10 8、(1.1±1.6)×10 9和(1.4±1.8)×10 9拷贝/ml;ALT异常率分别为44.0%、52.0%和42.6%;ALT水平分别为(58.6±79.0)、(57.1±75.2)和(62.6±90.3)IU/L,以上各组之间比较差异无统计学意义.结论msPCR技术检测nt 1 762～1 764突变灵敏特异.nt 1 762～1 764突变与肝病严重性有密切关系,这种相关性与突变株的e系统状态、高病毒复制无任何关系.     

慢加急性肝衰竭中IL-10的表达及其启动子甲基化状态的研究

目的探讨慢加急性肝衰竭患者血清IL-10的表达及其甲基化状态的意义。方法慢加急性肝衰竭组25例,慢乙肝组25例,正常对照组10例,ELISA法测定血清IL．10的水平,MSP方法检测IL-10启动子甲基化状态,三组间比较。结果肝衰竭组、慢乙肝组较正常对照组血清IL．】0水平明显升高,有统计学意义（P〈0．05）;肝衰竭组较慢乙肝组升高,无统计学意义（P〉0．05）。肝衰竭组IL-10水平与TBIL、MELD明显正相关（分别为r：0．566,r=0．443,P〈0．05）,与PTA呈明显负相关（r=-0．58l,P〈0．05）,与ALT、HBV．DNA无明显相关性（分别为r=-0．022,r=0．033,P〉0．05）。肝衰竭组甲基化分布状态较慢乙肝组和正常对照组有统计学差异（P〈0．05）。结论肝衰竭、慢乙肝患者IL-lO水平明显升高。IL-lO水平随肝衰竭的严重程度升高。IL-10启动子区甲基化可能是基因失活的重要机制。     

乙型肝炎病毒核心蛋白小干扰RNA载体的构建与验证

目的 构建针对乙型肝炎病毒核心蛋白的小干扰RNA真核表达载体,并验证其干扰效果.方法 设计针对HBV基因组（ayw亚型）HBc基因的小干扰RNA序列（位于2147 bp ～2165 bp序列）,目的片段与载体成功连接后转入大肠埃希菌DH5α,酶切及测序验证其正确性.最后以脂质体转入HepG2.2.15细胞中,检测其干扰效果.结果 成功构建针对乙型肝炎病毒核心蛋白的小干扰RNA真核表达载体,并命名为pshRNA-HBc..酶切及测序验证正确,pshRNA-HBc对HepG2.2.15细胞中HBc表达具有特异性干扰效果.结论 构建针对乙型肝炎病毒核心蛋白的小干扰RNA真核表达载体,为后续研究HBc的生物学功能奠定良好基础.     

慢加急性肝衰竭大鼠动物模型的建立

目的 研究大鼠慢加急性肝衰竭模型的建立方法.方法 SD大鼠给予50%四氯化碳植物油溶液腹腔注射,每3天1次,连续6或10周.分别于6周和10周时将大鼠随机分为2组,分别腹腔注射2 g/kg体重D-氨基半乳糖、100 μg/kg体重脂多糖联合0.5 g/kg体重D-氨基半乳糖.通过观察大鼠饮食,测定血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)和总胆红素(TBil)水平,以及观察大鼠肝组织病理形态改变来评价成模效果.结果 给予四氯化碳植物油溶液腹腔注射6或10周,分别可出现肝纤维化和肝硬化表现.在此基础上给予上述2种试剂急性攻击,可出现肝组织片状、大块或亚大块肝坏死.结论 四氯化碳腹腔注射诱导的慢性肝纤维化,肝硬化基础上分别给予D-氨基半乳糖、脂多糖联合D-氨基半乳糖可诱导慢加急性肝衰竭.     

环孢素A促进HepG2.2.15细胞中乙型肝炎病毒核心蛋白入核的研究

目的观察磷酸脂酶抑制剂环孢素A（CSA）对HBcAg表达和定位的影响，了解HBV生活的周期。方法以30μg/ml的CSA处理HepG2．2．15细胞2d或4d；共聚集显微镜观察细胞内HBcAg和HBsAg亚细胞定位；TUNEL方法检测细胞凋亡。结果对照组HepG2．2．15细胞中HBcAg主要定位于胞质。CSA处理2d后可使胞质中HBcAg和HBsAg水平下降，细胞核内HBcAg水平升高，可见约5％细胞发生凋亡。CSA处理4d后细胞核内HBcAg水平进一步提高，约25％细胞核内表达强度明显高于胞质，并约有30％细胞发生凋亡。结论CSA可促使HepG2．2．15细胞内HBcAg进入细胞核。HBcAg的核内表达定位增强可能与蛋白磷酸化水平增高，及细胞衰老或者凋亡有关。     

乙型肝炎病毒增强子Ⅰ/X基因启动子变异与HBV慢性感染疾病谱的关系

目的 探讨乙型肝炎病毒增强子Ⅰ (HBV Enh Ⅰ)/X基因启动子变异与乙型肝炎病毒慢性化感染疾病谱的关系.方法 随机收集275例HBV感染者的血清标本,包括慢性乙型肝炎( CHB) 100例,肝硬化(LC)74例,肝细胞癌(HCC) 101例.以入选病例的基因型为分组,采用半巢式PCR的方法扩增HBV Enh Ⅰ/X基因启动子并测序,测序结果与HBV参照序列比对,确定变异位点,使用x2检验和多变量logistic回归进行数据分析.结果 ①HBV基因分型结果:HBV B基因型患者158例(61.48％),包括CHB 70例,LC 36例,HCC 52例;HBV C基因型患者117例(38.52％),包括CHB 30例,LC 38例,HCC 49例.②HBV B基因型A1123Y变异在LC组明显高于CHB组(30.56％vs.8.58％,x2=8.533,P=0.005,A=4.693,95％ CI[1.567～14.056]),HCC组明显高于CHB组(28.85％ vs.8.58％,x2 =8.607,P=0.003,OR=4.324,95％ CI[1.544 ～ 12.109]);A1317G变异在HCC组明显高于CHB组(30.77％ vs.7.14％,x2=11.687,P=0.001,A=5.778,95％ CI[1.955～17.076]).HBV C基因型T1323C变异在HCC组明显高于CHB组(30.61％ vs.6.67％,x2 =6.318,P=0.012,A=6.176,95％ CI[1.301～29.331]).③多变量logistic回归分析发现A1317G(A =5.706,95％ CI[1.770 ～ 18.837],P =0.004)和T1323C (A =5.810,95％ CI[1.114 ～30.306],P =0.037)变异是HCC发生的独立危险因素.结论 乙型肝炎病毒增强子Ⅰ/x基因启动子突变与肝硬化、肝癌的发生有关,对变异位点的检测有助于预测肝硬化和肝癌的发生.     

Hepatitis B virus genotype B with G1896A and A1762T/G1764A mutations is associated with hepatitis B related acute-on-chronic liver failure

The existence of statistical associations between hepatitis B‐related acute‐on‐chronic liver failure and both hepatitis B virus (HBV) genotype and mutations in the basal core promoter (BCP) and precore (PC) regions needs to be confirmed. A total of 322 patients with a chronic HBV infection, including 77 with hepatitis B‐related acute‐on‐chronic liver failure, 109 with hepatocellular carcinoma (HCC) and 136 with chronic hepatitis B (CHB) were enrolled. The HBV genotype and the presence of mutations in the BCP/PC regions were determined by direct sequencing, and the frequencies were compared in the three patient groups. Overall, 198/322 (61.5%) were infected with genotype B and 124/322 (38.5%) with genotype C. Genotype B was significantly more frequent in patients with acute‐on‐chronic liver failure than CHB (92.2% vs. 60.3%, P < 0.001). As a contrast, genotype C was more common in patients with HCC than CHB (58.7% vs. 39.7%, P = 0.003). In genotype B patients, the A1762T/G1764A, A1846T, and G1896A mutations were significantly more prevalent in patients with acute‐on‐chronic liver failure than CHB (50.7% vs. 28.0%, P = 0.004; 59.2% vs. 34.1%, P = 0.002; 69.0% vs. 41.5%, P = 0.001, respectively). In multivariate analysis, the risk factors for acute‐on‐chronic liver failure were genotype B, A1762T/G1764A, and G1896A. In conclusion, CHB patients with genotype B, G1896A, and A1762T/G1764A had a higher tendency to develop liver failure than patients with genotype C. Therefore, HBV genotyping and detecting G1896A and A1762T/G1764A mutations might have important clinical implications as predictive risk factors for hepatitis B‐related acute‐on‐chronic liver failure. J. Med. Virol. 83:1544–1550, 2011. © 2011 Wiley‐Liss, Inc.     

核苷类似物治疗早中期HBV-ACLF患者随机对照临床研究

目的 研究核苷类似物抗病毒治疗慢加急性乙型肝炎肝衰竭（HBV-ACLF）早中期患者的疗效和安全性.方法 采用前瞻性随机开放平行对照临床实验设计,设基础治疗组、拉米夫定＋基础治疗组及恩替卡韦＋基础治疗组三组进行对照观察研究.结果 治疗1个月时三组生存率相近,但拉米夫定、恩替卡韦治疗组临床好转率显著高于基础治疗组;治疗随访6个月拉米夫定及恩替卡韦累积生存率分别为65.8%、60.1%,显著高于基础治疗组42%（P〈0.05）.治疗前HBV DNA〉107IU/L患者接受抗病毒治疗组累积生存率高于基础治疗组,治疗前MELD评分〉30患者累积生存率低于MELD评分≤30患者,但MELD评分〉30患者抗病毒治疗的疗效反应性更好.结论 核苷类似物恩替卡韦及拉米夫定治疗HBV-ACLF早中期患者可以显著提高治疗1个月的好转率,降低3个月、6个月的病死率,提高患者生存率.     

C基因型乙型肝炎病毒前C区及基本核心启动子突变与疾病进展的相关性研究

目的探讨HBV前C区（PreC）及基本核心启动子（BCP）突变与慢性乙型肝炎病毒（HBV）感染者疾病进展的关系。方法收集88例慢性HBV感染者血清标本，包括36例无症状携带者（其中24例为HBV携带者，12例为HBsAg携带者）、36例慢性乙型肝炎（慢性乙肝）和16例肝硬化患者。所有标本均经型特异性引物PCR法鉴定为HBVC基因型，并用巢氏PCR法扩增HBVPreC和BCP基因片段，用PCR产物直接测序法测序，然后用ClustalW1．8软件进行序列分析。结果在50例HBeAg阳性患者中，无症状携带者、慢性乙肝和肝硬化组的T1762／A1764双突变率和T1846突变率分别为12．5％、42．1％、100％和0％、5．3％、28．6％，差异均有统计学意义（P值分别为0．03和0．02）。在38例HBeAg阴性HBV感染者中，无症状携带者、慢性乙肝和肝硬化组的T1762／A1764双突变率分别为16．7％、58．8％和66．7％，差异无统计学意义（P=0．08）。肝硬化组的C／G1753突变率显著高于无症状携带者及慢性乙肝组（分别为55．6％、8．3％、11．8％，P=0．01），其A1896突变率也高于无症状携带者组（分别为55．6％、8．3％，P=0．01）。结论HBVT1762／A1764双突变与C基因型HBV慢性感染者的疾病进展有关。     

T1846 and A/G1913 are associated with acute on chronic liver failure in patients infected with hepatitis B virus genotypes B and C

The aim of this study was to determine whether mutations in the hepatitis B virus (HBV) genome are associated with the onset of acute on chronic liver failure (ACLF). For the longitudinal study, full‐length HBV genomes were cloned and sequenced from four ACLF patients and compared with sequences from matching samples collected before ACLF. For the cross‐sectional study, 166 serum samples were obtained, including 49 samples from patients with ACLF. The results of longitudinal study showed that C53T, A1846T, and G1896A were the most common mutations in association with ACLF. In the cross‐sectional study 61.2% patients with ACLF presented with T1846, which was higher than patients with chronic hepatitis B (CHB) (11.1%), liver cirrhosis (LC) (31.1%), and hepatocellular carcinoma (HCC) (33.3%). Prevalence of A/G1913 was 42.9% in patients with ACLF, also higher than patients with CHB (2.2%), LC (17.8%), and HCC (11.1%). There were no differences in HBV genotype and patients' HBeAg status among patients with ACLF, LC, and HCC. However, prevalence of T1846 was much higher in patients infected with genotype B (57.1%) than genotype C (30.4%). A/G1913 was higher in HBeAg negative patients (28%) than HBeAg positive patients (13.2%). Results of a multivariable analysis showed that T1846 and A/G1913 were independent factors for ACLF (OR = 3.373 and 4.244, respectively). Interestingly, T1846 destroys an ATG codon of a small open reading frame in the preC region, which may increase core protein expression. We conclude that T1846 and A/G1913 in the preC/C gene are closely associated with ACLF. J. Med. Virol. 83:996–1004, 2011. © 2011 Wiley‐Liss, Inc.     

HBV DNA PreC/C和BCP片段基因多态性研究

目的　研究乙肝患者HBVDNA核苷酸 (nt) 1735～ 196 5片段突变及其临床意义。方法　PCR扩增HBVDNAnt 1735～ 196 5片段 ,将产物进行HBVDNA测序。结果　在 6 8例乙肝患者中 ,nt 1735～ 196 5突变阳性率为 4 8 5 % ,检出点突变总数 16 8个 ,频率前 10位的是nt 176 4 (5 8.8% )、176 2 (44.1% )、1799(2 0.6 % )、176.6 (14.7% )、1896 (13.2 % )、175.4 (8.8% )、1899(8.8% )、176 8(7.4 % )、1814 (7.4 % )及 1913(7 4 % )。同时 ,首次检出nt 190 7、192 2、192 3位点突变。 5 4例慢性肝炎和 10例肝炎肝硬化患者HBVDNAnt 1896、176 4、176 2位点突变阳性率分别为 16.7%、35.2 %、35.2 %和 30.0 %、6 0.0 %、6 0. 0 % ,两者差异有统计学意义 (P <0.0 1)。结论　该研究提示 :(1)PreC C与BCP区基因突变可能与肝实质纤维化相关 ;(2 )HBVDNA突变发生率较高 ,且位点众多 ,基因测序对芯片探针设计有着十分重要的指导价值和临床意义     

HBV基因型、前S/S基因突变与HBV母婴传播免疫预防失败的关系

 目的：探讨乙型肝炎病毒（HBV）基因型、前S/S基因突变与HBV母婴传播免疫预防失败的关系。
方法：选择血清HBsAg阳性且HBV DNA定量≥1×1010 IU/L的孕妇及其新生儿,以新生儿是否发生免疫预防失败将孕妇分为免疫预防失败组（15例）和免疫预防成功组(45例),采用PCR扩增直接测序法对2组孕妇血中HBV进行基因分型和前S/S基因突变的检测,对比分析2组的不同。
结果：（1）基因型：2组孕妇血中HBV的基因型均为B和C型,均以B型为主,2组基因型分布相比,差异无统计学意义（P>0.05）。（2）突变率：HBV 前S/S基因2个片段的突变率在免疫预防失败组与免疫预防成功组间相比,差异均无统计学意义（P>0.05）;而B基因型与C基因型间相比,差异均有统计学意义（P<0.05）;但在同一基因型内,2个片段的突变率在免疫预防失败组与免疫预防成功组相比,差异均无统计学意义（P>0.05）。对HBV 前S/S基因2个片段的遗传树分析也显示不同基因型的基因突变率不同,但同一基因型的突变率在2组间差异无统计学意义（P>0.05）。（3）突变热点：在免疫失败组的4个病例中发现4个突变热点：529G-A、530A-G、826A-G1和166het-dupC各1例;在免疫成功组的6个病例中发现3个突变热点：A530T 1例,A530G 2例, T531C 3例。
结论：（1）不同HBV基因型间前S/S基因突变率不同。（2）HBV前S/S基因突变普遍存在,但并不是每个突变都与母婴传播免疫失败有关,仅分析基因突变率对研究免疫失败并无意义,寻找与免疫失败有关的特异性突变位点可能更有意义。     

HBV基因型与HBV感染慢性化、重症化的关系

Objective To explore the association between HBV genotype and chronic/severe liver disease with HBV infection in Chinese patients.Methods Serum samples were collected from 2922 patients with HBV infection.HBV genotyping was performed with type-specific primers polymerase chain reaction,and the virological and biochemical markers were detected,which differences in the genotypes between various clinical types of HBV infection and liver function and virological markers between various HBV genotyping were analyzed.Results The genotype B,C,BC combinations,D of 2922 patients with HBV infection accounted for 15.9%,83.5%,0.41%,0.21% respectively.The ratio of genotype B in acute hepatitis group was higher(P=0.003),which the ratio of genotype C in the cirrhosis group and the hepatocellular carcinoma group was higher(P=0.000,0.000).The difference in ratio of genotype C was not statistically significant between acute-on-chronic liver failure group and chronic hepatitis group.HBeAg-positive rate,viral load and liver function markers of B,C genotype group in acute hepatitis group and chronic hepatitis group were not significant different.HBeAg-positive rates of genotype C in acute-on-chronic liver failure group,cirrhosis group,hepatoeellular carcinoma group were higher than that of genotype B(P=0.000,0.024,0.003).Viral load of genotype C in hepatocellular carcinoma group was higher than that of genotype B(P=0.025).Cholinesterase levels of genotype C in the acute-on-chronic liver failure group and the hepatocellular carcinoma group was lower than that of genotype B(P=0.0004、0.02).Conclusion There were HBV genotype B,C,B/C combinations and D in Chinese patients with HBV infection,with genotype B and C being the major ones.Compared with HBV genotype B,genotype C in Chinese patients with HBV infection was more likely to chronic infection,evolved to cirrhosis and hepatocellular carcinoma, but genotype difference was not observed in occurrence of acute-on-chronic liver failure.Genotype was not significant effect in acute and chronic hepatitis B,but HBeAg-positive rate/viral load was higher and liver damage was more severe in severe and end-stage genotype C HBV infection patients.