肝病患者肝细胞内HBV DNA原位杂交研究——形态、分布及意义

应用生物素标记HBV DNA(乙肝病毒脱氧核糖核酸)作探针,对129例肝病患者肝组织作原位杂交研究。发现HBV DNA主要存在肝细胞浆内,可分为胞浆致密型、疏松型和包涵体型。HBV DNA阳性肝细胞在肝实质中分为三种型:小叶型、局灶型与散在点状分布。HBV DNA在慢性活动型肝炎中检出率最高(81％),显著高于肝硬化,慢性小叶型肝炎、急性肝炎及原发性肝癌组。乙肝复制指标阳性患者肝细胞内HBV DNA检出率明显高于非复制组;并观察到HBV DNA阳性肝细胞与肝细胞坏死灶关系密切,多数紧紧毗邻肝细胞坏死灶/或和位于坏死灶中间,尤以局灶型分布的HBV DNA阳性肝细胞为显著。     

应用原位杂交及免疫组化检测肝活检组织中流行性出血热病毒RNA及抗原

本文应用原位杂交及免疫组化技术对25例流行性出血热(EHF)患者肝活检组织进行了病毒RNA及其囊膜G_2蛋白的定位检测,结合光镜,认为肝细胞的变性、胞浆疏松化、点状坏死是EHF病毒直接侵犯并在其胞浆内增殖表达所致,肝组织的灶状坏死则主要是肝窦狭窄、枯否氏细胞增生导致微循环障碍引起的缺血性梗死。急性脂褐素沉积是病毒侵犯肝细胞的间接证据。研究还发现,肝细胞内病毒的多少与病程关系不明确,而与临床分型有一定相关,为探讨EHF的发病机制提供了分子水平的依据。     

我国25省市自治区丁型肝炎病毒感染的流行病学调查研究

应用本室先后建立的酶联免疫吸附法和核酸打点杂交法,检测我国25省布自治区、16个不同民族共9 758例乙型肝炎病毒感染者中的丁型肝炎病毒抗原、抗体和核酸。其中乙型肝炎病人4 714例,HBsAg慢性携带者5 044例。病人和携带者中丁型肝炎抗原阳性率分别为4.25％和3.0％,丁型肝炎抗体阳性率分别为1.46％和1.18％,丁型肝炎病毒核酸阳住率分别为3.70％和2.2％。在不同地区不同民族丁型肝炎的这些指标有一定差异。在16个民族中,维吾尔族、蒙族和藏族丁型肝炎抗体阳性率明显高于其他民族,黎族丁型肝炎核酸阳性率高于其他民族。但在1136例不同临床型乙型肝炎病人中,丁型肝炎感染率无明显区别。丁型肝炎感染与暴发性肝炎的关系有待进一步研究。     

丁型肝炎病毒抗原的表达纯化和抗原性分析

丁型肝炎病毒(Hepatitis D virus, HDV)是一种缺陷负链RNA病毒,其表面被乙肝病毒抗原(HBsAg)所包裹,内为丁型肝炎抗原(HDAg)及其基因组RNA。HDV基因组中有多个开放读码框架(ORF),其中只有抗基因组RNA链上的一个ORF编码的蛋白与HDAg相关[1,2]。HDV的抗原、抗体的测定是HDV感染诊断的主要标志,为了改进和提高HDV ELISA诊断试剂的质量,制备高纯度和高效价的HDAg尤为重要。为此,我们构建了HDAg基因表达株,以pQE表达系统为载体,利用该载体本身具备的6个组氨酸序列结构(6×histag)来纯化蛋白,直接提取高度纯化的HDAg,并应…     

不同肝病变组织中CD34、CD31、Ki-67的表达及意义

目的比较正常肝组织、慢性肝炎、肝硬化、肝细胞肝癌组织及肝转移腺癌中CD34、CD31、Ki-67不同表达,寻找有助于鉴别不同性质病变的生物学标记物.方法正常肝及病变肝组织标本共104例;其中,正常肝组织10例;慢生C型肝炎组织73例;肝硬化组织7例;肝细胞肝癌7例;结肠癌肝转移5例;乳腺癌肝转移2例.73例慢性C型肝炎组织全部为肝穿活检标本,其余组织均为手术切除标本.所有病例标本分别行CD34、CD31、Ki-67免疫组织化学染色,半定量评分系统评价染色结果.统计学分析结果数据.结果在非肿瘤组织,抗CD34阳性染色主要存在于汇管区,亦可见于汇管区周围的肝实质内血窦.阳性染色内皮细胞呈点状、线状、半环状及环状,散在或簇状分布.肿瘤组织内抗CD34阳性染色特征与非肿瘤组织相似,阳性染色血管在肿瘤组织内散布分布.CD34指数在各病变组中的表达排列顺序依次为:肝细胞肝癌>乳腺癌肝转移>结肠癌肝转移>肝硬化>慢性C型肝炎>正常肝组织,从正常肝组织至慢性肝炎至肝细胞肝癌,CD34表达明显增强.组织中,抗CD31阳性染色分布、定位、形态特征与CD34相似.CD31在慢性肝炎、肝硬化、肝细胞肝癌、结肠癌肝转移及乳腺癌肝转移组织中阳性表达率分别为:6.8%(5/73)、100%(7/7)、100%(7/7)、100%(5/5)、100%(2/2);肝癌组织中CD31染色强度明显大于非癌组织中,组间比较具有显著差异(P<0.05).Ki-67阳性染色细胞呈棕黄色核着色,散在分布于肝实质内.阳性染色细胞无形态特殊性,亦无分布上的特殊性.Ki-67在各病变组间的阳性表达率分别为:64.4%(47/73)、28.6%(2/7)、100%(7/7)、100%(5/5)、100%(2/2),其中以在结肠癌肝转移组织中表达最明显;组间比较具有非常显著差异(P<0.05).在正常肝脏、慢性C型肝炎、肝硬化、肝细胞肝癌CD34、CD31、Ki-67三种生物学标记物在同一标本同时表达的阳性率分别为:0%(0/0)、4.1%(3/73)、28.6%(2/7)、100%(7/7),CD34、CD31、Ki-67其中任两种同时表达的阳性率分别为0%(0/10)、63.0%(46/73)、100%(7/7)、100%(7/7).结论 CD34是慢性肝病、肝癌临床病理评价的指标之一,CD34与CD31、Ki-67同时分析有助于建立可靠的诊断.     

丁型肝炎病毒抗原的表达纯化和抗原性分析

丁型肝炎病毒(Hepatitis D virus,HDV)是一种缺陷负链RNA病毒,其表面被乙肝病毒抗原(HBsAg)所包裹,内为丁型肝炎抗原(HDAg)及其基因组RNA.HDV基因组中有多个开放读码框架(ORF),其中只有抗基因组RNA链上的一个ORF编码的蛋白与HDAg相关[1,2].     

慢性重度乙型肝炎患者肝组织中NF-κ Bp65和Caspase-3的表达及其意义

目的:探讨慢性重度乙型肝炎患者肝组织中NF-κBp65和Caspase-3的表达及其意义.方法:选择慢性重度乙型肝炎肝组织标本68例(慢重肝组),正常肝组织标本20例(对照组).采用免疫组化方法检测肝组织中NF-κBp65和Caspase-3的表达.结果:慢重肝组患者肝组织中NF-κBp65和Caspase-3阳性表达程度均明显强于对照组(P＜0.05);慢重肝患者死亡组肝组织中NF-κBp65和Caspase-3阳性表达程度均明显强于存活组(P＜0.05).Spearman相关分析发现,慢重肝组患者肝组织中NF-κBp65和Caspase-3表达之间有明显的正相关性(r=0.449,P＜0.05).结论:NF-κBp65和Caspase-3可能参与了慢性重度乙型肝炎的肝细胞凋亡过程,且具有一定相关性,并对判断患者预后有重要临床价值.     

CCl4和脂多糖诱导的急性肝损伤中肝细胞凋亡病理特点比较

目的比较CCl4和脂多糖诱导的两种急性肝损伤模型中肝细胞凋亡的病理特点。方法雄性BABL/c小鼠随机分为正常组、CCl4模型组与脂多糖模型组。CCl4和脂多糖模型小鼠分别腹腔注射0.03 mL/kg 50?l4/橄榄油混合液和10μg/kg脂多糖与900μg/kg半乳糖胺。9 h后采集标本,检测肝细胞凋亡,血清ALT、AST活性,肝组织SOD活性、MDA含量。结果与正常组比较,CCl4和脂多糖模型小鼠血清ALT与AST活性均明显升高(P<0.01),且脂多糖模型高于CCl4模型(P<0.01);模型小鼠均有明显肝细胞凋亡(P<0.01),且两组间无显著差异,但CCl4模型呈局灶性,以中央静脉周围为主;脂多糖模型呈弥漫性,肝细胞坏死与肝组织炎症更显著(P<0.01)。CCl4和脂多糖模型肝组织均SOD活性升高、而MDA含量下降(P<0.01),但CCl4模型更显著(P<0.05)。结论CCl4与脂多糖诱导的急性肝损伤小鼠模型均有明显的肝细胞凋亡,但CCl4模型以中央静脉区为主,脂质过氧化损伤更为明显;脂多糖模型呈弥漫性,肝细胞坏死与肝组织炎性反应较重。     

慢性重度乙型肝炎患者肝组织中NF-κBp65和Caspase-3的表达及其意义

目的：探讨慢性重度乙型肝炎患者肝组织中NF-κBp65和Caspase-3的表达及其意义。方法：选择慢性重度乙型肝炎肝组织标本68例（慢重肝组）,正常肝组织标本20例（对照组）。采用免疫组化方法检测肝组织中NF-κBp65和Caspase-3的表达。结果：慢重肝组患者肝组织中NF-κBp65和Caspase．3阳性表达程度均明显强于对照组（P〈0．05）;慢重肝患者死亡组肝组织中NF-κBp65和Caspase．3阳性表达程度均明显强于存活组（P〈O．05）。Spearman相关分析发现,慢重肝组患者肝组织中NF-κBp65和Caspase-3表达之间有明显的正相关性（r=0．449,P〈0．05）。结论：NF-κBp65和Caspase-3可能参与了慢性重度乙型肝炎的肝细胞凋亡过程,且具有一定相关性,并对判断患者预后有重要临床价值。     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布。结果表明（1）HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40％（55／136）和82％（112／136）。HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;（2）HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81％（133／164）及86％（141／164）。C33c抗原定位于癌细胞及肝细胞的浆内;核心抗原毁定位于癌细胞核中,又可定位于胞浆中。C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细胞在癌组织以弥漫核阳性常见,在癌旁肝组织以胸浆阳性为主;（3）HBxAg在肝细胞肝 癌中的检出率为75％（123／164）,C33c和HBxAg二者同时阳性占63％（103／164）。HCV感染在我国肝细胞肝癌中比较普遍,HCV和HBV重叠感染占相当比例,可能在肝细胞肝癌的发生中起着重要作用。     

Prevalence of HBV genotypes among Egyptian hepatitis patients

Phylogenetic analysis has led to the classification of hepatitis B virus into eight genotypes, designated A to H. The genotypes have differences in biological properties and show heterogeneity in their global distribution. These attributes of the genotypes may account not only for differences in the prevalence of hepatitis B virus mutants in various geographic regions, but also makes them responsible for differences in the clinical outcome and response to antiviral treatment in different population groups. Africa is one of the highly endemic regions of HBV with five genotypes (A–E) identified. Almost all patients in the Mediterranean area are infected with genotype D. However, there is little information of genotype distribution in Egypt. A total of 140 Egyptian patients with hepatitis B surface antigen (HBsAg) positive were enrolled in this study. Of the 140 patients, only 100 patients were HBV DNA positive and only these were included in the study. They were classified into 20 patients with acute hepatitis (AH), 75 patients with chronic active hepatitis (CAH) and 5 patients with hepatocellular carcinoma (HCC). HBV genotypes were determined using INNO-LiPA methodology which is based on the reversed hybridization principle. This study showed that genotype D constituted 87% of the total infections (75 CAH cases, 7 AH cases and 5 HCC cases). The other 13% showed mixed infections of D/F. These findings show that the most prevalent genotype in Egypt is genotype D especially in CAH and HCC patients while the mixed type D/F is only encountered in AH.     

Increased severity and morbidity of acute hepatitis in drug abusers with simultaneously acquired hepatitis B and hepatitis D virus infections.

Hepatitis D virus (delta agent) markers were present in 111 (36%) of 308 intravenous drug abusers who were positive for hepatitis B surface antigen (HBsAg), 52 of these having hepatitis D virus antigenaemia. IgM antibody to hepatitis B core antigen (anti-HBc IgM) was present in 92 out of 95 subjects tested, indicating that hepatitis D virus and hepatitis B virus infections had been acquired simultaneously. Hepatitis D virus markers were present in three out of four patients with fulminant hepatitis, and in 80 of 223 (36%) with mild or moderate hepatitis compared with four of 29 (14%) of those who were asymptomatic. These proportional differences were significant (p less than 0.001). Hepatitis D virus markers were present in twice as many patients positive for anti-HBc IgM requiring admission to hospital with acute hepatitis compared with outpatients attending a drug treatment centre. Tests on one patient showed complete disappearance of HBsAg, but hepatitis D antigen (HDAg or delta antigen) and hepatitis B e antigen (HBeAg) were still present in serum samples. All five patients with chronic active hepatitis had hepatitis D antibody (anti-HD) compared with seven of 24 (29%) with chronic persistent hepatitis (p = 0.008). Blocking anti-HD persisted for long periods after simultaneous infections with hepatitis B virus and hepatitis D virus but at lower titres than in patients with chronic liver disease.     

Prognosis for the patients with alcoholic and nonalcoholic liver disease

The purpose of this investigation was to determine the role of alcohol in development of progressive liver disease. For this purpose, 41 alcoholic patients were followed up for 5 years. Criteria for alcohol abuse was that the patients were enjoying 20 g alcohol daily in a period of 5 years for females and respectively 60 g daily for males. In the same time a group of 51 nonalcoholic patients with histologically proven chronic liver disease were investigated. In all 92 patients chronic liver disease and progression of the disease was proven by liver biopsy during a 5-years follow-up. In sera of all patients the markers of hepatitis viruses B, D and C were continuously determined and chronic viral hepatitis was excluded. Also, autoimmune chronic hepatitis was excluded. The results of the investigation showed that alcoholics develop cirrhosis hepatitis, in most cases 78.04%. The most progressive chronic liver diseases--cirrhosis and hepatocellular carcinoma--are significantly present among nonalcoholics (p < or = 0.05). In the mentioned investigation a large group of 51 patients with severe chronic hepatitis without a proven etiology of disease was found and it deserves priority in future research.     

应用酶联免疫吸附试验检测不同类型乙型肝炎中激活补体类HBsAg循环免疫复合物

本文以抗人C_(?)的羊IgG为包被抗体,以HRP-HBs抗体为指示抗体,建立了可检测激活补体类HBsAg循环免疫复合物(HBsAg/C3-CIC)的C_3捕捉法酶联免疫吸附试验。检测了236例六种类型临床诊断为乙型肝炎的病人血清标本,其阳性率分别为:无症状携带者(ASC)12.9％(4/31),急性肝炎(AH)36.7％(22/60),慢性迁延性肝炎(CPH)33.3％(7/21),慢性活动性肝炎(CAH)59.6％(34/57),重型肝炎(SH)77.8％(14/18),肝炎后肝硬化(PLC)67.3％(33/49),阳性率与肝损严重程度明显相关(P<0.01)。认为HBs-Ag/C3-CIC可能在乙型肝炎病毒引起的慢性活动性肝炎、重型肝炎和肝炎后肝硬化的发病过程中起重要作用,并可作为乙型肝炎的诊断、临床分型和预后判断的指标之一。     

Tumor necrosis factor-alpha and interleukin-4 in the blood sera of chronic hepatitis patients

The concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) in the blood sera of chronic hepatitis patients was, on the average, reliably higher than in the control group. A more pronounced increase in the concentration of TNF-alpha in the blood sera was observed in patients with chronic hepatitis of viral etiology in comparison with toxic hepatitis. The pronounced cytokine response of type Th2, manifested by the excessive production of IL-4, was typical for hepatitis B virus and hepatitis C virus infections, but not characteristic of hepatitis D virus infection. The replication activity of hepatotropic viruses induced a powerful cytokine response. In the presence of active virus replication in patients with chronic hepatitis B the levels of both TNF-alpha and IL-4 in their blood sera proved to be reliably higher than in patients with hepatitis B virus in the interactive phase.     

Detection of hepatitis B virus antigens in paraffin-embedded liver specimens from the Amazon region, Brazil

Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in difFerent other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4%) histological samples were HBsAg reactive and 5 (6.3%) were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens.     

Detection of a new antibody system reacting with Dane particles in hepatitis B virus infection.

Antibodies in the serum reacting with antigens on the surface of radiolabelled Dane particles distinct from hepatitis B surface and core antigens (HBsAg and HBcAg) were detected, using a double antibody precipitation assay, in 12 out of 15 patients early in the course of acute type B hepatitis and at the time of disappearance of circulating Dane particles. No such antibody activity was found in 15 of the 16 patients with HBsAg-positive chronic active hepatitis, 13 of whom had complete Dane particles in the serum. In a group of 16 asymptomatic HBsAg carriers (without Dane particles in serum) antibody activity was shown in nine. This demonstration of antibodies precipitating Dane particles may be relevant to the clearance of circulating hepatitis B virions and the termination of infection in acute type B hepatitis. Their absence in all but one of the cases of chronic active hepatitis might explain why the virus infection persists in this group of patients.     

慢性乙型肝炎病毒感染对妊娠期糖尿病及妊娠结局的影响

目的:探讨慢性乙型肝炎病毒(HBV)感染对妊娠期糖尿病(GDM)及妊娠结局的影响,为妊娠期产妇慢性HBV感染预防提供参考。方法:回顾性分析2015年2月-2017年2月在我院住院分娩的2615例慢性HBV感染产妇的临床病历资料,根据《慢性乙型肝炎防治指南》(2015年版)诊断标准,将所有产妇分为4组:慢性HBV携带者(A组)1128例、乙型肝炎e抗原(HBe Ag)阳性慢性乙型肝炎(B组)406例、HBe Ag阴性慢性乙型肝炎(C组)307例、非活动性乙型肝炎表面抗原(HBs Ag)携带者(D组)774例,并收集同期入院的823例HBV阴性产妇为对照组(E组)。比较各组的GDM发生率及不良妊娠结局发生率。结果:2615例慢性HBV感染产妇中,共发生GDM 866例,发生率为33.12%。B组与C组GDM发生率分别为38.92%、37.46%,均大于E组的30.74%(P0.05)。A组、B组、C组的妊娠期高血压疾病(PIH)发生率分别为7.98%、8.87%、9.77%,均高于E组的3.52%(P0.05);A组、B组、C组及早产发生率分别为3.10%、3.94%、4.56%,均高于E组的0.49%(P0.05)。C组的新生儿窒息发生率为1.63%,高于E组的0.36%(P0.05)。结论:产妇慢性HBV感染若合并肝功能受损或肝组织学病变,可能增加GDM的发生率,若HBV病毒复制活跃,可能导致PIH及早产发生风险增加。     

Trace elements in viral hepatitis

In this study, serum trace elements, including selenium (Se), zinc (Zn), copper (Cu), were determined by using Atomic Absorption Spectrophotometer (SpectrAA 250 Plus Zeeman, Varian, Australia) in sera of patients with viral hepatitis (A, B, C, D, E) cases (n = 102), and statistically compared with the controls (n = 52). In viral hepatitis, Cu levels were found as 3.23 ± 1.02 mg/L, and this value was significantly higher than the control group (1.13 ± 0.21) (p < 0.01). Both, Se and Zn levels found to be significantly low in viral hepatitis cases (p < 0.01). While Se level was 81.4 ± 26.01 μg/L in viral hepatitis (n = 101), it was found to be 166.15 ± 4.58 μg/L in healthy individuals. Meanwhile, Zn levels were 0.230 ± 0.081 mg/L and 0.748 ± 0.392 mg/L in hepatitis cases (n = 101) and the control group, respectively. There was no difference amongst viral hepatitis groups classified in regard with agents and clinical manifestation, such as A, acute hepatitis B, chronic hepatitis B, C, D and E. Previously, it was indicated that absorption disorders in gastrointestinal system, especially in chronic cases, were not main causes of decrease of trace elements by iron and several other parameters in sera of the cases. Therefore, we suggest that decrease in Zn and Se levels and elevation in Cu levels are probably resulted from defence strategies of organism and induced by the hormone-like substances.     

Chronic active hepatitis in alcoholics

In 42 chronic alcohol abusers liver biopsy was performed and chronic active hepatitis was diagnosed in 11 cases. In 4 cases etiology could be attributed to chronic HBV infection--they were positive for HBsAg in serum, three were positive for HBeAg and one case had anti-delta antibodies. In 7 cases etiology was obscure, at least in some of them alcohol could have been the underlying factor. Liver disease in these particular cases was clinically more severe than chronic active hepatitis due to infection with HBV and non-A, non-B viruses in non-drinkers. Two cases progressed into liver decompensation despite 1 and 2 years of abstinence, respectively. Chronic active hepatitis in alcoholics constitutes a frequent pathology, its etiology is variable, in some cases obscure.