应用表达谱芯片技术筛选HBeAg反式调节基因

目的 筛选与克隆HBeAg反式调节基因，了解其在体内的调节功能线索及机制。方法 以分子生物学技术构建HBeAg的真核表达载体pcDNA3．1(-)-HBeAg，转染HepG2细胞，以空载体pcDNA3．1(-)为平行对照，制备转染后的细胞裂解液，提取mRNA。应用基因表达谱芯片技术对差异表达mRNA进行检测和分析。结果 HBeAg表达质粒pcDNA3．1(-)-HBeAg经酶切鉴定和DNA测序鉴定正确。经基因表达谱芯片分析，5种基因的表达水平上调，7种基因的表达水平下调。结论 筛选到一些与细胞信号传导、免疫调节、蛋白翻译合成、肿瘤、神经系统发生相关的HBeAg反式调节的靶基因。     

HBsAg浓度与HBV复制水平的相关性分析

目的探讨HBsAg浓度与HBV复制水平的相关性。方法用时间分辨免疫荧光法(TRFIA)定量测定的乙型肝炎患者血清HBsAg浓度,比较HBV DNA复制或HBeAg患者HBsAg的滴度,同时进行HBsAg与DNA复制或HBeAg相关性分析;应用ROC曲线分析单独应用HBsAg或联合HBeAg,判断HBV DNA结果及复制状态的可靠性,分析判断DNA结果的符合率。结果HBsAg滴度和HBeAg水平或HBV DNA复制水平呈正相关。ROC曲线分析表明HBsAg滴度可用于HBV DNA阴性或阳性的判断,并且能够提高单独应用HBeAg滴度的ROC曲线下面积。以HBsAg 150ng/ml评估HBV DNA水平具有较高的阳性、阴性预测值和符合率。结论HBsAg浓度与HBV复制水平密切相关。     

聚乙二醇干扰素α-2a治疗慢性乙型肝炎疗效观察

目的：探讨聚乙二醇α-2a（PEG INFα-2a）治疗阳性慢性乙型肝炎的疗效和安全性。HBeAg方法：按随机对照原则选择80例HBVDNA、HBeAg阳性的慢性乙型肝炎患者，按1：1随机分配进入Fα-2a组和IFNα-1b组。结果：治疗6月时，PEG INFα-2a组HBeAg血清转换率（54．3％）高于IFNα-1b组（29．7%），P＜0．05。停药6月后，持续的HBeAg血清转换率分别为57．1%和32．4%．P＜0．05。停药6月后，持续的HBV DNA阴转率分别为62．9％和37．8%，P＜0．05。治疗后，两组的丙氨酸转氨酶（ALT）复常率差异无显著性，为62.9%和45.9%，停药后6月，两组的联合应答率分别为60．0%和35．1%，差异有显著性，P＜0．05。PEG INFα-2a组有3例病人HBsAg阴转，而IFN α-1b组仅有1例病人HBsAg阴转。两组有相似的不良反应，不良反应间差异无显著性，两组治疗过程中均无发生重要的不良事件。结论：PEG INF α-2a治疗HBeAg阳性的慢性乙型肝炎疗效优于普通干扰素IFN—1b．耐受性和安全性好。     

慢性乙型肝炎患者外周血HBsAg与HBV DNA相关性分析

目的探讨HBeAg（＋）和HBeAg（-）慢性乙型肝炎患者外周血HBsAg与HBV DNA的关系。方法定量检测HBeAg（＋）55例和HBeAg（-）36例慢性乙型肝炎患者血清HbsAg和HBV DNA的水平。结果 HBeAg（＋）患者血清HBV DNA、ALT和AST水平较HBeAg（-）患者高（P〈0.05）;HBeAg（＋）患者血清HBsAg水平较HBeAg（-）患者低（P〈0.05）;高水平血清HbsAg患者血清HBV DNA水平低（F=10.096,P〈0.01）;HBeAg（＋）慢性乙型肝炎患者HBsAg与HBV DNA存在负相关（r=-0.796,P〈0.01）,而HBeAg（-）慢性乙型肝炎患者HBsAg与HBV DNA无相关性（r=0.289,P〉0.05）。结论定量检测慢性乙型肝炎患者血清HBsAg水平有一定的临床意义。     

Serum hepatitis B virus RNA is a predictor of HBeAg seroconversion and virological response with entecavir treatment in chronic hepatitis B patients

BACKGROUND Characteristics of alterations of serum hepatitis B virus(HBV) RNA in different chronic hepatitis B(CHB) patients still cannot be fully explained. Whether HBV RNA can predict HBeAg seroconversion is still controversial.AIM To investigate whether HBV RNA can predict virological response or HBeAg seroconversion during entecavir(ETV) treatment when HBV DNA is undetectable.METHODS The present study evaluated 61 individuals who were diagnosed and treated with long-term ETV monotherapy at the Department of Infectious Diseases of Peking University First Hospital(China) from September 2006 to December 2007.Finally, 30 treatment-naive individuals were included. Serum HBV RNA were extracted from 140 μL serum samples at two time points. Then they were reverse transcribed to cDNA with the HBV-specific primer. The product was quantified by real-time quantitative PCR(RT-PCR) using TAMARA probes. Statistical analyses were performed with IBM SPSS 20.0.RESULTS Level of serum HBV RNA at baseline was 4.15 ± 0.90 log10 copies/mL. HBV RNA levels showed no significant difference between the virological response(VR)and partial VR(PVR) groups at baseline(P = 0.940). Serum HBV RNA significantly decreased among patients who achieved a VR during ETV therapy(P < 0.001). The levels of HBV RNA in both HBeAg-positive patients with seroconversion group and those with no seroconversion increased after 24 wk of treatment. Overall, HBV RNA significantly but mildly correlated to HBsAg(r =0.265, P = 0.041), and HBV RNA was not correlated to HBV DNA(r = 0.242, P =0.062). Furthermore, serum HBV RNA was an independent indicator for predicting HBeAg seroconversion and virological response. HBeAg seroconversion was more likely in CHB patients with HBV RNA levels below4.12 log10 copies/mL before treatment.CONLUSION The level of serum HBV RNA could predict HBeAg seroconversion and PVR during treatment. In the PVR group, the level of serum HBV RNA tends to be increasing.     

E抗原和E抗体阳性的慢性乙型肝炎组织病理学研究

目的探讨HBeAg阳性和抗-HBe阳性的慢性乙型肝炎患者的肝组织病理改变的差异性.方法分别对151例HBeAg阳性和62例抗-HBe阳性的慢性乙型肝炎患者进行肝活检,观察肝组织病理分级和分期情况,同时检测血清HBV DNA.结果 (1)肝组织病理分级、分期与血清HBeAg/抗-HBe的出现情况密切相关,抗-HBe阳性者中肝组织病理为G3～G4、S3～S4者明显多于G1～G2、S1～S2者;而在HBeAg阳性者中则相反;两组比较差异有显著性(P＜0.005);(2)在HBeAg阳性患者中HBV DNA检出率为83.4%(126/151);而在抗-HBe阳性患者中HBV DNA检出率为29%(18/62).结论 (1)抗-HBe阳性的慢性乙型肝炎患者中肝组织炎症、坏死及肝纤维化的程度较HBeAg阳性者严重;(2)HBeAg与HBV DNA存在良好的相关性,但在部分抗-HBe阳性的患者中仍可检出HBV DNA.     

Hepatitis B virus DNA levels, precore mutations, genotypes and histological activity in chronic hepatitis B

The present study aimed to clarify how viraemia levels reflect the clinical stages of chronic hepatitis B virus (HBV) infection, in particular studying whether 'healthy carriers' can be identified by analysing HBV DNA levels with a highly sensitive quantitative assay. Histology activity index (HAI), alanine aminotransferase (ALT) level, genotype and precore mutations were compared with the HBV DNA level, as measured using the Amplicor HBV Monitor assay in a prospective study. In 124 hepatitis B e antigen-negative (HBeAg-) patients, the majority with mild liver disease, log HBV DNA levels showed a Gaussian distribution around a geometric mean of 33 000 genome copies ml-1, and increasing HBV DNA level was associated with significantly higher inflammation (HAIinfl) and fibrosis (HAIfibr) scores and higher ALTi (ALT / the upper reference value). Severe inflammation (HAIinfl > or = 7) was seen in 83% (five of six), 36% (eight of 22) and 3% (one of 37) of HBeAg- patients with HBV DNA > 107, > 2 x 105 and < 104 copies ml-1, respectively. In severe HBeAg- hepatitis, patients with precore wild-type infection had lower HBV DNA levels than those with precore mutants. In 36 HBeAg-positive (HBeAg+) patients, no correlation between HBV DNA level and liver damage was seen. Ninety-six per cent of HBeAg- patients with ALTi < 0.5 had HAIinfl < or = 3. In HBeAg- carriers with ALTi 0.5-1.0, the relative risk for severe inflammation, comparing HBV DNA > 2 x 105 copies ml-1 vs < 2 x 105 copies ml-1, was 14.7. In conclusion, in HBeAg- carriers, HBV DNA < 104 copies ml-1 or ALTi < 0.5 indicates mild inflammation, while > 2 x 105 copies ml-1 of HBV DNA may justify further investigations. Precore status may be relevant for the interpretation of viraemia.     

Interferon-alpha therapy in chronic hepatitis B: early monitoring of hepatitis B e antigen may help to decide whether to stop or to prolong therapy

Hepatitis B e antigen (HBeAg) was quantified before, during and after interferon-α administration in a trial of 162 chronic hepatitis B patients treated for 16 or 32 weeks. In 139 of these patients we examined the prognostic value of the pretreatment level of HBeAg and the reduction in HBeAg level at weeks 4 and 8 for response at week 16. Multivariate analysis showed that the HBeAg pretreatment level is a highly significant predictor of response (judged as HBeAg and hepatitis B virus [HBV] DNA negativity), followed by a decrease in HBeAg from the start of therapy to week 8. During the first 8 weeks of therapy, a decrease in HBeAg of less than 40%, as observed in 30% of the patients, consistently resulted in non-response. After 16 weeks of treatment, non-responding patients were randomly assigned to receive no further treatment ( n =57) or prolonged treatment for another 16 weeks ( n =61). In both groups, changes in the HBeAg level from the start of (the first) therapy to week 8, but not the pretreatment HBeAg level itself, were significantly related to the response at week 52 (the end of follow-up). Changes in the HBV DNA level had no additional predictive value for response at weeks 16 or 52. Therefore, instead of sequential HBV DNA assessment, we recommend monthly monitoring of HBeAg during IFN-α therapy.     

Add‐on peginterferon alfa‐2a to nucleos(t)ide analogue therapy for Caucasian patients with hepatitis B ‘e’ antigen‐negative chronic hepatitis B genotype D

Nucleos(t)ide analogues (NAs) and peginterferon have complementary effects in chronic hepatitis B, but it is unclear whether combination therapy improves responses in genotype D‐infected patients. We conducted an open‐label study of peginterferon alfa‐2a 180 μg/wk added to ongoing NA therapy in hepatitis B e antigen (HBeAg)‐negative, genotype D‐infected patients with hepatitis B virus DNA <20 IU/mL. The primary endpoint was proportion of patients with ≥50% decline in serum HBsAg by the end of the 48‐week add‐on phase. Seventy patients received treatment, 11 were withdrawn at week 24 for no decrease in HBsAg, and 14 withdrew for other reasons. Response rate (per‐protocol population) was 67.4% (29/43) at week 48 (95% confidence interval [CI]: 51, 81) and 50.9% (28/55) at week 96 (95% CI: 38, 66). Median serum HBsAg decreased throughout peginterferon alfa‐2a treatment and was significantly lower than baseline at weeks 48, 72 and 96 (P < 0.001). Decreases in HBsAg of ≥0.5‐log₁₀ and ≥1‐log₁₀ were documented in 19 (44.2%) and 6 (14.0%) patients at week 48 and 6 (10.9%) and 17 (30.9%) patients at week 96. The proportion of patients with HBsAg <1000, <500, <100 and <10 IU/mL at ≥1 timepoint during treatment was 78.6% (n = 44), 57.1% (n = 32), 21.4% (n = 12) and 7.1% (n = 4). Interferon gamma‐induced protein 10 increased from baseline up to week 48, with week 12 levels significantly associated with response at week 48. Addition of peginterferon alfa‐2a to ongoing NA therapy significantly decreased HBsAg levels in HBeAg‐negative patients with genotype D infection (ClinicalTrials.gov NCT01706575).     

Relationship between hepatitis B core‐related antigen levels and sustained HBeAg seroconversion in patients treated with nucleo(s)tide analogues

Hepatitis B e antigen (HBeAg) seroconversion experienced during nucleo(s)tide analogue (NUC) therapy is often not sustained. We aimed to study whether hepatitis B core‐related antigen (HBcrAg) levels predict sustained HBeAg seroconversion in patients treated with NUCs. We studied HBeAg‐positive patients treated with NUCs for at least 6 months. We quantified HBcrAg at baseline and at the time of HBeAg seroconversion and studied the relationship with HBeAg seroconversion and subsequent relapse. HBcrAg was quantified at baseline in 196 patients; levels varied significantly by HBV genotype and correlated with HBsAg, HBV DNA and HBeAg. Baseline HBcrAg levels were lower in patients who achieved HBeAg seroconversion than in those who did not; the unadjusted hazard ratio (HR) was 0.802 (95% CI: 0.656‐0.980, P = 0.031); and this association was not sustained in multivariate analysis. HBcrAg remained detectable in all patients at the time of HBeAg seroconversion. Higher HBcrAg at the time of seroconversion was an independent predictor of relapse (adjusted HR: 1.855 (95% CI: 1.099‐3.133, P = 0.021), and none of the patients with HBcrAg < 4.90 log U/mL experienced relapse. Baseline HBcrAg is not an independent predictor of HBeAg seroconversion during NUC therapy. HBcrAg remains detectable in patients after HBeAg seroconversion. Patients with lower levels at the time of seroconversion have a higher probability of sustained HBeAg seroconversion.