乙型肝炎病毒核心DNA疫苗研究进展

第一代乙型肝炎疫苗是从乙型肝炎表面抗原(HBsAg)无症状携带者血清中分离HBsAg颗粒,经灭活后制成的乙型肝炎血源疫苗;第二代乙型肝炎疫苗即为HBSAg基因重组疫苗,该疫苗可诱导体液免疫应答产生中和抗体,但不能诱导细胞免疫应答,     

甲肝灭活疫苗在特殊人群中的应用

摘要：随着我国甲型肝炎疫苗计划免疫的实施,甲型肝炎的发病率在迅速下降。对于特殊人群如静脉注射
毒品者、男同性恋、ＨＩＶ 感染者、慢性肝病患者等的免疫策略仍未明确。国内外相关研究显示,特殊人群
往往暴露甲型肝炎病毒后发病风险较高,接种甲型肝炎疫苗后应答反应较差,且疫苗保护时间相对较短,
对此类人群应按何种免疫程序接种才能达到较好的保护效果,仍需进一步研究。建议特殊人群的甲型肝炎
疫苗免疫列入国家免疫策略之中。
关键词：甲型肝炎灭活疫苗;吸毒人群;慢性肝病患者;ＨＩＶ 感染者;男男性行为者
中图分类号：Ｒ１８６　　文献标识码：Ａ　　文章编号：１００９ ６６３９ （２０１４）０４ ０３６８ ０４     

肝组织中HBV cccDNA荧光定量聚合酶链反应检测法的建立

目的 建立和优化一种灵敏、特异的检测肝组织中乙型肝炎(乙肝)病毒(HBV)共价闭合环状DNA(cccDNA)荧光定量聚合酶链反应(荧光定量PCR).方法 设计检测HBV DNA(tDNA)和cccDNA特异性引物及探针,用3.44×100-3.44×109copies/μl含HBV全基因组序列(C基因型)质粒作为标准品,建立荧光定量PCR检测标准曲线.取33例乙肝肝癌患者肝组织标本、13例慢性乙肝患者肝活检组织标本和10例非乙肝患者肝组织标本,验证该法灵敏度和特异度.提取肝组织中DNA,取一部分进行质粒安全ATP依赖的DNA酶(PSAD)酶切;另一部分不酶切作为tDNA和β-globin检测样本,分别进行HBV cccDNA、tDNA和p-globin定量检测,以β-globin为参比,对每个细胞的HBV cccDNA和tDNA含量进行标准化.结果 检测肝组织中HBV cccDNA和tDNA定量的线型范围均为3.44 × 100-3.44×109 copies/μl.检测HBV cccDNA和HBV DNA下限均为3.44×100copies/μl.33例乙肝肝癌患者和13例慢性乙肝患者的肝组织中HBV cccDNA最低含量分别为0.003 copies/cell和0.031 copies/cell;检测10份非乙肝肝癌患者的肝组织标本均为阴性.应用PSAD消化肝组织中提取的DNA可减少假阳性,提高cccDNA检测法特异度达7.24× 102倍.对2例乙肝肝癌患者的肝组织标本重复检测5次,Ct值的变异系数为0.224%～0.609%.结论 该方法灵敏度和特异度高,重复性好,可用于检测肝组织中HBV cccDNA.

Abstract：

Objective To establish and optimize a sensitive and specific quantitative realtime polymerase chain reaction(PCR)method for detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA)in liver tissue. Methods Specific primers and probes were designed to detect HBV DNA(tDNA)and cccDNA. A series of plasmids(3.44 × 100-3.44 × 109 copies/μl)containing a full double-stranded copies of HBV genome(genotype C)were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma(HCC), 13 Chronic hepatitis B patients(CHB)and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase(PSAD)for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount(copies/cell)of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. Results The standard curves of real-time PCR with a linear range of 3.44 × 100 to 3.44 × 109 copies/μl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 100 copies/μl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 102 times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficience of variations on cycle threshold (Ct)were between 0.224%-0.609%. Conclusion A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.     

HBV cccDNA检测临床应用研究进展

乙型肝炎病毒共价闭合环状DNA(hepatitis Bvirus covalently closed circular DNA,HBVcccDNA)是嗜肝病毒复制过程中产生的一种重要复制中间体,在宿主细胞核内积聚,形成HBV cccDNA池,半衰期长,现有抗病毒药物不能清除,是宿主肝细胞持续     

慢性乙肝患者石蜡包埋肝组织中HBV cccDNA定量检测方法的建立

目的建立慢性乙肝患者微量石蜡包埋肝组织共价闭合环状DNA(HBV cccDNA)定量检测方法。方法以37例慢性乙肝患者甲醛固定石蜡包埋肝组织为研究对象,提取肝组织HBV DNA,经不降解质粒的ATP依赖的DNA酶(PSAD)消化后,利用滚环扩增加跨缺口实时荧光PCR扩增技术检测肝组织中HBV cccDNA含量,以β-actin为内参照。通过已知浓度的模板DNA进行梯度稀释鉴定该方法的灵敏度,并对该方法进行批内和批间重复性检测。应用该方法分析37例慢性乙肝患者肝组织HBV cccDNA与血清总HBV DNA、肝组织总HBV DNA的关系,以及肝组织HBV cccDNA和HBV DNA与血清HBeAg表达之间的关系。结果成功建立了微量石蜡包埋肝组织HBV cccDNA的定量检测方法,该方法具有较好的特异性、灵敏度和稳定性。HBeAg阳性者肝组织中cccDNA含量明显高于HBeAg阴性者,HBV cccDNA水平与血清总HBV DNA水平(R2=0.48,P=0.042)及肝组织总HBV DNA水平(R2=0.63,P=0.001)呈正相关。结论该方法可特异灵敏地定量检测微量石蜡包埋组织切片中的HBV cccDNA。     

猴痘病毒感染

猴痘是一种罕见的病毒性疾病 ,主要发生于中部与西部非洲热带雨林国家。该病于 1 958年首次在丹麦哥本哈根一实验室绿猴中发现 ,可引起实验用灵长类动物发病 ,因而得名为猴痘。此后在非洲动物血样检测时 ,发现许多非洲啮齿动物有猴痘病毒感染。该病毒也可感染小鼠、大鼠和兔。1 970年刚果首次报告猴痘病毒感染人类的病例[1 3 ] 。一、美国猴痘流行情况2 0 0 3年 6月 1 3日美国疾病预防控制中心 (CDC)报告 ,美国境内数个州发生人类猴痘爆发。 2 0 0 3年 6月 1 0日美国CDC接到威斯康星、伊利诺伊和印第安纳州共报告 53例在接触发病的宠物…     

中波紫外线对人前臂皮肤致红斑效应的实验研究

本文着重研究了中波紫外线对人前臂皮肤的急性损伤作用即红斑生成作用，将受试者的前臂分成五个不同剂量的照射点，观察照射后２４ｈ内红斑发生情况，求红斑发生率与受照剂量关系的回归曲线，得出ＭＥＤ５０为１８３．８Ｊ／ｍ２，其９５％可信限为１６８．６～２００．６Ｊ／ｍ２。     

肝组织中HBV cccDNA荧光定量聚合酶链反应检测法的建立

Objective To establish and optimize a sensitive and specific quantitative realtime polymerase chain reaction(PCR)method for detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA)in liver tissue. Methods Specific primers and probes were designed to detect HBV DNA(tDNA)and cccDNA. A series of plasmids(3.44 × 100-3.44 × 109 copies/μl)containing a full double-stranded copies of HBV genome(genotype C)were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma(HCC), 13 Chronic hepatitis B patients(CHB)and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase(PSAD)for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount(copies/cell)of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. Results The standard curves of real-time PCR with a linear range of 3.44 × 100 to 3.44 × 109 copies/μl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 100 copies/μl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 102 times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficience of variations on cycle threshold (Ct)were between 0.224%-0.609%. Conclusion A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.     

紫外线致电焊作业工人皮肤损伤的调查

本调查探讨了电弧光辐照与电焊工皮肤损伤的剂量 反应关系以及各危险因素的作用强度。1.对象与方法 :选择北京、天津、齐齐哈尔市 8个工厂接触电焊弧光≥ 1年的作业人员 82 4人为接触组 ,各厂从未接触电弧光的工作人员 2 2 3人为对照组。采用问卷和体检相结合的方法 ,内容包括 :年龄、性别等一般情况 ,职业史 ,疾病史 ,面罩、眼镜、工作服、手套等防护措施的使用情况及皮肤的症状、体征等。紫外线辐照度测定 :用中国计量科学院监制的ＳＵＶ 3型紫外辐射照度计 ,分别测量工作位面罩内、外眼及面部紫外线 ( 36 5、2 90～ 32 0和 2 5 4ｎｍ)的…     

脂肪肝的流行病学研究进展

脂肪肝是一种常见的临床现象,但不是一种独立的疾病。正常人的肝脏中,脂质含量占肝湿重的2％～4％,其中磷脂占50％以上,甘油三酯(TG)占20％,游离脂肪酸(FFA)占20％,胆固醇约占7％,其余为胆固醇酯。当肝细胞内脂质蓄积超过肝湿重的5％,或组织学上每单位面积有1/3以上肝细胞脂肪变时,称为脂肪肝。根据肝细胞内脂肪含量,可将脂肪肝分为轻、中、重三型。根据是否有过量酒精摄入,可将脂肪性肝炎分为酒精性脂肪性肝炎(ASH)和非酒精性脂肪性肝炎(NASH)。本文对脂肪肝的流行病学研究进展作一简要综述。