TTV DNA在肝组织中表达研究

目的 探索TTV DNA在肝组织表达特点。方法 选择TTV DNA高保守区设计一对引物，采用PCR扩增法，以地高辛标记TTV ORF1部分基因序列，合成Gla，G2b两种亚型的双链TTV DNA探针。应用巢氏PCR法筛选出TTV DNA阳性的慢性肝炎、肝硬化、原发性肝癌病人的肝组织标本共60份，同时用两种探针进行原位杂交。结果 TTV DNA主要见于肝细胞、肝癌细胞的细胞核内，极少数肝细胞胞浆内TTV DNA呈弱阳性。TTV DNA阳性细胞在慢性肝炎、肝硬化、肝癌组织中均呈散在分布。肝癌组织与癌周组织中TTV DNA阳性细胞密集程度差别不明显。结论 TTV是一种嗜肝病毒，TTV DNA在被感染肝组织及肝癌的细胞核内表达及复制。     

聚合酶链反应评价原位杂交检测慢性HBV感染者肝内HBV DNA的结果

用地高辛探针原位杂交检测慢性HBV感染者肝内HBV DNA,聚合酶链反应(PCR)检测血清HBV DNA评价病毒复制状态。发现肝内HBV DNA阳性肝细胞,在14例肝组织病变不活动的HBeAg阳性慢性无症状HBV携带者(ASC)呈弥漫性分布;而在     

原位杂交检测肝组织中TTV DNA研究

目的:研究输血传播病毒(transfusion transmitted virus,TTV)DNA在肝炎患者肝组织中的存在,证实TTV是一种新型嗜肝病毒。方法:用地高辛标记TTV DNA(Dig-TTV DNA)探针,以原位杂交技术进行血清PCR检测,发现单一TTV感染13例,TTV、HBV混合感染4例,非甲～非庚和非TTV感染的肝炎患者6例。与此同时对肝组织进行检测,进行肝组织学、免疫组化、肝功能和临床特点等分析。结果:23例肝组织中TTV DNA阳性15例(65.22％),其中单一TTV感染11例(84.62％),TTV DNA混合感染者3例(75.00％),非甲～非庚和非TTV感染1例,临床分型急性肝炎5例(55.56％),慢性肝炎10例(71.43％)。TTV DNA阳性细胞在急性肝炎患者系弥漫分布于肝小叶内,在慢性肝炎于汇管区附近较为密集。TTV DNA主要存在于肝细胞核,少数位于肝细胞浆内,以核型为主。23例肝组织都有不同程度病理改变,大多数较轻,12例肝组织免疫组化检测HBsAg、HBcAg、HCVNS_3、HGVNS_5均阴性,肝功能均有轻～中度损害,大多数病人有乏力、纳差、尿黄症状,部分病人有肝脾肿大、皮肤或巩膜黄染体征。结论:TTV可能是一种新型嗜肝病毒。     

肝细胞癌患者血清TT病毒感染检测分析

目的 调查TT病毒在肝细胞癌患者及南通地区正常人群中的感染状况。方法 采用巢式聚合酶链反应(n—PCR)法，对145例肝细胞癌患者、143例献血员和375例体检者血清进行了检测分析。结果 145例肝细胞肝癌患者中有48例TTV DNA阳性(33．1％)，献血员中有13例TT病毒DNA阳性(9．1％)，正常体检人群中有39例TT病毒DNA阳性(10．4％)。肝细胞癌患者中TT病毒感染率明显高于献血员和正常人群。结论 TT病毒与肝细胞癌发生有一定的关系，是导致肝癌的一个危险因素。     

慢性肝病患者肝组织中HGV抗原的表达及意义

目的探讨慢性肝病患者组织中庚型肝炎病毒（ＨＧＶ）表达与意义。方法应用免疫组织化学方法以鼠抗ＨＧＶＮＳ５甲克隆抗体检测１４２例慢性肝病患者肝组织中ＨＧＶ抗原，部分患者采用ＲＴ－ＰＣＲ方法检测其血清中ＨＣＶＲＮＡ。结果１４２例肝病患者中，２９例（２０．４％）组织中检出ＨＧＶ抗原，肝硬化组（４２．９％，１２／２８）较慢性肝炎（１５．９％８／１１）和肝癌（１４．３％，９／６３）组检出率高。阳性信号位于胞浆中，阳性细胞可成散在、簇状或弥漫性分布。阳性细胞周围可有炎性坏死；肝癌患者抗原阳性细胞主要位于癌旁肝组织，癌巢中仅偶见少数散在分布的阳必细胞；绝大多数组织抗原阳性者其血清ＨＧＶＲＮＡ为阳性。有４例患者组织中ＨＧＶ抗原阳性但其血清ＨＧＶＲＮＡ阴性。结论ＨＧＶ可在慢性肝病包括肝细胞肝癌患者肝组织中表达，ＨＧＶ感染在慢性肝炎病期进展及肝癌发生中具有一定意义。     

山东地区输血传播病毒(TTV)检测及部分基因克隆和序列测定

目的 了解TTV山东分离株感染状况及基因变异的情况。方法应用逆转录聚合酶链反应(PCR)检测26例山东地区非甲-庚型肝炎和12例肝细胞癌患者血清中TTV DNA,并对阳性扩增的产物利用PCR技术直接克隆和测序,分析其基因变异的情况。结果26例非甲-庚型肝炎中11例TTV DNA阳性(42％)。对其中两株(TTVSD4、TTVSD5)部分基因克隆测序,并与日本株(ABOO8394)相比较,其核苷酸旬同源性为99.9％和100％。而12例肝细胞癌患者中3例TTV DNA阳性(25％),对其中一株(TTVSD6)部分基因克隆与测序,与日本株(ABOO8394)相比较,其核苷酸序列同源性为99％;TTV山东株三株间核苷核同源性均为99％。结论本研究证实山东地区非甲-庚型肝炎和肝细胞癌患者中存在着较高TTV感染,TTV感染可能具有嗜肝性,而且可能与肝功能损害有关,是引起非甲-庚型肝炎的重要病原。     

地高辛素探针原位杂交用于肝病及肝癌的分子原理研究

建立了敏感性高、特异性强的地高辛素探针原位杂交技术,并用于肝病和肝癌分子病理学的研究。我们发现慢性乙型肝炎与肝轻微病变52例中43例,肝硬化6例中5例,12例肝癌中10例癌周组织,8例癌组织内可检测出HBV DNA。上述结果揭示了慢性HBV感染与肝癌的分子病理联系。结果还发现原位杂交与转移杂交或PCR检测的结果有较好的一致性。地高辛素探针原位杂交技术可广泛用于消化系疾病分子病理研究。     

经血传播病毒在肝脏及肝外组织的感染状况

目的探讨TTV在肝脏及肝外组织中的定位、分布及其意义。方法通过PCR方法扩增TTV基因组ORF2中123bp的DNA片段，构建TTVDNA质粒并克隆，序列分析表明与日本ABO11494序列具有高度同源性。采用地高辛标记TTVDNA探针并与组织进行原位杂交，检测了22例因肝病死亡患者的肝脏、脾脏、肾脏、胃、小肠等组织内TTVDNA感染状况。结果8例肝脏检出TTVDNA，5例肾脏、４例脾脏、２例小肠、２例胃检出TTVDNA。阳性信号在组织中分布呈局灶性或散在胞浆型。肝脏感染度较其它组织高。阳性细胞与组织炎症坏死无固定病理解剖学关系。结论支持TTV有嗜肝性的观点；TTV可以感染肝外多种组织并可能导致TTV持续感染。     

经输血传播病毒在肝外组织中的检测及其意义

目的 探讨经输血传播病毒（TTV）在肝及肝外组织的定位、分布及其意义。方法 采用PCR及原位杂交方法,对13例非甲－非庚型病毒性肝炎患者的尸解肝、胰、肾、脾、睾丸和心脏石蜡包埋组织中的TTV DNA进行了检测。结果 5例肝组织、3例肾脏及胰腺、腺脏组织各2例中检出了TTV核酸杂交信号,2例睾丸及心肌组织各检出1例,阳性信号主要定位于肝及肝外组织实质细胞的核内,这些肝外组织未见明显的病理损伤改变。PCR的检出率与原位杂交基本一致。结论 TT病毒能感染肝及肝外组织,肝外组织中的感染可能与TT病毒的再度感染及在不同人群中的较高感染率有关。     

慢性肝病患者血清和肝组织乙,丙型肝炎标志物的检测及意义

目的:探讨血清免疫学标志物与血清、肝组织HBV DNA、HCV RNA的关系。方法:对病理诊断为慢性肝炎、肝炎后肝硬化的22例患者进行了血清免疫学标志物、斑点杂交HBV DNA的检测,并采用PCR技术对血清、肝组织的HBV DNA、HCV RNA分别进行了检测。结果:8例病理诊断为慢性肝炎的患者血清HBsAg、抗-HBc、HBeAg、抗-HBe、抗-HBs、抗-HCV阳性者分别为3例、4例、0例、1例、2例、0例,斑点杂交HBV DNA阳性1例,血清、肝组织PCR HBV DNA阳性分别为4例、6例,HCV RNA阳性分别为3例、4例,14例病理诊断为肝炎后肝硬化的患者血清HBsAg\抗-HBc、HBeAg、抗-HBe、抗-HBs、抗-HCV阳性数分别为14例、4例、2例、7例、0例、3例,斑点杂交 HBV DNA阳性7例,血清、肝组织PCR HBV DNA阳性分别为10例、10例,PCR HCV RNA阳性4例、4例。结论:对乙、丙肝免疫学标志物的分析应结合PCR检测结果;慢性肝病病理学改变与感染HBV和/或HCV有关,以HBV感染为多见,少数为HCV感染;血清和肝组织PCR HBV DNA、HCV RNA具有一致性,血清检测结果基本可反映肝内病毒复制状况。     

Immunohistochemical analysis of retinoblastoma gene product (pRB) expression in malignant and non-malignant liver diseases

ABSTRACT: The retinoblastoma gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To analyze the expression of retinoblastoma gene product in the process of liver regeneration and the initiation of hepatocellular carcinoma, we studied immunohistochemically the expression of retinoblastoma gene product and DNA polymerase alpha (DPA) in 33 patients with various liver diseases. Only a few hepatocytes positive for retinoblastoma gene product were found in undamaged, nonregenerating liver tissues, whereas many hepatocytes positive for retinoblastoma gene product were detected in specimens of regenerating liver obtained from patients with acute or chronic liver diseases. Similarities were found between distribution patterns of hepatocytes positive for retinoblastoma gene product and those of hepatocytes positive for DPA, and a highly significant positive correlation was found between the number of hepatocyte nuclei stained for retinoblastoma gene product per 1000 nuclei examined (R-LI) and the number of hepatocyte nuclei stained for DPA per 1000 nuclei examined (D-LI) in tissues obtained from patients with nonmalignant liver disease. Hepatocellular carcinoma cells positive for DPA were detected in the 14 hepatocellular carcinoma specimens tested. In ten of these specimens, hepatocellular carcinoma cells positive for retinoblastoma gene product were found but not in the other four. For all hepatocellular carcinoma specimens, R-LI was proportional to D-LI. Thus in both nonmalignant and malignant liver, retinoblastoma gene product increased in proportion to proliferation of hepatocytes or hepatocellular carcinoma cells.     

Infection with a novel human DNA virus (TTV) has no pathogenic significance in patients with liver diseases

BACKGROUND/AIMS: A recently identified DNA virus, termed TT virus (TTV), has been associated with post-transfusional hepatitis, and a high prevalence of TTV infection in patients with acute or chronic liver disease of unknown etiology has been reported from Japan, but few data are available about TTV infection in other countries. METHODS: Using hemi-nested-PCR amplification to detect TTV-DNA sequences in serum, we investigated TTV infection in blood donors and in patients with liver diseases of varied etiology. RESULTS: The prevalence of TTV infection was 13.7% in blood donors (23/168), 18.6% in chronic hepatitis C (19/102), 28.6% in chronic hepatitis B (16/56), 29.9% in hepatocellular carcinoma (20/67), 9.1% in cryptogenic chronic liver disease (2/22) and 39.6% in fulminant hepatitis (19/48). The prevalence of TTV infection in patients with virus-induced or idiopathic fulminant hepatitis was similar. Comparison of TTV-infected and non-infected patients did not reveal significant differences concerning demographic, epidemiological or histopathological features. In patients with hepatitis C, response to interferon therapy was not related to TTV infection. Phylogenetic analysis of TTV isolates showed that at least three different types of TTV are present in Spain. CONCLUSIONS: Our data suggest that TTV infection is frequent among blood donors and patients with acute liver disease. However, pathogenic effects associated with TTV infection were not observed.     

Poor association of TT virus viremia with hepatocellular carcinoma

AIM: The aim of this study was to clarify the relationship between TT virus (TTV) infection and the development of hepatocellular carcinoma. METHODS: TTV from serum was examined in 224 patients with hepatocellular carcinoma (HCC) and 106 patients with chronic liver disease (CLD) but without HCC who were admitted to our hospital between 1995-1997. As controls, 48 patients without liver disease were also examined. TTV DNA was detected using nested PCR method after extraction of DNA from serum. RESULTS: TTV DNA was detected in 29/224 (13%) of patients with HCC; in 14% (4/28) of HCC patients negative for both hepatitis B virus surface antigen (HBsAg) and anti-hepatitis C virus antibody (anti-HCV), in 9% (2/22) of HCC patients positive for HBsAg, and in 12% (21/170) of HCC patients positive for anti-HCV. The prevalence of TTV DNA in HCC patients (13%) was not significantly higher than in CLD patients (22%). There were no significant differences in age, gender, liver function, tumor biology (size, TNM classification), other viral markers, or amount of alcohol intake between TTV-positive and -negative HCC patients. Only a history of blood transfusion was significantly more frequent in TTV-positive HCC patients than in TTV-negative cases (p= 0.02). Coinfection with TTV did not correlate with the severity of HCV-positive liver disease. There was no significant difference in prognosis between TTV-positive and -negative HCC patients. CONCLUSIONS: TTV does not seem to contribute to the development of HCC from chronic liver disease and is not correlated with severity of liver disease.     

肝组织中TTV DNA原位检测及临床病理分析

目的：探讨非甲－非庚型急慢性病毒性肝炎患者肝细胞中ＴＴＶ（ｔｒａｎｓｆｕｓｉｏｎ－ｔｒａｎｓ－ｍｉｔｔｅｄ ｖｉｒｕｓ，ＴＴＶ）ＤＮＡ表达与临床病理的关系．方法：采用地高辛素标记ＴＴＶ ＤＮＡ探针以原位杂交技术对１９例血清学病毒标记非甲－非戊型、免疫组化检测肝细胞中ＨＧＶ ＮＳ５阴性的病毒性肝炎患者石蜡炎患者石蜡包埋肝组织进行了检测，并对其中７例相应血清进行了ＰＣＲ检测。结果：急慢性病毒性肝炎肝组     

健康人群和肝病患者中检测TTV的意义

目的了解新型肝炎病毒－ＴＴＶ的致病性和在健康人群和肝病患者中的流行情况．方法收集１８０份健康体检患者血清和１５６份不同类型肝病患者血清,采用ＰＣＲ方法检测ＴＴＶ的ＤＮＡ．同时检测ＨＡＶ,ＨＢＶ,ＨＣＶ,ＨＥＶ和ＨＧＶ感染标志,比较分析ＴＴＶ在健康人群和不同类型肝病患者中流行情况及其致病性．结果健康体检人群和肝病患者中,ＴＴＶＤＮＡ检出率分别为２２％和４５％,两组间无显著性差异（Ｐ＞００５）．体检人群中,ＡＬＴ正常和升高者的检出率分别为１７％和１４３％．急性肝炎,慢性肝炎和肝硬变者的检出率分别为４８％,４３％和４７％．１１例阳性患者中,３例ＡＬＴ正常,８例ＡＬＴ异常．在８例ＡＬＴ异常患者中,６例为ＨＢＶ现行感染,１例为ＨＣＶ现行感染,仅１例为ＮＡ－Ｇ肝炎患者．结论在中国健康体检人群和肝病患者中能检出低水平的ＴＴＶ现行感染．但似乎仅引起个别患者的转氨酶轻度升高．ＴＴＶ的致病性可能较弱或需要其他因素协同致病．     

High prevalance of TT virus infection in Japanese patients with liver diseases and in blood donors.

BACKGROUND/AIMS: Although a novel DNA virus, TT virus (TTV), has been isolated from a patient with cryptogenic post-transfusion hepatitis, its pathogenic role remains unclear. To elucidate its prevalence and clinical impact in patients with liver diseases, the presence of TTV DNA was assessed in patients with liver diseases and blood donors (BDs) in Japan using two primer sets, one conventional and the other new and highly sensitive. METHODS: We studied 261 samples, 72 with chronic hepatitis associated hepatitis C virus (HCV-CH), 57 with hepatocellular carcinoma associated HCV (HCV-HCC), 12 with HCC without either HCV or hepatitis B virus (NBNC-HCC), and 120 of BDs. RESULTS: Using two primer sets, TTV DNA was detected in 68 (94.4%), 53 (93.0%), 12 (100%), and 98 (81.7%) HCV-CH, HCV-HCC, NBNC-HCC, and BDs, respectively. The prevalence was not significantly different between HCV-CH and HCV-HCC, or between HCV-HCC and NBNC-HCC. Comparison between patients with and without TTV revealed no significant differences in backgrounds or biochemical findings. Histopathological findings in patients with HCV-CH, and number, maximum diameter, and histological differentiation of HCC also did not demonstrate any relation to TTV infection. TTV strains can be divided into five groups using phylogenetic analysis, but no disease-specific group appears to exist. CONCLUSIONS: Our data suggest that: 1) TTV is very prevalent among patients with liver diseases and even among BDs in Japan, 2) TTV infection does not impact on liver damage with HCV infection, and 3) TTV infection also does not affect the development or progression of HCC.     

Transfusion-transmissible virus and hepatocellular carcinoma: a case-control study

Although transfusion-transmissible virus (TTV) is often present in the serum of patients with acute and chronic non-A–C liver diseases, its hepatotropism, pathogenicity to the liver and hepatocarcinogenicity have not been proven. We used a case-control format to compare the prevalence of TTV infection among 148 southern African Blacks with hepatocellular carcinoma and 148 matched hospital-based controls, and to test for possible interactive effects between this virus and hepatitis B virus (HBV) and hepatitis C virus (HCV) in the development of the tumour. We also determined the prevalence of TTV in 988 blood donors in Gauteng province of South Africa. The presence of TTV DNA in serum samples was detected by using the polymerase chain reaction, Southern hybridization and nucleotide sequencing. Individuals infected with TTV did not have an increased risk of developing hepatocellular carcinoma (relative risk 1.1; 95% confidence limits 0.5–2.4). Moreover, co-infection with TTV did not further increase the risk of tumour development in patients chronically infected with HBV and/or HCV. TTV was present in the serum of 2.2% of blood donors: 4.0% in Black and 1.5% in White donors. We conclude that TTV is unrelated to the development of hepatocellular carcinoma in Black Africans.     

Clinical application of the measurement of serum asialoglycoproteins to estimate residual liver function in patients with chronic liver diseases with or without hepatocellular carcinoma

The correlation between the amount of asialoglycoproteins and results of conventional liver function tests was studied in patients with chronic liver diseases, with or without hepatocellular carcinoma. The objective was to determine the clinical significance of the measurement of levels of serum asialoglycoproteins. The levels were elevated in accordance with the progress of liver diseases, and correlated with the decrease in albumin content, cholinesterase activity, the ratio of esterified cholesterol to total cholesterol and to the increase of indocyanine green retention at 15 min (p less than 0.001). There was no correlation with values of glutamic oxaloacetic and pyruvic transaminases. The amount of serum asialoglycoproteins also correlated with survival time in fatal cases of cirrhosis and/or hepatocellular carcinoma. Bilirubin and bile acids did not interfere with the measurement of serum asialoglycoproteins in cases of hyperbilirubinemia. Serum asialoglycoprotein levels are a good indicator of hepatic functional reserve in patients with chronic liver diseases, with or without hepatocellular carcinoma.     

Clinicopathological study on TTV infection in hepatitis of unknown etiology

AIM: To investigate the state of infection, replication site, pathogenicity and clinical significance of transfusion transmitted virus (TTV) in patients with hepatitis, especially in patients of unknown etiology. METHODS: Liver tissues taken from 136 cases of non-A non-G hepatitis were tested for TT virus antigen and nucleic acid by in situ hybridization (ISH) and nested-polymerase chain reaction (PCR). Among them, TT virus genome and its complemental strand were also detected in 24 cases of autopsy liver and extrahepatic tissues with ISH. Meanwhile, TTV DNA was detected in the sera of 187 hepatitis patients by nested-PCR. The pathological and clinical data of the cases infected with TTV only were analyzed. RESULTS: In liver, the total positive rate of TTV DNA was 32.4% and the positive signals were located in the nuclei of hepatocytes. In serus, TTV DNA was detected in 21.4% cases of hepatitis A-G, 34.4% of non-A non-G hepatitis and 15% of healthy donors. The correspondence rate of TTV DNA detection between liver tissue with ISH and sera with PCR was 63.2% and 89.3% in the same liver tissues by ISH and by PCR, respectively.Using double-strand probes and single-strand probes designed to detect TTV genome, the correspondence rate of TTV DNA detected in liver and extrahepatic tissues was 85.7%. Using single-strand probes, TTV genome could be detected in liver and extrahepatic tissues by PCR, but its complemental strands (replication strands) could be observed only in livers. The liver function of most cases infected with TTV alone was abnormal and the liver tissues had different pathological damage such as ballooning, acidophilia degeneration, formation of apoptosis bodies and focus of necrosis, but the inflammation in the lobule and portal area was mild. CONCLUSION: The positive rate of TTV DNA among cases of hepatitis was higher than that of donors, especially in patients with non-A non-G hepatitis, but most of them were coinfected with other hepatitis viruses. TTV can infect not only hepatocytes, but also extrahepatic tissues. However, the chief replication place may be liver. The infection of TTV may have some pathogenicity. Although the pathogenicity is comparatively weak, it can still damage the liver tissues. The lesions in acute hepatitis (AH) and chronic hepatitis (CH) are mild, but in severe hepatitis (SH), it can be very serious and cause liver function failure, therefore, we should pay more attention to TTV when studying the possible pathogens of so-called "liver hepatitis of unknown etiology".     

Prevalence of TT virus before and after blood transfusion in patients with chronic liver disease treated surgically for hepatocellular carcinoma

BACKGROUND: To examine the prevalence of TT virus (TTV) before and after blood transfusion, we retrospectively examined serum samples obtained from 55 patients who received blood transfusions before, during and after resection of hepatocellular carcinoma. METHODS: TT virus DNA was extracted from serum samples and detected by nested polymerase chain reaction. Before transfusion, seven (12.7%) were positive for TTV. Patients were transfused whole blood or separated blood components (fresh frozen plasma, platelet and/or red blood cells), the total amount of transfused fresh frozen plasma ranging from 12 to 271 (median 38) units. RESULTS: Seven (14.6%) of the 48 TTV-negative patients became positive for TTV-DNA 1 month after transfusion. Only one of the seven patients, who was already positive for HCV-RNA, exhibited elevation of alanine aminotransferase. Five of the newly infected seven patients become negative for TTV during a 2 year follow up. CONCLUSIONS: Our findings suggest that the proportion of patients with TTV was relatively high in this sample, and that the prevalence of TTV transmission by blood components was also relatively high (14.6%). Although TTV persisted for more than 6 months in some patients, infection was not noticeable during the course of chronic liver disease.