应用聚合酶链反应技术检测肝组织中的HBV－DNA

采用聚合酶链反应（ＰＣＲ）技术，对４２例肝活切组织石蜡切片中乙型肝炎病毒（ＨＢＶ）ＤＮＡ进行检测，并与乙肝表面抗原（ＨＢｓＡｇ）的免疫组织化学及血清学检测进行比较，ＨＢＶ－ＰＣＲ阳性率为７３．８％，高于组织及血清ＨＢｓＡｇ阳性率（分别为５９．５％和５０．０％）。３例病理形态呈肝炎改变，而血清ＨＢｓＡｇ（─）的肝组织中有２例检出ＨＢＶ－ＤＮＡ，提示ＰＣＲ的高度敏感性和准确性。８３．３％的门脉性肝硬变和８７．５％的肝细胞癌组织中ＨＢＶ－ＰＣＲ呈阳性，进一步证实了上述两病与ＨＢＶ的关系密切。我们还发现肝细胞淤胆患者ＨＢＶ感染率较高，ＨＢＶ－ＤＮＡ及组织ＨＢｓＡｇ阳性比例各为６／９和４／８。     

Detection of micronuclei formation and nuclear anomalies in regenerative nodules of human cirrhotic livers and relationship to hepatocellular carcinoma

Human cirrhosis is considered an important factor in hepatocarcinogenesis. The lack of substantial genetics and cytogenetics data in human cirrhosis led us to investigate spontaneous micronuclei formation, as an indicator of chromosomal damage. The analysis was performed in hepatocytes of regenerative, macroregenerative, and tumoral nodules from 30 cases of cirrhosis (paraffin-embedded archival material), retrospectively selected: cryptogenic, hepatitis C virus, and hepatitis C virus associated with hepatocellular carcinoma (HCC). Thirteen control liver samples of healthy organ donors were included. Micronucleated hepatocytes were analyzed with Feulgen-fast-green dyeing techniques. The spontaneous frequency of micronucleated hepatocytes in both regenerative and macroregenerative nodules of all cirrhotic patients was significantly higher than for the normal control group. There was no significant difference in frequency of micronucleated hepatocytes in regenerative nodules compared with macroregenerative nodules for all cases analyzed, whereas a significantly higher frequency of micronucleated hepatocytes was detected in tumoral nodules, compared with cirrhotic regenerative nodules and normal parenchyma. A higher frequency of the nuclear anomalies termed broken-eggs was observed in hepatitis C virus-related samples. Chromatinic losses and genotoxicity already existed in the cirrhotic regenerative nodules, which might predispose to development of HCC.     

Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues

The hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). The relationship was examined between HBV antigens and IAP (inhibitor of apoptosis) family in development of HCC. The expression levels of HBV antigens (HBsAg, HBcAg, and HBxAg) and members of the IAP family (survivin, XIAP, cIAP-1, and cIAP-2) were detected immunohistochemically in tissues from 34 cases of HCC and 30 cases of liver cirrhosis. The positive rate of survivin was higher than these three molecules in all three tissue types (P < 0.05). The positive rates of HBxAg and survivin were high in HCC (76.5% and 88.2%), paratumor (85.3% and 91.2%), and liver cirrhosis (100% and 93.3%) tissues, with no significant differences between the survivin- and HBxAg-positive rates (each P > 0.05). To examine the effect of HBx on survivin expression, plasmid pCMV-X (encoding the HBx gene) was transfected transiently with or without plasmid pcDNA3-sur (encoding the survivin gene) into H7402 hepatoma cells and L-O2 human normal liver cells. Cells over-expressing HBx alone showed increased apoptosis along with a dose-dependent increase in survivin levels. However, co-expression of survivin inhibited the HBx-induced apoptosis. To examine the effect of HBx on survivin in hepatoma cells without apoptosis, plasmid pCMV-X was transfected stably into human hepatoma H7402 cells and L-O2 cells. These H7402-X and L-O2-X cells showed high-level expression of both HBx and survivin, but did not show apoptosis. The addition of pSilencer 3.0-X, an RNAi vector targeting the HBx gene, reduced the expression levels of survivin protein in H7402-X cells. Collectively, these data demonstrate that HBx upregulates survivin expression in hepatoma tissues, suggesting that HBx and survivin may both be involved in carcinogenesis of HCC.     

Clonal analysis of micronodules in virus C-induced liver cirrhosis using laser capture microdissection (LCM) and HUMARA assay

Most hepatocellular carcinomas (HCC) arise from malignant transformation of regenerative cirrhotic nodules. Because HCC has a very poor prognosis, detection of these premalignant lesions may improve the management of patients with cirrhosis. In this regard, clonal analysis of liver micronodules should be of particular interest in order to differentiate polyclonal regenerative micronodules from monoclonal neoplastic potentially malignant micronodules. To address this issue, 112 micronodules from 15 cases of explanted liver cirrhosis were carefully microdissected from paraffin-embedded tissue using a laser capture microscopy system. Clonal analysis was performed by analyzing X-chromosome inactivation, as indicated by the methylation status of the human androgen receptor gene (HUMARA). For each microdissected micronodule, a large set of pathological features was evaluated and correlated with their clonal status. Clonal analysis showed that 57 micronodules (51%) were monoclonal and 55 (49%) were polyclonal. Prevalence of monoclonal nodules ranged from 25% to 71% according to cases. In all cases, mono- and polyclonal nodules were randomly distributed in the cirrhotic liver. Although the clonal status was not significantly affected by the presence or absence of macronodules in the adjacent liver, size of monoclonal micronodules was significantly larger than size of polyclonal micronodules (mean size of the monoclonal nodules: 3 + 0.1 mm vs mean size of the polyclonal nodules: 2.5 +/- 0.1 mm, p = 0.007). Among the elementary pathological features evaluated, only the presence of iron overload was correlated with a monoclonal status (p = 0.04). In conclusion, clonal analysis of liver cirrhosis shows that 51% of micronodules are monoclonal lesions, supporting the notion that liver cirrhosis is a multineoplastic lesion. Because monoclonality is a marker of neoplasia, cirrhosis with accumulation of monoclonal nodules may be carefully followed, and monoclonal nodules should be screened for additional markers to assess their biological behavior.     

Clinical usefulness of alpha-1-antitrypsin in the diagnosis of hepatocellular carcinoma.

Serum levels of alpha-1-Antitrypsin(AAT) were determined in 42 patients with hepatocellular carcinoma(HCC), 5 patients with metastatic liver cancer from stomach adenocarcinoma, 10 patients with liver cirrhosis, 10 patients with chronic hepatitis, and 66 controls by rocket immunoelectrophoresis using rabbit antiserum. The mean level of serum AAT was 225.5 +/- 73.0 mg/dl in 66 controls. The serum AAT in patients with HCC was 428.7 +/- 123.3 mg/dl, which was significantly higher than those in the controls and in patients with liver cirrhosis or chronic hepatitis(p less than 0.02). The level of AAT in metastatic liver cancer was similar to that in HCC. The positive cut-off value for elevation of serum AAT in this study was determined as above 445 mg/dl, the mean plus 3 standard deviations in the controls. Elevations of serum AAT were observed in 54.8%, 60.0%, and 10.0% of patients with HCC, metastatic liver cancer, and liver cirrhosis, respectively, while none of the patients with chronic hepatitis or the controls was positive. The serum AAT levels in 42 patients with HCC were analyzed with regard to sex, age, serum albumin, HBsAg, alpha-fetoprotein(AFP), and diameter of HCC, with no significant differences being observed between these factors and the serum AAT levels except for the diameter of the HCC. The positive rate in the HCC with a diameter of 10 cm or more was 74.1%, which was a significantly higher rate compared with 20.0% in the HCC with diameters less than 10cm. The positive rate of AFP for HCC was 61.9%, when 500 ng/ml of AFP was used as the cut-off value.(ABSTRACT TRUNCATED AT 250 WORDS)     

CAS基因在原发性肝癌组织中的表达及其与HBV感染的关系

目的 探讨细胞凋亡易感基因(CAS)可否作为肝癌的病理诊断标志物,以及肝细胞癌中CAS蛋白的表达与HBV感染之间的关系.方法 应用免疫组化法检测肝癌、癌旁组织及未发生肿瘤的肝硬化、肝炎组织中CAS蛋白的表达情况,同时应用免疫组化法、核酸原位杂交法检测HBV感染的肝细胞癌组织、癌旁组织中HBsAg、HBcAg以及HBV DNA的表达情况,分析肝细胞癌中CAS蛋白的表达与HBV感染之间的关系.结果 CAS蛋白在肝癌组织中的表达较癌旁组织明显升高(P＜0.01),而癌旁组织中CAS蛋白表达较未发生肿瘤的肝硬化、肝炎组织显著增强(P＜0.01).低分化型肿瘤细胞的CAS蛋白表达明显高于中分化型和高分化型(P＜0.01).CAS蛋白在HBV感染的肝细胞癌组织中的表达明显高于非HBV感染的癌组织(P＜0.01),其中HBV DNA阳性的肝细胞癌组织中CAS蛋白表达水平显著高于HBV DNA阴性的肝细胞癌组织(P＜0.05).结论 CAS蛋白在肝细胞癌组织中呈高表达,肝细胞癌分化愈低其表达愈强,表明CAS蛋白可作为肝细胞癌的病理诊断与分化程度的评价标志物.并推测HBV DNA可能通过上调CAS的表达,在HBV感染相关性肝癌的发生发展过程中发挥重要的作用.     

CD54和HLA-ABC在肝癌、肝硬化组织中的表达

目的：研究ＣＤ５４ 及ＨＬＡＡＢＣ 在肝细胞肝癌（ ＨＣＣ） 及肝硬化（ＬＣ） 组织中的表达、定位及其意义。方法：以免疫组化ＡＰＡ 法,检测ＣＤ５４ 和ＨＬＡＡＢＣ 在ＨＣＣ 及ＬＣ 组织中的表达和定位。结果：ＣＤ５４ 在ＨＣＣ 组织中的阳性率为７７ ．１ ％ （３７／ ４８） , 且以膜表达为主, 呈蜂窝样排列; 在ＬＣ 组织中的阳性率为４０ ．０ ％ （１２／ ３０） , 以核表达为主。ＨＬＡＡＢＣ 在ＨＣＣ 组织中的阳性率为３０ ．０ ％ （１２／４０） ,在ＬＣ 组织中的阳性率为６３ ．３ ％ （１９／３０） 。ｘ２ 检验,ＨＣＣ 组织中ＣＤ５４ 的表达高于ＬＣ,两者相差显著（ Ｐ＜ ０ ．０５） ,Ｐｏ ｗｅｒ ＝ ０ ．９２ 。ＨＣＣ 组织中ＨＬＡＡＢＣ 的表达低于ＬＣ,两者相差显著（ Ｐ ＜ ０ ．０５） ,Ｐｏｗｅｒ ＝ ０ ．８０ 。结论：ＣＤ５４ 在ＨＣＣ 组织中的高度表达并不能引起有效抗肿瘤免疫应答,原因之一可能是ＨＬＡＡＢＣ 在ＨＣＣ 组织中的表达率降低所致。     

p53 immunoreactivity in hepatocellular adenoma, focal nodular hyperplasia, cirrhosis and hepatocellular carcinoma

The prolonged half-life of mutant p53 makes feasible its immunocytochemical detection. In order to assess the pathogenetic role of mutant p53 in regenerative and neoplastc liver disease we studied its immunohistochemical expression in cases of hepatic cirrhosis, hepatocellular carcinoma (HCC), cirrhosis with areas of HCC, hepatocellular adenoma and focal nodular hyperplasia. The study included needle and wedge biopsies of 50 cirrhotic livers, 59 HCCs (36 of them with associated cirrhosis), six adenomas and two focal nodular hyperplasias. Sixty-five HCC fineneedle cytology specimens were also included in the study. There was no immunohistochemical evidence of mutant p53 expression in any of the cases of cirrhotic liver (except for one instance associated with HCC) adenoma or focal nodular hyperplasia. In contrast p53 was detected in 8.5% of HCC cases in the biopsy series and 24% of HCC cases in the fine needle aspiration series. In addition, mutant p53 expression in HCC was positively correlated with tumour grade. According to grade, the distribution of p53 positive immunoreactivity among HCCs was as follows: Grade I-II, 0% of cases in the biopsy series and 9% in the fine needle aspirates; Grade III, 18% in the biopsy series and 55% in the fine needle aspirates; and Grade IV, 40% in the biopsy series. Therefore, mutant p53 expression does not seem to be associated with benign liver lesions but seems to correlate with the progression of HCC through various grades of increasing malignancy.