Association of p16INK4A hypermethylation with hepatitis B virus X protein expression in the early stage of HBV-associated hepatocarcinogenesis.

The aim of the present study was to explore the relationship between methylation status of the p16(INK4A) promoter and some HBV-related factors, and the role of these factors in p16(INK4A) hypermethylation and hepatocellular carcinoma (HCC) progression. Twenty-three cases of surgically resected HBV-associated HCC and 25 fine-needle aspiration biopsy cases of chronic hepatitis B (CHB) were studied. The methylation status of the p16(INK4A) promoter was determined by methylation-specific polymerase chain reaction (PCR). Two-step immunohistochemical staining showed the expression of viral antigens in situ. Tissue HBV-DNA levels were determined by fluorescence quantitative real-time PCR. PCR and the direct sequencing method were used for mutation analysis. In peritumoral tissues (P = 0.025) and CHB samples (P = 0.029), the expression of hepatitis B virus X protein (HBx) was higher in methylated groups of p16(INK4A) promoter than in unmethylated groups. Other HBV factors including hepatitis B surface antigen and hepatitis B core antigen, tissue HBV-DNA levels and HBV x gene mutations had no relation to the methylation status of p16(INK4A) promoter. The data indicate that p16(INK4A) promoter hypermethylation correlated closely with higher HBx expression in the precancerous lesions, suggesting that HBx may play an important role in the early stage of HBV-associated hepatocarcinogenesis via induction of hypermethylation of p16(INK4A) promoter.

Changes of integrin expression in rat hepatocarcinogenesis induced by 3'-Me-DAB.

AIM:To investigate the expression of integrins in rats liver during 3'-Me -DAB induced hepatocarcinogenesis and to find out the relationship between integrins and liver cancer metastasis.METHODS:The expressions of integrins alpha(1), alpha(2), alpha(3) and alpha(5) and epidermal keratin (EK) were observed by immunohisto chemical PAP method.RESULTS:In the normal liver tissues, hepatocytes express integrins alpha(1) and alpha(5) and in the bile duct epithlium, EK. In liver cirrhosis, hepatocytes highly express integrins alpha(1), alpha(2), alpha(3) and alpha5and in hyperplastic bile duct epithelium, integrins alpha(1), alpha(5) and EK. Expression of integrins alpha(1), alpha(2), alpha(3) and alpha(5) were obviously decreased in the preneoplastic nodules and primary carcinoma but expressions of integrins alpha(1) and alpha(5) in metastasis in the lung and diaphragma were higher than those in primary carcinoma.CONCLUSION:Integrins alpha(1) and alpha(5) may play a major role in chemically induced hepatocarcinogenesis and metastasis in rats.

Dynamic changes of type I,III and IV collagen synthesis and distribution of collagen-producing cells in carbon tetrachloride-induced rat liver fibrosis.

AIM:To find out the relationship between the gene transcription of different types of procollagen and the deposition of the relevant collagens in the liver tissue and to confirm the types of collagen producing cells in liver fibrogenesis.METHODS:Dynamic changes of the expression of alpha1(I), alpha1(III) and alpha1(IV) procollagen mRNA and relevant collagens and the distribution of collagen producing cells during liver fibrogenesis of rat induced by CCl(4) (20 weeks) were investigated with Northern blot analysis, in situ hybridization and immunohistochemical techniques.RESULTS: The increased expression of alpha 1(III) procollagen mRNA by Northern blot analysis was the most predominant one among the three mRNAs during fibrogenesis. However, the enhanced expression of alpha1(IV) procollagen mRNA occurred very early while the expression of alpha1(I) mRNA was not enhanced much until the middle stage of the experiment. Desmin (Dm) positive hepatic stellate cells (HSCs) and few myofibroblasts (MFs) in and around the necrotic areas expressed alpha1(I), alpha1(III) and alpha1(IV) procollagen mRNA signals detected by in situ hybridization at the early stage of the experiment. All the three procollagen mRNA signals thereafter mainly localized in fibroblasts (Fbs) and MFs in fibrotic septa during the middle and late stages of fibrosis, which distributed parallel to the corresponding collagens detected by immunohistochemical study. In addition, the endothelial cells of sinusoids and the small blood vessels within the septa also showed alpha1(IV) procollagen mRNA and type IV collagen expression.CONCLUSION:It is considered that "HSC-MF-Fb effect cell system is the major cellular source of collagen production in liver fibrosis, in which HSCs are collagen producing precursor cells in the early liver fibrogenesis, thereafter the synthesis of type I, III and IV collagens (Col I, Col III and Col IV) mainly derives from MFs and Fbs, which play a very important role in the progress of liver fibrosis. The endothelial cells along sinusoids, as another source of Col IV production, might participate in the capillization of liver sinusoids.

Study of heteroserum-induced rat liver fibrosis model and its mechanism.

AIM:To investigate the morphological changes in the process of heteroserum induced rat liver fibrosis and the mechanism of fibrogenesis of this model.METHODS:A model of heteroserum-induced rat liver fibrosis was established by intraperitoneal injection of porcine serum. In addition to the observation of the morphological changes of this model, the infiltration of eosinophils and mast cells were measured quantitatively and the deposition of IgG and complement C(3) was detected by immunofluorescence.RESULTS:The rat liver fibrosis was induced successfully at the end of the 8th week after the injection of heteroserum.Besides the increase of hepatic stellate cells (HSC) during the process of liver fibrosis,proliferation and activation of primary mesenchyma cells (PMCs) were also found.In the early stage, the infiltration of eosinophils and mast cells was significantly increased and the deposition of IgG and complement C(3) was positive in the portal tracts and septa, while gradually reduced after the injection was stopped.CONCLUSION:This model is suitable for the research on liver fibrogenesis; the pathogenesis of this model may be related with the allergen-induced late phase reaction (LPR) caused by the injection of heteroserum, and the HSCs and the PMCs are important sources of ECM-producing cells.

Replicative efficiency and pathogenicity of hepatitis B virus e-minus precore variant.

To study the replicative efficiency and pathogenicity of hepatitis B virus precore variant (A1896), anti-hepatitis B virus e antigen (HBe) titre was studied in naturally occurring wild-type virus infection, A1896 variant infection and dual infection. Higher titre of anti-HBe was found in patients with no virus replication and in patients coinfected with the wild-type virus and A1896 variant, which suggest that anti-HBe may either act as an inhibitor of virus replication or as selective pressure for the A1896 variant. Three site-directed mutants were constructed in the duck hepatitis B virus (DHBV) precore region. A frame shift in the encapsidation signal region abolished replication of DHBV; mutation in the initiation codon of the precore and mutation to generate a termination codon at the distal region of the precore resulted in decreased replication in the duck model. More significant pathological changes were found in the liver tissues of ducks infected with the mutant which mimicked the HBV A1896 variant.