肝硬化患者乙肝表面抗原的定量检测及意义

目的：探讨HBsAg定量测定在乙肝相关性肝硬化病程中的变化和意义。方法：选择乙肝相关性肝硬化患者60例纳入实验对象,根据2000年9月（西安）第10次全国病毒性肝炎学术会议修订的《病毒性肝炎防治方案》中的诊断标准分为代偿期组和失代偿期组,其中代偿期组35例,失代偿期组25例。另选取20例乙型肝炎病毒携带者作为对照组。应用电化学发光免疫分析法测定患者血清中HBsAg和HBeAg滴度,免疫荧光定量PCR法检测HBVDNA载量。结果：对照组、肝硬化代偿期组和肝硬化失代偿期组HBsAg滴度分别为：2574.73±3252.27COI、5494.35±2129.84COI和6921.25±1957.60COI,三组之间差别均有统计学意义（P〈0.05）。肝硬化代偿期组中,HBsAg滴度与HBVDNA、HBeAg水平呈负相关性（P〈0.05（）r=-0.350;r=-0.514）。肝硬化失代偿期组中,HBsAg滴度与HBVDNA及HBeAg水平均无明显相关性（r=-0.020;r=0.154）。结论：肝硬化失代偿期HBsAg滴度明显高于肝硬化代偿期,代偿期HBsAg滴度高于HBV携带者组,即HBsAg滴度随肝脏疾病进展呈阶梯型递增。肝硬化代偿期,HBsAg滴度与HBVDNA、HBeAg水平呈负相关性,HBsAg水平可以作为评估病毒复制的参考指标。肝硬化失代偿期,HBsAg滴度与HBVDNA和HBeAg无相关性,不能反映病毒复制水平,不能作为评估病毒复制的参考指标。     

Novel evidence suggests Hepatitis B virus surface proteins participate in regulation of HBV genome replication

Naturally occurring mutations in surface proteins of Hepatitis B virus (HBV) usually result in altered hepatitis B surface antigen (HBsAg) secretion efficiency. In the present study, we reported two conserved residues, M75 and M103 with respect to HBsAg, mutations of which not only attenuated HBsAg secretion (M75 only), but also suppressed HBV genome replication without compromising the overlapping p-gene product. We also found M75 and M103 can initiate truncated surface protein (TSPs) synthesis upon over-expression of full-length surface proteins, which may possibly contribute to HBV genome replication. However, attempts to rescue replication-defective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful, which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.     

Genetic polymorphisms of CYP2E1 and DNA repair genes HOGG1 and XRCC1: Association with hepatitis B related advanced liver disease and cancer

A population based case–control study was designed to explore the genetic risk factors for hepatitis B virus (HBV) related liver disease susceptibility. A total of 424 subjects comprising 210 controls, 50 acute HBV (AVH), 84 chronic HBV (CHBV), 25 HBV related cirrhosis and 55 HBV related hepatocellular carcinoma (HCC) cases were included in the study. PCR-RFLP was used for the genotyping of Cyp2E1*5B, hOGG1 codon 326 and XRCC1 codon 399. Compared to controls, Cyp2E1 rsaI variant c2 genotype increased the risk of HBV related liver disease severity by 2.68 fold, the highest for HCC cases (3.981 folds, p = 0.106); and was associated with higher histology activity index (HAI) (p < 0.001) in CHBV patients. Cyp2E1 and hOGG1 variants were independently associated with a significantly higher fibrosis score in CHBV group. Analysis of gene–gene interaction studies showed an increased risk of HCC, cirrhosis and CHBV in a Cyp2E1 variant + XRCC1 variant combination (p < 0.001); and hOGG1 variants + XRCC1 variants. A mutually independent heterozygous hOGG1 and XRCC1 combination resulted in a decreased risk of HBV related liver disease. On the other hand, a wild-type hOGG1 and XRCC1 combination was associated with a significantly higher risk of AVH (p = 0.010) but a lower risk of CHBV (p = 0.032) and HCC (p = 0.006). The gene–gene interactions were also associated with a significant increase in HAI and fibrosis score in CHBV patients. Cyp2E1, hOGG1 and XRCC1 genotypes significantly alter the risk of HBV related liver disease susceptibility and severity, independently or through gene–gene interaction.     

一种联合检测乙型肝炎病毒前S1抗原与核心抗原方法的建立及其与病毒核酸检测结果的一致性

本研究以与血清中HBV DNA含量高度相关的两种HBV抗原(前S1抗原与核心抗原)为靶标,建立了联合检测这两种HBV核酸相关抗原(NRAg)的双抗体夹心法ELISA试剂.对系列稀释血清的检测表明,该试剂的平均分析灵敏度为103.2基因组拷贝/mL(95%可信限102.2-4.2基因组拷贝/mL),显著高于前S1抗原或核心抗原的单独检测.对994份HBsAg阴性血清的检测结果表明NRAg ELISA的特异性为99.7%(95%可信限:99.1%～99.9%).对271份临床慢性肝炎血清进行检测,结果NRAg ELISA与HBV DNA结果的总符合率达96.3%(95%可信限:93.3%～98.2%),NRAg ELISA的读值/临界值比(S/CO)与HBV基因组拷贝数呈正相关.利用NRAg试剂,发现了1例HBsAg"a"抗原表位突变的变异株.这些结果显示HBV NRAg ELISA与HBV DNA具有高度相关性,并能够检测出HBsAg抗原变异株,有望成为HBsAg变异株筛选的有力工具,并为广大基层医疗单位提供一种便捷的替代HBV DNA定性检测的手段.     

Genetic variants of TNFα, IL10, IL1β, CTLA4 and TGFβ1 modulate the indices of alcohol-induced liver injury in East Indian population

Alcohol induced liver disease or alcoholic liver disease (ALD), a complex trait, encompasses a gamut of pathophysiological alterations in the liver due to continuous exposure to a toxic amount of alcohol (more than 80g per day). Of all chronic heavy drinkers, only 15-20% develops hepatitis or cirrhosis concomitantly or in succession. Several studies revealed that inter-individual as well as inter-ethnic genetic variation is one of the major factors that predispose to ALD. The role of genetic factors in ALD has long been sought for in ethnically distinct population groups. ALD is fast emerging as an important cause of chronic liver disease in India; even in populations such as "Bengalis" who were "culturally immune" earlier. While the genetic involvement in the pathogenesis of ALD is being sought for in different races, the complex pathophysiology of ALD as well as the knowledge of population level diversity of the relevant alcohol metabolizing and inflammatory pathways mandates the need for well designed studies of genetic factors in ethnically distinct population groups. An array of cytokines plays a critical role as mediators of injury, inflammation, fibrosis and cirrhosis in ALD. We, therefore, studied the association of polymorphisms in five relevant cytokine genes with "clinically significant" ALD in an ethnic "Bengali" population in Eastern India. Compared with "alcoholic" controls without liver disease (n=110), TNFα -238AA genotype, IL1β -511CC genotype, TGFβ1 -509CC genotype and IL10 -592AA genotype were significantly overrepresented in ALD patients (n=181; OR=2.4 and 95% CI 1.2-5.5, P(genotype)=0.042, P(allelic)=0.008; OR=2.7 and 95% CI 1.2-5.9, P(genotype)=0.018, P(allelic)=0.023; OR=4.7 and 95% CI 1.7-13.1, P(genotype)=0.003, P(allelic)=0.014; and OR=2.2 and 95% CI 1.1-4.8, P(genotype)=0.04, P(allelic)=0.039 respectively). Moreover a cumulative genetic risk analysis revealed a significant trend for developing ALD with an increase in the number of risk alleles on IL10 and TGFβ1 loci among alcoholics. The risk genotype of IL1β and TGFβ1 also influences the total bilirubin, albumin and alanine aminotransferase levels among alcoholic "Bengalis". The present study is the first case-control study from Eastern India that comprehensively identified polymorphic markers in TNFα, IL10, IL1β and TGFβ1 genes to be associated with ALD in the Bengali population, accentuating the significance of genetic factors in clinical expressions of ALD.     

超声波清洗杀灭微生物效果的实验研究

目的:观察超声波清洗在相等条件及不同的时间对微生物杀灭作用的影响。方法:采用悬液法在相同时间内分别对大肠埃希菌;白色假丝酵母菌及HBsAg阳性血清进行了杀灭效果测试。结果:超声波清洗1min-15min对大肠埃希菌杀灭率分别为18.2%-94.2%;对白色假丝酵母菌为1.4%-49.7%。而超声波清洗1min-15min对HBsAg阳性血清的抗原性无完全破坏性作用。结论:单一超声波清洗杀灭微生物的效果是随时间的延长杀灭微生物的效果越好。扫描电镜显示,随着超声时间的延长,细菌的细胞壁破坏越严重。而对HBsAg阳性抗原在短时间内不能被完全破坏。     

荧光素标记检测乙型肝炎病毒表面抗原特异性T淋巴细胞方法的探讨

用绿色荧光前体化合物Calcein AM标记靶细胞,经过与杀伤性T淋巴细胞(CTL)效应细胞数小时的孵育,通过分析培养上清中的荧光强度检测CTL效应.通过对一系列标记条件的研究:不同浓度Calcein AM标记靶细胞、不同洗涤缓冲液、不同标记细胞浓度、不同洗涤次数、不同裂解液配方,确定CalceinAM荧光素标记法的最佳条件.用该方法检测乙型肝炎病毒表面抗原的DNA疫苗免疫Balb/c小鼠对P815S细胞的CTL效应,E/T比为10时,杀伤效率接近饱和,达到65%.通过荧光显微镜直接观察杀伤后的P815S细胞,细胞破裂程度与计算出的杀伤效率有一定相关性.     

新疆野生啮齿动物嗜肝病毒的感染

[目的]为调查新疆野生啮齿动物的血清中嗜肝病毒的存在状态。[方法]应用乙肝表面抗原诊断试剂盒和乙肝表面抗体诊断试剂盒,对野生啮齿动物进行了血清学调查。[结果]灰旱獭(Marmotabaibaci-na)的阳性率为17.8%(24/135)、长尾黄鼠(Citellusundulatus)阳性率为17.4%(12/69)和赤颊黄鼠(Citelluserythrogenys)阳性为(8/15)。作者认为赤颊黄鼠可以作为乙型肝炎病毒潜在的动物模型。     

消毒剂过氧化氢脲性能的实验研究

过氧化氢脲为过氧化氢类消毒剂,实验表明,其5%水溶液25℃时有良好的的杀菌作用。杀灭细菌营养繁殖体需2min,作用15min可将细菌芽孢全部杀灭。作用30min可将HbBsAg完全灭活。提高浓度、温度,延长作用时间或降低pH值,可加强其杀菌作用。