Дисертації з теми "Hepatitis B; human haemopoietic cells"

Tc17 cells and the semi-invariant human mucosal associated invariant T (MAIT) cells are important CD8+ tissue-homing cell populations. Both are characterized by high expression of CD161 (++) and type-17 differentiation, yet their origins and relationships remain poorly defined. By transcriptional and functional analyses it is demonstrated that a pool of polyclonal, pre-committed type-17 CD161++CD8αβ+ T cells exists in cord blood, from which a prominent MAIT cell (TCR Vα7.2+/Vβ2 or 13.2) population emerges post-natally. During this expansion, CD8αα T-cells appear exclusively within CD161++CD8+/MAIT subset, sharing cytokine production (IL17, IL-22 and IFN-γ), chemokine-receptor expression (CCR2, CCR6 and CXCR6), TCR-usage and transcriptional profiles with their CD161++CD8αβ+ counterparts. These data demonstrate the origin and differentiation pathway of MAIT cells from a naïve type-17 pre-committed CD161++CD8+ T cell pool and the distinct phenotype and function of CD8αα cells in man. The CD161++CD8αβ and CD8αα T cell subsets are reduced in the peripheral circulation in chronic hepatitis B and C and are enriched in the liver in chronic hepatitis C. Their potential role in immunity to chronic viral hepatitis B and C is demonstrated by their expression of activation/exhaustion markers CD69, CD25, HLA-DR and PD-1. In addition a substantial distinct CD161-CD8β^low population is demonstrated in chronic hepatitis B, co-characterised by a CD28^low, HLA-DR^high phenotype and high expression of IFN-γ, with important implications for the development of immunotherapy and vaccination.

Les lymphocytes T CD4 (LT CD4) jouent un rôle central dans la régulation des réponses immunitaires vis-à-vis des agents infectieux et des vaccins. Cependant, leur différenciation in vivo est encore mal comprise et les caractéristiques des LT CD4 capables de persister à long terme tout en assurant une réponse immunitaire protectrice sont mal définies. L’approfondissement de ces connaissances est indispensable pour le développement de nouveaux vaccins.

Pour approcher cette question, nous avons utilisé deux approches expérimentales. La première est un suivi de la différenciation des LT CD4 au cours de la réponse immune primaire chez des sujets vaccinés contre l’hépatite B ;la deuxième est la caractérisation phénotypique et fonctionnelle des LT CD4 mémoires antigène(Ag)-spécifiques pendant la phase d’état. Cette analyse a été réalisée au sein des LT CD4 spécifiques d’Ag vaccinaux, l’Ag de surface du virus de l’hépatite B (HBs) et la toxine tétanique (TT), ainsi que ceux spécifiques des Ag du cytomégalovirus (CMV). Les LT CD4 Ag-spécifiques ont été mis en évidence par cytométrie de flux après marquage intracytoplasmique du ligand du CD40 (CD40L) exprimé en réponse à une stimulation de courte durée par l’Ag. Des expériences basées sur la stimulation par la toxine du syndrome du choc toxique et le marquage du segment Vbeta2 du récepteur des LT ont démontré la bonne sensibilité et spécificité de cette méthode.

Le suivi de la réponse primaire chez 11 donneurs jusqu’à plus d’un an après immunisation par le vaccin anti-hépatite B a permis d’établir un modèle de différenciation des LT CD4 Ag-spécifiques in vivo chez l’homme. Nous avons mis en évidence des LT CD4 spécifiques d’un nombre limité de peptides immunodominants de la protéine HBs suggérant une réponse de type oligoclonale. Grâce à l’utilisation d’un cytomètre neuf couleurs, nous avons mené une analyse détaillée de l’hétérogénéité de la population mémoire HBs-spécifique. L’expression du CCR7 permet de distinguer des cellules de type mémoire centrale (LTCM, CCR7+) et effectrice (LTEM, CCR7-) se distinguant notamment par leur capacité à migrer vers les ganglions lymphatiques ainsi que par leurs propriétés fonctionnelles. Nous avons montré l’existence de ces deux sous-populations au sein des cellules HBs-spécifiques mais par opposition à leur définition initiale, ces LTCM sont capables de produire des cytokines effectrices. La proportion importante de LTCM exprimant le Ki67 témoigne d’une activité proliférative persistante in vivo et suggère la capacité de ces cellules à s’auto-renouveler et éventuellement à alimenter le pool des LTEM. La proportion importante de LTCM exprimant la chaîne alpha du récepteur à l’IL-7 (CD127) suggère que ces cellules sont sensibles aux signaux émanant de l’IL-7, une cytokine dont le rôle dans le maintien de la mémoire lymphocytaire T est connu. Compte tenu de la relevance potentielle de ces caractéristiques uniques pour le développement de vaccins et de l’accumulation de travaux montrant l’avantage sélectif des LTCM à conférer une immunité protectrice, nous avons focalisé la dernière partie de ces recherches sur cette sous-population. Une étude transversale des LTCM spécifiques de plusieurs types d’Ag (éliminés (HBs et TT) ou persistants (CMV)) a été menée. Nos résultats montrent une hétérogénéité, variable selon l’Ag, de la capacité de ces cellules à produire des cytokines effectrices et de leur phénotype de différenciation. Cette donnée nouvelle soulève la possibilité que les LTCM soient hétérogènes dans leur capacité à conférer une immunité protectrice. L’acquisition du marqueur KLRG1 par une fraction des LTCM s’associe à une capacité accrue à produire des cytokines effectrices et à une expression élevée du CD127. La possibilité que ces cellules soient particulièrement aptes à conférer une immunité protectrice et durable est discutée, tout comme les mécanismes menant à leur génération et l’intérêt de ces connaissances pour la conception de nouveaux vaccins.

Doctorat en Sciences médicales
info:eu-repo/semantics/nonPublished

This thesis studied aspects of the interaction of hepatitis B virus (HBV) with haemopoietic cells and cell lines, to address the reported tropism of HBV for haemopoietic tissues. Emphasis was directed at demonstrating specific attachment of HBV to defined subpopulations of peripheral blood leucocytes (PBL) and bone marrow cells (BM), and the distribution of receptors for HBV on well-defined haemopoietic cell lines. Biochemical characterisation of the virus-cell interaction was also performed, and the question of infectivity of haemopoietic cell lines was addressed' Firstly, a quantitative assay of HBV binding to liver plasma membranes (PM) was adapted to show that isolated PBL PM bound serum-derived FIBV particles to a similar degree, based on their protein content. Using synthetic peptides representative of various amino acid sequences of the preSl and preS2 regions of L HBsAg to inhibit HBV binding to the pM, it was found that peptide pres l(12-32) inhibited binding to PBL PM by 60-80% and peptide preSl(21-47) inhibitedby 0-30% (depending on the source of PM), while peptides preSl(32-49) and preS2(120-145) did not inhibit binding. This contrasts with results obtained using liverPM, where peptide preSl(12-32) did not inhibit binding, while peptide preSl(21- 47) inhibited by 70%, and preSl(32-49) inhibited by approximately 12%. Peptide preS2(120- 145) had no effect on binding. Thus, different regions of the L surface protein appear to mediate attachment to PBL and hepatocytes. HBV particles isolated from serum are complexed with serum proteins including IgG. To test the involvement of receptors for IgG and complement fragments (opsonins) in the HBV-PM interaction, a panel of ligand-blocking monoclonal antibodies (MAbs) to opsonin receptors was used, and it was shown that the three classes of receptors for IgG (FcγRI, FcγRII and FcγRIII) and CR3, are not major receptors for HBV on PBL or hepatocytes, as MAbs to these did not inhibit HBV binding. It was also shown that HBV does not utilise the receptor for IgA, FcαR, for attachment to PBL, despite reported sequence homology between the large envelope protein of HBV and the Fc portion of human IgA. In contrast to a published report that IL-6 mediates binding of HBV to hepatocytes, IL-6 was shown not to mediate attachment to either liver or PBL PM, by virtue of pre-incubation with a blocking polyclonal anti-serum to IL-6. Glycosaminoglycans (GAGs) were found to influence HBV binding to PM: soluble heparin (HE) inhibited binding to liver PM by up to 80%, and to leucocyte PM by up to 40%; chondroitin sulphare C (CS-C) enhanced virus binding (approximately 1.5-fold) to leucocyte pM only. Chondroitin sulphate A and hyaluronate had no effect on binding to either PM, arguing that simple electrostatic properties of GAGs were not responsible for the observed effecrs. The incomplete inhibition by HE and enhancement by CS-C could indicate the presence of more than one class of binding site for HBV on the respective PM, and coupled with the differential pattern of inhibition in the presence of synthetic peptides, argues that receptors for HBV on pBL and hepatocytes may be either different, or altered forms of the same molecule(s). To extend these studies, whole cell binding assays were developed in order to accurately define which subsets of pBL and BM cells could bind HBV. Using purified HBV particles as the first stage in an immunofluorescence-based detection system, followed by detection of bound HBV using anti-preS1 MAbs F35.25 or MAl8/7, and a FlTC-conjugated third-stage antibody, specific membrane staining of peripheral blood monocytes from 8/9 donors was observed. In addition, binding of HBV to the erythroleukaemia cell line K562 was observed, while other myeloid cell lines did not appear to bind virus. This assay was then adapted to a suspension cell assay with analysis by flow cytometry, using phycoerythrin as the detecting fluorochrome. The parameters of binding were optimised for K562 cells and these were then applied to analyse HBV binding to PBL and BM cells obtained from healthy volunteers, whose serum was free of HBV markers. Based on their light scattel characteristics, monocytes and neutrophils were the only cell types in the peripheral blood that bound HBV' while binding to lymphocytes was not observed. This was confirmed by two-colour immunofluorescence to simultaneously detect bound HBV and subset-specific leucocyte markers. Similarly, in the BM, only monocytes bound HBV. Importantly, haemopoietic stem cells (cD34+) did not bind HBV. Binding was tested to 'activated' populations of lymphocytes (pHA-treated), monocytes (LPS-treated), and neutrophils (fMLP-treated). The pattern of HBV binding was not affected by these treatments. Monocytes cultured in vitro, bound significantly more virus than freshly isolated monocytes. Taken together, these results indicate that only monocytes, and to a lesser extent neutrophils, express potential receptors for HBV, and a differentiation-dependent upregulation of receptor sites for HBV is observed on Monocytes The distribution of potential HBV receptors was determined on a number of haemopoietic cell lines, representative of various haemopoietic lineages. K562 (erythroid), and the monocyte cell line THP-l, were the only haemopoietic cell lines which bound HBV, while binding was also observed to the human hepatoma cell line HepG2. A number of other erythroid and monocyte cell lines, as well as T and B tymphoid, and a megakaryocytic line, all failed to bind HBv. A comparison of the surface immunophenotypes of all the cell lines tested excluded all known CD-classified molecules (including opsonin receptors), as candidate HBV receptors. The biochemical characteristics of the interaction of HBV with all of these cell types were then examined. On K562 and THP-1, HBV binding was sensitive to the protease chymopapain but insensitive to trypsin, indicating that the molecule was a glycosylated protein. Pre-treatment of these cell lines with tunicamycin, to inhibit post-translational addition of N-linked carbohydrate to surface glycoproteins, did not influence HBV binding, indicating that these moieties are not important for virus attachment. Enzymatic removal of cell surface sialic acids with neuraminidase significantly enhanced HBV binding to K562 and THP-1 cells but did not confer binding to otherwise 'negative' cell lines. Binding of HBV to cultured monocytes and HepG2 cells was trypsin and chymopapain sensitive' and was not increased by neuraminidase pre-treatment. Cation chelation demonstrated that HBV binding of to all cell types was Ca²⁺/Mg²⁺-independent, and acid elution of cells showed that binding was not mediated by peripherally-bound molecules. Binding of HBV to monocytes and to HepG2 cells was significantly reduced by pre-treatment of the cells with PI-PLC, implying that the molecule responsible for binding to these cells is GPl-linked. In this case, a comparison with HBV binding to K562 was not informative due to the resistance of the GPI linkages on these cells, and possibly also on THP-I cells (based on CD59 cleavage), to hydrolysis by PI-PLC. Thus, cells expressing potential receptor(s) for HBV, whose characteristics do not correlate with any other proposed candidate, have been identified. Immunoprecipitation analysis using HBV particles covalently cross-linked to the surface of ¹²⁵I-labelled K562 cells, and anti-S MAb coupled to goat anti-mouse IgG-Sepharose, resulted in the identification of a 50 kDa species as a putative HBV receptor. Contrary to reports that HBV inhibits colony formation by myeloid cell lines in semi-solid media, no inhibitory effect by HBV was seen on clonal cell growth in liquid culture. K562 cells were found to be able to efficiently internalise HBV particles, which accumulated in a perinuclear compartment. In infection studies, K562 cells positive for HBsAg aftet 2-4 days post-infection became enlarged, and their numbers decreased steadily over an 11 day period. It is not clear whether these cells represent a transient or differentiated cell type. Similarly, it appears that the level of HBV DNA in these cells declines steadily during the infection course.
Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology and Immunology, 1998

碩士
國立陽明大學
生物化學研究所
89
Abstract HE-145 has been shown to suppress HBsAg production and stimulate cell proliferation on human hepatoma Hep3B/T2 cells. Similarly, TPA and insulin suppress HBsAg and stimulate cell growth. In this study, we investigated the effects of HE-145, TPA and insulin on the activities of human protein promoter in Hep3B/T2 cells, while suppressing the HBsAg production. At the same time, the HBV gene expression and replication of HepES2 cells were examined to identify the effect of HE-145, TPA, and insulin on HBV genome. HE-145, TPA and insulin suppressed HBsAg production in a does — dependent manner on Hep3B/T2 cells. The promoter activities were assayed by transient transfection with various plasmids, containing either the promoter region of HBV or human proteins with a reporter gene of luciferase, into the Hep3B/T2 cells. HE-145, TPA and insulin were found to suppress SPII luciferase and CP luciferase activity in a dose-dependent manner on Hep3B/T2 cells. This suggested that the HBsAg production might mediate SPII promoter. Addition of 1.5 M HE-145 increased cyclin A promoter activity three folds while addition of TPA and insulin had no effect. This suggested that the growth stimulatory effect was due to activate cyclin A promoter at the transcription level. HE-145 was also shown to suppress the promoter activity of CRE and NFB in a does — dependent manner. The NFB promoter activity was increased by 16 folds when treated with 200 nM TPA. At the same time, the stimulated promoter activity was decreased to four times higher than control under the combination treatment of TPA and HE-145. This result showed HE-145 down-regulation of NFB promoter activity. Northern blotting analysis was performed on HBV RNA transcripts. There are three major transcripts of approximately 3.5, 2.4, and 2.1 kilo-bases in the total cellular RNA extract on human hepatoma HepES2, Hep3B/T2, and HepG2/A2 cells. The HBV-specific transcripts were reduced among 3.0M HE-145, 200nM TPA, and 100 nM insulin treatment. This result corresponds to previous experiments using SPII and core promoter assay. A rational approach to the development of drugs for the treatment of HBV infection is to identify the compounds that specifically inhibit HBV DNA extracted from HepES2 cells exposed to 3.0M HE-145, 200nM TPA, and 100 nM insulin. The decrease in HBV DNA replication by HE-145 and TPA shows their potential to be an anti-HBV agent. The evidences revealed in this mechanism-based study may provide us with a clue to possible approaches for drug development in the future.

碩士
國立陽明大學
生化暨分子生物研究所
101
Hepatitis B virus (HBV) infection has been documented to associate with diabetes and hepatocellular carcinoma. The prevalence of HBV is common, especially in Asia. The aim of the study is screening Chinese herb medicine to identify agents which can suppress gluconeogenesis, enhancing hepatitis B viral gene expression and further to understand the signaling pathway. Firstly, the combination of 8-bromo-cAMP (cAMP) and dexamethasone (DEX) was used to mimic the fasting and stress condition in Hep3B/T2 cells. It has been found a synergistic effect on the induction of HBV core promoter activity and the key enzymes of gluconeogenesis: phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and peroxisome proliferators-activated receptor-γ coactivator-1α (PGC-1α) mRNA and protein expression of PGC-1α using quantitative real-time PCR and Western Blotting respectively. It was found that Chinese herb Duzhong (Eucommia ulmoides) and one of its active flavonoid components, Quercetin dose-dependently suppressed G6Pase, PEPCK and PGC-1α mRNA; protein expression of PGC-1α and HBV core promoter together identifying nt1656-1675 as binding sequences of core promoter. This identified nt1656-1675 as a response element in the HBV core promoter using serial deletion as well as point mutation at different regions of the core promoter. In addition, the overexpression of PGC-1α reversed the core promoter activity by Duzhong and Quercetin. To examine which intracellular signaling pathway is crucial for Duzhong and Quercetin, it had been seen that PI3K inhibitor wortmannin blocked AKT activation induced by Duzhong and Quercetin and abolished suppressive activity of Duzhong and Quercetin on cAMP/DEX activated HBV core promoter activity and gluconeogenetic enzyme expression. Therefore, it is proposed that Duzhong and Quercetin inhibit core promoter and gluconeogenic gene expression via activation of PI3K and AKT pathway. Finally, the Duzhong and Quercetin treatment significantly reduced HBV mRNA expression and core protein in 1.3ES2 cells. Overall, Chinese herb Duzhong and Quercetin may inhibit HBV gene expression through phosphorylation of AKT to down-regulate the host gluconeogenesis. This study which HBV is controlled by the hepatic metabolic gluconeogenesis may be the references of new drug developing of anti-HBV agents. The coming out rule of the close link between HBV and liver metabolism can be further exploited for host-targeted therapeutic strategies.

碩士
國立陽明大學
生化暨分子生物研究所
97
Hepatitis B virus (HBV) infection causes acute and chronic hepatitis, and the affected patients have an increased risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). Although a preventive vaccine for HBV is available, effective drugs to eradicate HBV in chronic carriers are still urgently needed.The Chinese herbal medicine Xiao-Chai-Hu-Tang consists of Bupleuri radix and Scutellaria radix (HD-1S) and is widely used for treatment of liver disease. In searching of compounds on suppression of hepatitis B surface antigen (HBsAg) production, our laboratory previously have shown that HD-1S strongly suppressed hepatitis B surface antigen (HBsAg) in human hepatoma cells. In this study, I demonstrated that the aqueous extract of HD-1S and one of its components Wogonin had potent anti-HBV activity. Both HD-1S and Wogonin not only suppressed HBsAg production but also decreased HBV transcripts and HBV viral particles in cultured human hepatoma cells. To understand the molecular mechanism of suppression of HBV gene expression by HD-1S and Wogonin, the effects of HD-1S and Wogonin on four viral promoter activities using luciferase as a reporter were examined. This study showed clearly that both HD-1S and Wogonin selectively suppresses core promoter (CP) of HBV. Such suppressive effects of HD-1S and Wogonin are liver-specific because no suppressive activity of HD-1S and Wogonin was observed when CP activity was assayed in non-liver 293T cells. Furthermore, ectopic expression of PGC-1�� and PPARr/CEBP�� abolished the suppressive effect of HD-1S and Wogonin on HBV CP activity, respectively. Therefore, HD-1S and Wogonin may selectively modulate transcriptional machinery of human liver cells to suppress HBV gene expression and replication. Results from this study also suggested that some components other than Wogonin might also have novel anti-HBV activity and deserve futher investigation in the future.

碩士
國立陽明大學
生物化學研究所
87
HE-145 was isolated from the heartwood of the stem of Taiwania cryptomerioides Hayata. HE-145 suppressed endogenous HBsAg production in long-term cultured human hepatoma Hep3B/T2 cells, but has no cytotoxicity on the cells. HE-145 suppressed the HBsAg production on Hep3B/T2 cells in a dose-dependent manner with IC50 of 56 nM for short-term culture. Previously Northern blotting analysis showed that the suppression of HBsAg gene expression by HE-145 were mainly at the m-RNA level. So, it is interested to know the effect of HE-145 on the promoter activity of HBV genome in Hep3B/T2 cells. In these studies, we investigate the promoter activity of HBV genome in human hepatoma cells Hep3B/T2, and HuH-7. Human hepatoma Hep3B/T2 cells carry an integrated HBV genome in its chromosome, and continuous secretion of HBsAg into culture medium. Human hepatoma cells, Huh-7, carry no endogenous HBV genome. The promoter activity assay was performed after transient transfection with various plasmids that contains promoter region of CAT with the HBV genome into Hep3B/T2, and HuH-7 cells. HE-145 suppressed SPII promoter, but stimulated core promoter on Hep3B/T2, and HuH-7 cells both in a dose-dependent manner. In contrast to the promoter activities of SPI and X do not show dramatically different by HE-145 concentration up to 2.8 μM. Additionally, HE-145 not only stimulated human protein cyclin A promoter activity, but also dominantly suppressed SPII promoter in Dexamethasone-treated cells. It reported that the suppression of HBsAg production by insulin mediated through PI3-Kinase. In order to observe the effect of suppression of HBsAg production by HE-145, the PI3-Kinase inhibitor wortmanin was used in human hepatoma Hep3B/T2 cells. The result suggests that PI-3 kinase may involve the suppression mechanism of HBsAg production by HE-145 in Hep3B/T2 cells. In this thesis, these results can be used to the further investigative approaches for the regulatory mechanism of HE-145 in human hepatoma cells in the future.

碩士
東海大學
生物學系
91
Hepatocellular carcinoma (HCC) is one of the ten major leading death diseases in Taiwan. The causal agent of HCC is hepatitis B virus (HBV) which encodes a protein (HBx). X gene is the smallest open reading frame (ORF) among hepatitis B virus (HBV) genome and it encodes the HBV X protein (HBx). The HBx is a multifunctional protein that is implicated in the pathogenesis of hepatocellular carcinoma. Very little is known about its functional mechanisms although interactions with several nuclear and cytoplasmic proteins have been demonstrated in vitro, there is no clear consensus as to the location of HBx in infected hepatocytes. So quantification and cellular distribution of the hepatitis B virus X protein in human hepatocellular carcinoma were studied. Western immunodetection of HBx antigen in liver extracts of twenty HCC patients was performed using the anti-HBx monoclonal antibody. The results show that 85% liver tissues of HCC contain a specific and comigrated HBx antigen. To further elucidate the role of HBx, the distribution of HBx was also examined by immunohistochemical staining. Eight fresh liver tissue samples, normal and tumor parts of four HCC patients were analysed. HBx was detected in both normal and tumor tissues using anti-HBx monoclonal and polyclonal antibodies. HBx was mainly located in cytoplasm. This distribution analysis of HBx will provide the basis for the study of the functional identification of the HBx.

碩士
國立陽明大學
生化暨分子生物研究所
98
The Chinese herbal medicine, Xiao-Chai-Hu-Tang, is a seven components prescription used for liver disease treatment. Our laboratory has found Scutellaria radix (1S), a major component of Xiao-Chai-Hu-Tang, is in possession of suppressing hepatitis B virus surface antigen (HBsAg) secretion in HepG2/A2 cells. In this thesis, human hepatoma cell lines 1.3ES2, HepG2/A2 and Hep3B/T2 were used to elucidate the mechanism of anti-HBV effect of Scutellaria radix including two of its components, Chrysin and Wogonin. In this study, it was found that 1S, Chrysin and Wogonin not only suppressed HBsAg production in cultured HepG2/A2 cells but also decrease HBV transcripts of RNA, core protein and viral particles in 1.3ES2 cells. To further understand the molecular mechanism of suppression of HBV gene expression by 1S, Chrysin and Wogonin, luciferase reporter assay was used for examining the viral promoter activity. The results showed that 1S, Chrysin and Wogonin suppressed viral core promoter expression. Such suppressive effects are liver-specific because the repressive effect was not observed in human embryonic kidney 293T cells. Furthermore, ectopic expression of liver-specific transcription factors in HepG2/A2 cells could partially reverse the suppression effect of 1S, Chrysin and Wogonin on core promoter. Using western blotting, 1S, Chrysin and Wogonin not only showed the decrease of endogenous PPARγ protein but also 1S suppress endogenous HNF4α protein expression, therefore, these three compounds may inhibit hepatitis B virus through different transcription factors of core promoter in molecular mechanisms. Using the treatment of 8-Br-cAMP and dexamethasone of 3B/T2 cells to mimic the starvation state of cells, it was found the HBV core promoter activity was stimulated. Gluconeogenesis genes, glucose-6-phosphatase (G6Pase) and phosphenolpyruvate carboxykinase (PEPCK) were activated. The expression of transcription coactivator PGC1α proteins were elevated in starvation state of Hep3B/T2 cells. Results showed that 1S effectively suppressed 8-Br-cAMP and dexamethasone induced G6Pase mRNA expression and HBV core promoter activity, however Chrysin and Wogonin had no effect on G6Pase and PEPCK. Taken together, 1S, Chrysin and Wogonin may act through modulating hepatic transcriptional machinery to suppress HBV viral gene expression and virus production. However, turns on the gluconeogenic program, only 1S showed suppression of G6Pase.

碩士
國立陽明大學
生化暨分子生物研究所
102
Hepatitis B virus (HBV) infection has been documented to be associated with developing liver cirrhosis and hepatocellular carcinoma (HCC). Currently, the major strategy of treatment is to target viral polymerase/reverse-transcriptase activity. However, adverse side effects, drug resistance, and incomplete therapeutic effect of these anti-HBV drugs are reported. PGC-1α, a major metabolic regulator of gluconeogensis, is known to be strongly induced to coactivate HBV gene expression in the liver of fasting mice. In this study, we found that combination of 8-Br-cAMP and dexamethasone had synergistic effect on the induction of PGC-1α and its downstream genes, such as PEPCK and G6Pase. In addition, HBV core promoter activity was also synergistically induced by combination of 8-Br-cAMP and dexamethasone treatment. We set up drug screening and identify that Graptopetalum paraguayense (GP) and Thioridazine were the candidate novel anti-HBV drugs. The first part, I will report the result from GP and second part will disscuss thioridazine. GP, a herbal medicine, is commonly used in Taiwan to treat liver disorders. Here, we used partially purified fraction of GP, referred to as HH-F3, to study whether it could inhibit HBV via regulation of PGC-1α. HH-F3 could dose-dependently suppress 8-Br-cAMP/dexamethasone-induced G6Pase, PEPCK and PGC-1α expression as well as HBV core promoter activity. Moreover, HH-F3 blocked HBV core promoter activity via inhibition of PGC-1α expression. Ectopic expression of PGC-1α could partially reverse the inhibition of HH-F3 to HBV core promoter activity. In addition, HH-F3 treatment significantly reduced HBV surface antigen expression, HBV mRNA production, core protein level, and HBV replication. In the second part, Thioridazine, an anti-psychotic drug, could inhibit 8-Br-cAMP/dexamethasone-induced G6Pase, PEPCK expression as well as HBV core promoter activity. However, Thioridazine-mediated inhibition of gluconeogenesis and HBV expression was not significant as compared to HH-F3. In conclusion, HH-F3 could inhibit HBV replication and gluconeogenesis through down-regulation of PGC-1α. Targeting PGC-1α may be a novel therapeutic strategy for HBV infection. Together, HH-F3 might have the potential for the treatment of chronic hepatitis B patients associated with metabolic syndrome.

碩士
國立陽明大學
生物化學研究所
92
Infection by hepatitis B virus (HBV) frequently results in acute and chronic hepatitis and is associated with a high risk of developing primary hepatocellular carcinomas in human. Immunization and nucleoside analogs as drugs against HBV have been shown to be effective. Owing to the efficacy and resistance of drugs, effective drugs to eradicate HBV in chronic carriers are still not available. There is an urgent need to develop novel drug screening system to search for new anti-HBV therapeutics. Recently, cell culture system has been used to study the mechanisms of Chinese medicinal herbs, which are widely used as a resource for the development of new drugs. Human hepatoma cell lines, HepG2/A2 and Hep3B/T2, contain integrated HBV gnomes and continually secrete HBsAg. These cell lines provide an assay system for screening of anti-HBV agents from Chinese herb medicines. This study has examined the anti-HBV activities of several pure compounds extracted from traditional Chinese herbs. Clausena excavate has been used for snake biting. Schizandrae fructus has exhibited protective effect in the liver injury. TS95 and KP-19-1-401 were extracted from Clausena excavate and Schizandrae fructus respectively. Both TS95 and KP-19-1-401 suppressed the HBsAg production in a dose-dependent manner in Hep3B/T2 cells. In HepG2/A2 cells, these two pure compounds suppressed the HBsAg and HBeAg production both in time- and dose-dependent manners but had no obvious effect on cell growth. The cell line HepES2 is a clonal derivative of human hepatoblastoma, HepG2 in which the 1.3 copies of the entire HBV genome is stably transfected into the host genome, and in which HBV is replicated, resulting in the production of infectious virus. Northern blotting analysis was performed to study the HBV RNA transcripts. The HBV-specific transcripts both on 3.5/3.6 and 2.1/2.4 kb were reduced by 2 ug/ml TS95 and 1 ug/ml KP-19-1-401. We further investigated the regulatory mechanism by transiently transfecting various plasmids that contain a HBV promoter region and a luciferase reporter gene into Hep3B/T2 cells. The promoter activity was analyzed by luciferase assay. TS95 and KP-19-1-401 suppressed both CP-luciferase and SPII-luciferase activity in a dose-dependent manner. KP-19-1-401, but not TS95, suppressed SPI-luciferase activity in a dose-dependent manner. TS95 and KP-19-1-401 showed no obvious effect on X-luciferase activity. Furthermore, KP-19-1-401 suppressed cyclin A-luciferase activity in a dose-dependent manner in Hep3B/T2 cells. The protein level of cyclin A but not cyclin B1 and cyclin D was reduced by 1 ug/ml KP-19-1-401-treated Hep3B/T2 cells. 1.0 ug/ml KP-19-1-401 induced the occurrence of cell cycle arrest at the G1 phase in Hep3B/T2 cells.

碩士
國立陽明大學
生物化學研究所
83
Destruxins是一種環縮月太，本研究分離真菌Metarhiziumanisopliae萃取液，經由高效能液相層析儀純化，並利用質譜儀以及核磁共振技術等，定出兩種Destruxin類似物DestruxinE chlorohydrin和E2 chlorohydrin的結構，其化學式分別為C29H48CIN5O8以及C28H46CIN5O8，構造式分別是cyclo(2,4-dihydroxy-5-chloropentanoic acid -Pro-Ile-(Me-Val)-(Me-Ala)-β-Ala)depsipeptides和cyclo(2,4-dihydroxy-5-chloropentanoic acid-Pro-Val-(Me-Val)-(Me-Ala)-β-Ala)depsipeptides。其中後者尚未在此真菌培養液中被發現過，是一個新的天然物。人類肝癌細胞株經這兩種Destruxin類似物處理48小時後，乙型肝炎病毒表面抗原的產生會受到抑制，其抑制值IC50分別是0.15μM和6.5μM。另外本研究也比較M.anisopliae培養液中其它Destruxin結構類似物的抑制情形，顯示其效果與分子環的大小有關。 Destruxins也是vacuolar-type ATPase的抑制劑，人類肝癌細胞株經Destruxins處理後也可以觀察到胞器酸化的情形被抑，但5分鐘後即恢復原來的酸鹼值，即使再加入Destruxins，這個現象也不再重現。細胞處理Destruxins 40分鐘後，表面抗原會累積在細胞體內，但是經48小時的處理細胞體內的表面抗原並沒有累積的現象，顯示Destruxins可透過抑制vacuolar-type ATPase的活性而暫時延緩表面抗原的輸送。 這些結果顯示，Destruxins抑制乙型肝炎病毒表面抗原的活性可能由於其暫時抑制胞器酸化的情形所直接導致，也可能是透過其它途徑。 Destruxins are a series of cyclodepsipeptides. In this study, two destruxins, Destruxin E chlorohydrin and Destruxin E2 chlorohydrin, the later is a new compound were identified in this study. They were separated from the culture broth of Fungus, Metarhizium anisopliae and purified by HPLC. The structures were characterized by Mass spectroscopy and NMR techniques. he chemical formulas are C29H48CIN5O8 and C28H46CIN5O8, and the structural formulas are cyclo (2, 4-dihydroxy-5-chloropentanoic acid-Pro-Ile-(Me-Val)-(Me-Ala)-β-Ala) depsipeptides and cyclo (2, 4-dihydroxy-5-chloropentanoic acid-Pro-Val-(Me-Val)-(Me-Ala)-β-Ala) depsipeptides. Both of the compounds can suppress the Hepatitis B virus surface antigen (HBsAg) production on Hep 3B cells after 48 hours treatment. The value of inhibition of of HBsAg production with IC50 of Destruxin E chlorohydrin and Destruxin E2 chlorohydrin is 0.15 μM and 6.5 μM, respectively. The suppression of HBsAg production for other known Destruxins isolated from M. anisopliae culture broth has the same suppressive effect. The effect was affected by the size of the 19-membered ring. Destruxins inhibited the activity of vacuolar-type ATPase. The acidification of cell organelles was blocked while treating with Destruxins but recovering after 5 minutes in Hep 3B cell system. The HBsAg accumulated in the cells by treatment with Destruxinsfor 40 minutes, but it did not accumulate for 48 hours-treatment This indicates that destruxins can delay the secretion ofHBsAg by inhibiting the activity of vacuolar-type ATPase. The inhibitory effects of destruxins HBsAg production may be caused by the inhibition of acidification of cell organelles directly It may involve in some other ways.

碩士
國立陽明大學
生物化學研究所
88
HE-145 was isolated from the heartwood of Taiwania cryptomerioides Hayata. HE-145 suppressed HBsAg production and stimulated cell proliferation on human hepatoma Hep3B/T2 cells. Insulin had the same suppression of HBsAg and stimulation of cell growth effect as HE-145 on Hep3B/T2 cells. So in these studies, we are interested to know the difference effects between and Insulin on suppression of HBsAg production, HBV promoter activity, and cell proliferation on Hep3B/T2 cells. Both HE-145 and Insulin suppressed HBsAg production in a does — dependent manner on Hep3B/T2 cells. The suppression IC50 of HE-145 and Insulin were about 0.08 μM,and 0.1 nM respectively. The promoter activity was assayed by transient transfection with various plasmid that contains promoter region of HBV with CAT or luciferase reported genome into Hep3B/T2 cells. Both HE-145 and Insulin suppressed SPII-luciferase activity in a dose-dependent manner on Hep3B/T2 cells. It suggested that the suppression of HBsAg production it might mediate SPII promoter. We respectively transfected plasmid SPII-CAT, SPI-CAT, CP-CAT, and XP-CAT into Hep3B/T2 cells or treated with HE-145 and Insulin to analyze HBV promoter activity. HE-145 suppressed SPII promoter activity, but stimulated CP promoter activity at higher concentration, 9 μM. However, HE-145 didn’t affect SPI and XP promoter activity. In contrast, Insulin suppressed both SPII and CP promoter activities, but 100nM insulin stimulated SPI promoter activity. Insulin didn’t affect XP promoter activity same as HE-145. A selective PI3 kinase inhibitor, wortmannin was used to study whether PI3 kinase involving on suppression of HBsAg production by HE-145 or Insulin. We found that the suppression of both HBsAg production and SPII promoter activity by HE-145 were reversed on wortmannin-pretreated experiments. So PI3 kinase may play an important role in suppression of HBsAg production on HE-145 and Insulin. Furthermore, the cell proliferation increased on HE-145- or Insulin-treated Hep3B/T2 cells. Flow cytometric analysis indicated that it had more cells arrested at S phase after different time treatment of HE-145 or Insulin. It suggested the growth stimulatory effect was due to shorten G1 phase accompanied by increase in S phase of cell cycle. 中文摘要......................................3 壹、 緒論.....................................5-9 貳、 材料與方法..............................10-20 參、 結果....................................21-27 肆、 討論....................................28-32 伍、 參考文獻................................33-36 圖...........................................37-58 附錄.........................................59-63

碩士
國立陽明大學
生化暨分子生物研究所
96
Phorbol ester (TPA) is a natural product that can activate protein kinase C (PKC) through binding to C1 domain of PKC. In our laboratory, it has been shown that TPA can suppress hepatitis B surface antigen (HBsAg) secretion in human hepatoma cell line Hep3B/T2. In the present study, human hepatoma cell line, HepG2/A2 and 1.3ES2 were used to elucidate the mechanism of anti-HBV effect of TPA. HepG2/A2 cells continually serete HBsAg into the culture medium. It was found that TPA suppressed HBsAg secretion by HepG2/A2 cells in a dose- dependent manner. The effects of TPA on viral promoter activity using luciferase as a reporter were examined. It was found that TPA selectively suppressed HBV core promoter (CP) and surface antigen promoter II (SPII) but has no effect on surface antigen promoter I (SPI) and X promoter (XP). The suppressive effects of TPA on CP activity are liver-specific because no suppressive activity of TPA was observed when CP activity was assayed in non-liver human cells such as HeLa and 293T. The 1.3ES2 cells derived from HepG2 cells in which continually produced HBV viral particles, 3.5 kb, 2.4 kb and 2.1 kb RNA and core proteins. Suppression of Dane particles secretion by TPA was comfirmed using quantitative real-time PCR in 1.3ES2 cells. Northern blot analysis showed the suppression of HBV gene expression by TPA mainly at the mRNA level in 1.3ES2 cells. These findings suggest that TPA has good anti-HBV activity. To understand the molecule mechanism of TPA on CP activity, luciferase reporter analysis was performed and revealed that TPA decreased the CP activity via affecting specific cis-element of HBV promoter for core antigen, including hepatocyte nuclear factor 4 (HNF-4), peroxisome proliferator- activated receptors (PPARs), peroxisome proliferator-activated receptor coactivator-1a (PGC-1a) etc. Ectopic expression of HNF-4 or PGC-1a partially reversed the TPA-mediated suppression of HBV RNA. Western blotting showed that TPA suppressed HNF-4 but did not affect the level of PGC-1a. Overexpression of constitutively active PKC isoforms revealed that PKCa and PKCq could mimic the effect of TPA on CP activity. Knockdown assay of PKCa partially reversed the TPA-mediated suppression of CP activity in HepG2/A2 cells. Therefore, TPA suppress CP activity may modulate through PKCa- dependent pathway. In the present study, I have demonstrated a natural product TPA as an anti-HBV agent with the suppressive mechanism on CP. I found that TPA suppressed CP activity due to reduced HNF-4 expression and transcriptional coactivator PGC-1a is also involved in this process. Furthermore, TPA suppressed CP activity may modulate through PKCa-dependent pathway.

碩士
國立陽明大學
生化暨分子生物研究所
99
The current approved drugs against hepatitis B virus infections exhibited side-effects or drug resistance. It is important to develop biologically active small molecules with different modes of action for the new drug. Helioxanthin (HE-145), a natural lignan isolated from Taiwanese cryptomerioides. Previous studies from our laboratory showed that HE-145 can suppress human hepatitis B virus gene expression. A series of HE-145 analogues were synthesized and evaluated for their anti-hepatitis B virus activity. Among them, compound HE-145-111 showed the most effective suppression on the hepatitis B surface antigen (HBsAg) agent. In this study, the human hepatoma cell lines, 1.3ES2, M33, HepG2/A2 and Hep3B/T2, were used to elucidate the mechanism of anti-HBV effect of HE-145-111. The effects of HE-145-111 on four viral promoter activities using luciferase as a reporter were examined. It was found that HE-145-111 suppressed core promoter (CP), surface antigen promoter I and II (SPI and SPII) and promoter for X gene (XP). The suppressive effects of HE-145-111 on CP activity is liver-specific and HE-145-111 selectively suppresses the liver-specific prmoter activities because no suppressive activity of HE-145-111 was observed when CP or OCN promoter activity was assayed in human embryonic kidney 293T cells. Then, I identified the nt1656-1708 which contains hepatocyte nuclear factor 4α (HNF4α) binding sequence as a response element of HE-145-111 in HBV core prmoter using serial deletion mutants of core promoter. Furthermore, HE-145-111 could inhibit the expression of HNF4α mRNA and protein but not affect the HNF4α protein stability. In addition, the HNF4α promoter activity was repressed dose-dependently with treatment of HE-145-111. These results together suggested that HE-145-111 may represent a novel class of anti-HBV drug which suppresses the HBV core promoter activity by down-regulating HNF4α gene expression to suppress HBV gene expression and replication in human liver cells. Previous researches revealed that peroxisome proliferators-activated receptor-γ coactivator 1 (PGC-1α) is induced strongly to coactivate the HBV gene expression of fasting mice. PGC-1α is a major regulator for key gluconeogenic genes glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) acting through the liver-enriched nuclear receptor HNF4α. Interestingly, The similar transcription factors are involved in regulation of HBV core promoter activity and gluconeogenic genes expression. Therefore, I hypothesize that HE-145-111 inhibites the HBV gene expression through regulateing the host gluconeogenesis. It showed that combination of 8-bromo-cAMP and dexamethasone had a synergistic effect on the mRNA and protein expression of G6Pase, PEPCK, and PGC-1α as well as G6Pase and croe promoter activities. HE-145-111 also could suppress not only the G6Pase and PEPCK mRNA expression but also HBV core promoter and G6Pase promoter activity on 8-bromo-cAMP and dexamethasone-induced system. Nevertheless, HE-145-111 had no effect on the 8-bromo-cAMP/dexamethasone-induced PGC-1α and HNF4α protein expression as well as HNF4α mRNA and promoter activity. In conclusion, HE-145-111 represses the HBV gene expression by down-regulating HNF4α protein and possibly through regulating indirectly the host gluconeogenesis. There have lots of expections for the anti-HBV ability and regulation of gluconeogenesis with HE-145-111 on the period of discussion about drug development. The differentiation between HBV and gluconeogenesis is worthy of further investigation. HE-145-111 has potential to be a clinical drug for treating chronic hepatitis B and regulating blood sugar in the future.

碩士
國立陽明大學
生化暨分子生物研究所
100
Hepatitis B virus (HBV) infection has been documented to associate with developing liver cirrhosis and hepatocellular carcinoma. In previous study, we have identified Chinese herb Scutllariae radix (HD-1S) can suppress HBV gene expression and virus production in human hepatoma cells. The HBV suppressive effect of HD-1S is regulated mainly at the transcriptional level via core promoter by recruitment of cellular transcriptional factors and coactivator PPARγ coactivator-1α (PGC-1α) to play a role as anti-HBV agent. Comparison of the list of liver transcriptional factors participating in HBV transcriptional program with those targeted by PGC-1α reveals a considerable overlap. In this study, I reported that HD-1S potently suppresses core promoter activity is controlled by hepatic gluconeogenesis. Furthermore, I demonstrated that the suppression of HD-1S with respect to HBV core promoter activity is through Akt activation to restrain PGC-1α expression. The mechanistic studies were carried out as the followings: firstly, I showed that combination of cAMP and glucocorticoids (DEX) had a synergistic effect on the induction of phosphoenolpyruvate carboxykinase (PEPCK), or glucose-6-phosphatase (G6Pase), and PGC-1α mRNA respectively. In addition G6Pase or HBV core promoter activity, and PGC-1α protein expression were also synergistically induced by combination of cAMP and DEX treatment. Secondly, I demonstrated that HD-1S suppressed gluconegenesis and HBV core promoter together identifying nt 1656-1675 (19 nt) as binding sequences of core promoter using the deletion mutants of core promoters or the plasmid construct of three times of nt 1656-1675 (19 nt). The overexpression of PGC-1α partially reversed the HD-1S mediated suppression of HBV core promoter activity. Thirdly, I demonstrate that one of the major components, baicalein (5,6,7- trihydroxyflavone) had the same biological activities as HD-1S mentioned above. Fourthly, I showed that either HD-1S or baicalein suppressed PGC-1 expression through activation of Akt signaling pathway. In Hep3B/T2 cells, HD-1S or baicalein suppressed HBV core promoter and G6Pase promoter activity through inhibiting PGC-1α protein expression by phosphorylated Akt, and the inhibition was resembled with PI3K inhibitor, wortmanin which could block PI3K/Akt signally pathway and reverse HD-1S and baicalein mediated gluconeogenic gene and HBV core promoter regulation. Overexpression constitutively active Akt plasmid could reduce induction of HBV core promoter activity against combination of cAMP and DEX induction. On the other hand, dominant negative Akt could not block endogenous Akt activation against HD-1S or baicalein treatment. Fithly, I showed that HD-1S and baicalein could reduce CRE-SEAP activity against cAMP-induced Hep3B/T2 cells using alkaline phosphatase activity analysis. In conclusion, HD-1S and baicalein repress the HBV gene expression by down-regulation PGC-1α directly through regulating the host gluconeogenesis. This mechanism study which HBV is controlled by the hepatic metabolic gluconeogenesis may broaden our understanding of the regulation of HBV expression. The coming out rule of the close link between HBV and liver metabolism can be further exploited for host-targeted therapeutic strategies.

碩士
國立陽明大學
生化暨分子生物研究所
98
Our laboratory has shown previously that TPA and insulin suppress hepatitis B virus surface antigen (HBsAg) production and gene expression in human hepatoma Hep3B/T2 cells. However, the molecular mechanism of TPA and insulin action on HBV viral gene expression is poorly understood. Therefore, firstly, preliminary experiments were performed to demonstrate that TPA and insulin suppress HBV core promoter activity using luciferase as a reporter assay. Then, I identified nt1656-1675 which contains hepatocyte nuclear factor 4α (HNF-4α) binding sequence as a response element of TPA and insulin in the HBV core promoter using serial deletion mutants of core promoter. Secondly, it was found that TPA and insulin reduced protein level but not mRNA of HNF-4α. Thirdly, it was found that TPA activated PKC, ERK, p38 and c-jun in HepG2/A2 cells and insulin activated AKT, ERK and c-jun in Hep3B/T2 cells. In order to understand what signaling pathways of TPA and insulin are employed to suppress HBV gene expression, specific inhibitors were used. PD98059 (MEK1 inhibitor) treatment of the cells abolished TPA action on HNF-4α and HBV core promoter activity. In addition, insulin treatment of the cells suppressed HNF-4α and HBV core promoter activity were blocked by Wortmannin (PI3K inhibitor) treatment. This thesis further demonstrated that TPA and insulin reduced HNF-4α to suppress HBV core promoter activity through the activation of ERK and AKT pathway respectively. Previous researches have shown that peroxisome proliferators-activated receptor-γ coactivator 1 (PGC-1) is strongly induced to coactivate HBV gene expression in liver of fasting mice. PGC-1 is a major metabolic regulator of key gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) acting through the liver-enriched nuclear receptor HNF-4α. Intriguingly, similar transcription factors are involved in the regulation of the HBV core promoter activity and the gluconeogenic gene expression. I therefore hypothesize that TPA and insulin suppress HBV gene expression through regulating host gluconeogenesis machinery. Here, it was showed that TPA and insulin inhibited G6Pase mRNA expression through the activation of ERK and AKT pathway respectively in Hep3B/T2 cells. The combination of cAMP and glucocorticoid had a synergistic effect on the induction of the HBV core promoter activity and G6Pase mRNA expression. TPA and insulin also suppressed cAMP/glucocorticoid-induced HBV core promoter activity and G6Pase mRNA expression in Hep3B/T2 cells. This study has revealed that TPA and insulin have a novel mode of action to suppress HBV gene expression. Based on these observations, I proposed a working model to suggest that the anti-HBV activity of TPA and insulin may act through targeting on hepatic gluconeogenesis machinery.

碩士
國立陽明大學
生化暨分子生物研究所
101
Hepatitis B virus (HBV) infection may result in cirrhosis and hepatocellular carcinoma. Although currently there is hepatitis B vaccine can prevent HBV infection, for already infected patients, the clinical therapies for HBV have many side effects. Therefore, development of new anti-hepatitis B drugs is still one of the important research topics. Previous studies from our laboratory, it has showed that natural compound Helioxanthin, HE-145 can suppress both HBV gene expression and secretion of viral particles in human hepatoma HepG2/A2 cells. The role of inhibition by HE-145 is via interfering with the host transcriptional machinery of viral promoter. It also found that HE-145-111, one of derivatives of HE-145 may suppress HBV core promoter activity and gluconeogenesis genes, such as glucose-6-phosphatase (G6Pase) and phosphoenolcarboxykinase (PEPCK) expression in human hepatoma cells. In my study, it is interested to know whether the anti-HBV activity is through regulating of gluconeogenesis gene expression, and further to suppress HBV core promoter activity. It has been demonstrated that the inhibition of HBV by HE-145-111 is through down-regulating of the metabolic co-activator PGC-1α. It was found that HE-145-111 suppressed HBV surface antigen secretion and core protein in Hep3B/T2 cells and 1.3ES2 cells respectively. In order to understand the relationships between suppression of HBV and gluconeogenesis by HE-145-111 in Hep3B/T2 cells, the combination of 8-bromo-cAMP (cAMP) and dexamethasone had a synergistic effect on the induction of HBV core promoter activity and mRNA levels of G6Pase, PEPCK, and PGC-1α; and on the induction of PGC-1α protein levels. Also the identification of CPD2 (nt1656-1675) as a response element in the HBV core promoter using serial deletion as well as point mutation at different regions of the core promoter. The overexpression of PGC-1α construct plasmid can reverse the inhibition of core promoter activity. HE-145-111 suppressed gluconeogenesis, G6Pase, PEPCK and PGC-1αmRNAs and PGC-1α protein and HBV core promoter together identifying CPD2 (nt 1656-1675) as binding sequences of core promoter. In 1.3ES2 cells, it was confirmed that HE-145-111 not only suppressed HBV mRNA and core protein levels but also suppressed the mRNA levels of G6Pase, PEPCK and transcription co-activator PGC-1α. In conclusion, HE-145-111 represses the HBV gene expression by down-regulation PGC-1α protein and through regulating directly the host gluconeogenesis. This mechanism study which HBV is controlled by the hepatic metabolic gluconeogenesis may broaden our understanding of the regulation of HBV expression.

碩士
高雄醫學院
生物化學研究所
85
The association of hepatitis B virus (HBV) infection with hepatocellular carcinoma (HCC) is well established. However, no consensus regarding the etiology of HCC was elucidated. In order to investigate the variation of gene expression and chromosome stability of hepatocyte after integration of HBV DNA, three hepatoma cell lines (Hep3B , HepG2 and msG2) were employed for Transcript Profile Analysis-PCR and RT-PCR. We have identified the chromosome translocations and differential gene expressions in hepatoma cells after HBV DNA integration. The variations were evidenced no correlation with HBV DNA sequence by southern hybridization. Isolated RT-PCR products were cloned, sequenced, and identified by screening complementary deoxyribonucleic acid (cDNA) database. Five of these cDNA clones have not been identified because they show no homology to known database sequences. The sixth cDNA is 581 base pairs in length and exhibits 98 % homology to nucleotides 8094 to 8674 of human mitochondrial genome ( the Genebank number: #X62996). This location of the mitochondrial genome encodes ATP synthase 6 and cytochrome C oxidase III (Cyt C oxIII).

Yip, Wing Kit.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 100-119).
Abstract also in Chinese.
Abstract (English version) --- p.i
Abstract (Chinese version) --- p.iii
Acknowledgments --- p.v
Table of Contents --- p.vii
List of Tables --- p.x
List of Figures --- p.xi
List of Abbreviations --- p.xiii
Chapter CHAPTER 1 --- INTRODUCTION --- p.1
Chapter 1.1 --- Hepatocellular Carcinoma --- p.1
Chapter 1.1.1 --- Epidermiology --- p.1
Chapter 1.1.2 --- Etiology --- p.1
Chapter 1.2 --- Hepatitis B Virus --- p.3
Chapter 1.2.1 --- The Epidermiology of Hepatitis B Virus Infection --- p.3
Chapter 1.2.2 --- The Morphology and Genome of Hepatitis B Virus --- p.4
Chapter 1.2.3 --- HBV Genotypes and Their Significance --- p.8
Chapter 1.3 --- Hepatitis B Virus X Protein --- p.9
Chapter 1.3.1 --- HBx Alters Various Signal Transduction Pathways --- p.10
Chapter 1.3.2 --- HBx Interacts with Various Transcription Factors and Co-activators --- p.12
Chapter 1.3.3 --- HBx Induces Epigenetic Alterations --- p.14
Chapter 1.3.4 --- Identification of COOH-terminal Truncated HBx in Liver Tumors --- p.15
Chapter 1.4 --- MicroRNAs --- p.17
Chapter 1.4.1 --- Transcriptional Regulation and Biogenesis of MicroRNAs --- p.18
Chapter 1.4.2 --- MicroRNAs and Cancer --- p.21
Chapter 1.4.3 --- MicroRNAs and HCC --- p.25
Chapter 1.5 --- Hypothesis and Aims of the Study --- p.29
Chapter CHAPTER 2 --- MATERIALS and METHODS --- p.30
Chapter 2.1 --- Patients --- p.30
Chapter 2.2 --- Cell Lines --- p.30
Chapter 2.3 --- Cloning of Various HBx Constructs --- p.32
Chapter 2.3.1 --- PCR Amplification of HBx Fragments --- p.32
Chapter 2.3.2 --- Cloning of HBx Fragments into TA-vectos --- p.33
Chapter 2.3.3 --- Heat Shock Transformation --- p.33
Chapter 2.3.4 --- Sub-cloning of HBx Fragments into Lentiviral Vectors --- p.34
Chapter 2.4 --- Generation of Lentivirus --- p.37
Chapter 2.4.1 --- Lentivirus Infection --- p.37
Chapter 2.5 --- RNA Extraction --- p.38
Chapter 2.6 --- Western Blot Analysis --- p.39
Chapter 2.7 --- MiRNA Microarray --- p.40
Chapter 2.7.1 --- Cyanine3-pCp Labeling of RNA Samples --- p.40
Chapter 2.7.2 --- Sample Hybridization --- p.41
Chapter 2.7.3 --- Microarray Wash --- p.41
Chapter 2.7.4 --- Array Slide Scanning and Processing --- p.41
Chapter 2.8 --- Detection of HBx Gene Deletion by PCR --- p.43
Chapter 2.9 --- Immunohistochemistry --- p.44
Chapter 2.10 --- Quantitative Real-time PCR --- p.45
Chapter 2.11 --- Proliferation Assay --- p.47
Chapter 2.12 --- Cell Cycle Analysis --- p.48
Chapter 2.13 --- Annexin V Apoptosis Assay --- p.49
Chapter 2.14 --- Colony Formation Assay --- p.50
Chapter 2.15 --- Statistical Analysis --- p.51
Chapter CHAPTER 3 --- RESULTS --- p.52
Chapter 3.1 --- Detection of Full-length and COOH-terminal Truncated HBx in HCC Tissues --- p.52
Chapter 3.2 --- Confirmation of HBx Expression in HCC Tissues --- p.55
Chapter 3.3 --- Comparison of HBx from Different HBV Genotypes for Study --- p.61
Chapter 3.4 --- Functional Characterization of COOH-tterminal Truncated HBx --- p.64
Chapter 3.4.1 --- Selection of COOH-terminal Truncated HBx --- p.64
Chapter 3.4.2 --- Generation of Various HBx-expressing Hepatocyte Cell Lines --- p.66
Chapter 3.4.3 --- Effect of Full-length and COOH-terminal Truncated HBx on Cell Proliferation --- p.69
Chapter 3.4.4 --- Effect of Full-length and COOH-terminal Truncated HBx Cell Cycle --- p.34
Chapter 3.4.5 --- Effect of Full-length and COOH-terminal Truncated HBx on Apoptosis --- p.45
Chapter 3.5 --- MicroRNA Profiling of Various HBx-expressing Hepatocyte Cell Lines --- p.76
Chapter 3.5.1 --- Identification of Deregulated MicroRNAs by Microarray --- p.76
Chapter 3.5.2 --- Validation of Deregulated MicroRNAs by Real-time PCR Analysis --- p.80
Chapter 3.5.3 --- Confirmation of Deregulated MiRNAs in HCC and Adjacent Non-tumor Tissues --- p.84
Chapter 3.5.4 --- Potential Downstream Targets of the HBx-deregulated MiRNAs --- p.87
Chapter CHAPTER 4 --- DISCUSSION --- p.91
Chapter 4.1 --- The Impact of COOH-terminal Truncated HBx in HCC --- p.91
Chapter 4.2 --- The Biological Significance of COOH-terminal Truncated HBx Induced MiRNAs --- p.94
Chapter 4.3 --- Limitations of the Present Study --- p.97
Chapter 4.4 --- Future Studies --- p.98
Chapter CHAPTER 5 --- CONCLUSION --- p.99
REFERENCES --- p.100