HepG2.2.15细胞中联合应用siRNA抑制HBV复制和表达的研究

观察联合应用siRNA对HepG2．2．15细胞中HBV抗原表达和复制的抑制作用。应用ELISA方法检测HBeAg和HBsAg;HBVDNA水平用实时定量PCR测定;用RT—PCR检测HBVmRNA水平。结果显示,实验中应用的HBV特异性siRNA均具有明显的抗HBV抗原表达和病毒复制作用;联合应用siRNA较单独应用具有更强的抗HBV作用。可见,HepG2．2．15细胞中联合应用siRNA对HBV复制的抑制作用比单独应用siRNA更有效。     

Stable expression and integrated hepatitis B virus genome in a human hepatoma cell line

HepG2.2.15 cell is a widely used cell model for studying HBV (hepatitis B virus) in vitro. In these cells, the HBV genome is integrated in several sites of HepG2 cellular DNA. These multiple copies may have some influence on the cellular processes. We constructed a new plasmid, pSEH-Flag-HBV, and transfected it into HepG2 cells, and then screened it with hygromycin. We then used ELISA, PCR, and RT-PCR to detect the expression of HBV in these cell lines. A cell line that stably expressed hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) was established. Using Southern blotting analysis, we found that the HBV genome was integrated as a single copy in the cellular DNA. This cell line will be a useful alternative model for HBV studies.     

Differential cellular gene expression induced by hepatitis B and C viruses

Hepatitis B virus (HBV) is a hepatotropic virus that causes acute and chronic hepatocellular injury and hepatocellular carcinoma. To clarify how HBV proteins regulate host cellular gene expression, we used our in-house cDNA microarray and HepG2.2.15 cells, which are derived from HepG2 cells and produce all HBV proteins. Of 2304 genes investigated, several genes were differentially expressed in HepG2.2.15 cells compared with HepG2 cells. These genes included insulin-like growth factor II and alpha-fetoprotein, consistent with previous reports. Furthermore, we previously performed similar microarray analyses to clarify the effects of hepatitis C virus (HCV) proteins on host cells, using a HepG2-derivative cell line, which produces all HCV proteins. Using these two microarray results, we compared the differences in cellular gene expression induced by HBV and HCV proteins. The expression of the majority of genes investigated differed only slightly between HBV and HCV protein-producing cells. However, HBV and HCV proteins clearly regulated several genes in a reciprocal manner. Combined, these microarray results shed new light on the effects of HBV proteins on cellular gene expression and on the differences in the pathogenic activities of these two hepatitis viruses.     

Hepatitis B virus MHBs antigen is selectively sensitive to glucosidase-mediated processing in the endoplasmic reticulum.

Previous studies have shown that hepatitis B virus (HBV) secretion from HepG 2.2.15 cells is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase under conditions where secretion of cellular glycoproteins are not detectably affected. The 2.2.15 cells are derived from HepG2 and contain intact dimers of the viral genome. They produce and secrete infectious HBV. The secretion of the viral envelope polypeptide, MHBs, was selectively and quantitatively reduced from 2.2.15 cells in which glucosidase was inhibited, whereas the envelope polypeptide, SHBs, was relatively insensitive, being as resistant as were most host glycoproteins. Because 2.2.15 cells express all HBV ORFs, it seemed possible that the sensitivity of MHBs secretion involved its interaction with the viral nucleocapsid or other viral gene products. The work reported here showed that MHBs secretion from HepG2 cells transfected with a plasmid that expresses only the MHBs polypeptide was as sensitive to glucosidase inhibitors as it was from 2.2.15 cells. These data show that the sensitivity of the MHBs polypeptide secretion to glucosidase inhibitors is entirely encrypted within its structural gene. The reasons the MHBs polypeptide, but not SHBs, is so sensitive to glucosidase processing are discussed.     

Two-dimensional blue native/SDS-PAGE analysis reveals heat shock protein chaperone machinery involved in hepatitis B virus production in HepG2.2.15 cells

Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Despite the prevalence of infection, gaining a complete understanding of the molecular mechanisms of HBV infection has been difficult because HBV cannot infect common immortalized cell lines. HepG2.2.15, however, is a well established version of the HepG2 cell line that constitutively expresses HBV. Therefore, comparative proteomics analysis of HepG2.2.15 and HepG2 may provide valuable clues for understanding the HBV virus life cycle. In this study, two-dimensional blue native/SDS-PAGE was utilized to characterize different multiprotein complexes from whole cell lysates between HepG2.2.15 and HepG2. These results demonstrate that two unique protein complexes existed in HepG2.2.15 cells. When these complexes were excised from the gel and subjected to the second dimension separation and the proteins were sequenced by mass spectrometry, 20 non-redundant proteins were identified. Of these proteins, almost 20% corresponded to heat shock proteins, including HSP60, HSP70, and HSP90. Antibody-based supershift assays were used to verify the validity of the distinct protein complexes. Co-immunoprecipitation assays confirmed that HSP60, HSP70, and HSP90 proteins physically interacted in HepG2.2.15 but not HepG2 cells. We further demonstrated that down-regulation of HSP70 or HSP90 by small interfering RNA significantly inhibited HBV viral production but did not influence cellular proliferation or apoptosis. Consistent with these results, a significant reduction in HepG2.2.15 HBV secretion was observed when the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin was used to treat HepG2.2.15 cells. Collectively these results suggest that the interaction of HSP90 with HSP70/HSP60 contributes to the HBV life cycle by forming a multichaperone machine that may constitute therapeutic targets for HBV-associated diseases.     

Effects of 1,2,4,6-<Emphasis Type="Italic">tetra</Emphasis>-O-galloyl-β-D-glucose from <Emphasis Type="Italic">P. emblica</Emphasis> on HBsAg and HBeAg secretion in HepG2.2.15 cell culture

A polyphenolic compound, 1,2,4,6-tetra-O-galloyl-β-D-glucose (1246TGG), was isolated from the traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae) and assayed for its potential as an anti-hepatitis B virus (HBV) agent. The cytotoxicity of 1246TGG on HepG2.2.15 as well as HepG2 cells was determined by observing cytopathic effects, and the effects of 1246TGG on secretion of HBsAg and HBeAg in HepG2.2.15 cells were assayed by enzyme immunoassay. Results indicates that treatment with 1246TGG (6.25 μg/mL, 3.13 μg/mL), reduced both HBsAg and HBeAg levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. This study provides a basis for further investigation of the anti-HBV activity and possible mechanism of action of 1246TGG.     

Synthesis and biological assay of 4-aryl-6-chloro-quinoline derivatives as novel non-nucleoside anti-HBV agents

A series of 4-aryl-6-chloro-quinoline derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities, namely the abilities to inhibit the secretion of HBV surface antigen (HBsAg), HBV e antigen (HBeAg), and replication of HBV DNA in HepG 2.2.15 cells. Most of the compounds exhibited moderate inhibitory activity against the secretion of HBsAg and HBeAg. Nine compounds (3, 5, 6, 7, 10, 14, 17, 20, 24) showed significant inhibition against HBV DNA replication with IC(50) values in the range of 4.4-9.8 μM, which were comparative to that of positive control tenofovir. Of them, compounds 10, 17, and 20 had low cytotoxicities, resulting in high SI values, >551.2, >143.7, and >284.5, respectively.     

牡蛎多糖体外对HepG2.2.15细胞分泌HBsAg、HBeAg的影响

目的 探讨牡蛎多糖体外抗乙型肝炎病毒(HBV)的作用.方法 采用HepG2.2.15细胞为体外细胞模型,给予不同浓度牡蛎多糖进行混合培养,作用9d后收集上清液,用ELISA法测定上清液中HBsAg、HBeAg的水平,观察药物对HepG2.2.15细胞分泌病毒杭原的影响,同时以MTT法检测药物在体外对HepG2.2.15细胞生长的抑制作用.结果 牡蛎多糖浓度在0.1～1 000 μg/mL时,对细胞无明显的毒性作用;牡蛎多糖可明显抑制HBsAg、HBeAg的分泌,其半数有效浓度( IC50)分别为362.2、558.6 μg/mL,治疗指数(TI)为＞27.6和＞17.9.结论 牡蛎多糖体外具有一定的抗HBV作用.     

NIRF在体内外可明显抑制乙型肝炎病毒抗原的表达

随着对NIRF（Np95/ICBP-90 like RING finger protein）研究的深入,其功能已涉及细胞癌变进程以及表观遗传学等领域. 近期研究显示,NIRF能与HBc (hepatitis B virus core protein )相互结合,但其对乙型肝炎病毒(HBV)抗原表达的影响尚不明确. 本文通过转染pAAV-HBV1.3质粒和高压水动力法尾静脉注射BALB/C小鼠,建立乙型肝炎病毒的细胞和动物模型,研究NIRF对乙型肝炎病毒抗原表达的影响. ELISA检测细胞上清和小鼠血清中HBsAg、HBeAg的分泌和表达情况,Western 印迹或免疫组化染色技术检测HBcAg. 结果显示,乙型肝炎病毒抗原分泌的细胞以及小动物模型建立成功,并且无论在体内外,NIRF都能对它们的表达起抑制作用,期待能为后续的HBV致病机理以及治疗药物的研究提供支持与帮助.     

热休克蛋白70（HSP70）对HBV复制及抗原分泌的影响

目的了解慢性乙型肝炎患者（CHB）血清中热休克蛋白70（HSP70）与HBV—DNA的关系,并探讨HSP70能否影响乙型肝炎病毒（HBV）的复制与抗原分泌。方法选择安徽医科大学第二附属医院住院和门诊CHB患者60例为研究对象,并以30例健康体检人群为对照者,采用酶联免疫技术（ELISA）分析CHB患者和对照者血清HSP70表达水平,运用荧光定量PCR技术分析CHB患者血清HBV—DNA复制水平;以RNA干扰（RNAi）技术抑制HepG2．2．15细胞中HSP70基因的表达,同时分析细胞培养上清中HBVDNA以及相关抗原的表达情况。结果CHB患者血清中HSP70的表达水平明显高于其在健康对照者血清中的表达水平（P〈0．05）,且与HBV—DNA拷贝数呈正相关性（r=0．540,P〈0．05）;其中HBeAg阳性的CHB患者血清中HSPT0的表达水平也显著高于HBeAg阴性CHB患者血清中的表达水平（P〈0．05）。以RNAi技术特异性降低HSP70在HepG2．2．15细胞中的表达,HepG2．2．15细胞培养上清中HBV—DNA和相关抗原的分泌量明显降低（P〈0．05）。结论HSP70蛋白很可能在HBV的生命周期中发挥重要作用,从而影响HBV的复制和乙型肝炎的进展。     

Fibronectin is essential for hepatitis B virus propagation in vitro: may be a potential cellular target?

Previous studies in our laboratory strongly suggested that fibronectin was upregulated by hepatitis B virus (HBV) in HepG2.2.15 cells. Report by Budkowska A also indicated that human liver fibronectin could bind HBV in a species-restricted manner. Therefore, it is reasonable to ask whether inhibiting fibronectin expression might have anti-HBV activity and whether fibronectin might be developed as a new potential cellular target for anti-HBV drugs. By using fibronectin antisense oligonucleotide (ASODN), fibronectin antibody, and Protocatechuic aldehyde (PA), we were able to show that HBV productions in HepG2.2.15 cell culture were reduced in a dose-dependent manner by fibronectin inhibition. In addition, we found that treatment with ASODNs, fibronectin antibody, and PA did not affect HepG2.2.15 cell viability. Furthermore, we observed that fibronectin inhibition sensitized HBV to anti-HBV drugs. In summary, this study demonstrates that fibronectin is essential for HBV propagation and also provides some evidences for the potential of fibronectin as a new cellular target for HBV infection therapy.     

Mucroporin-M1 Inhibits Hepatitis B Virus Replication by Activating the Mitogen-activated Protein Kinase (MAPK) Pathway and Down-regulating HNF4α in Vitro and in Vivo

Hepatitis B virus (HBV) is a noncytopathic human hepadnavirus that causes acute, chronic hepatitis and hepatocellular carcinoma (HCC). As the clinical utility of current therapies is limited, new anti-HBV agents and sources for such agents are still highly sought after. Here, we report that Mucroporin-M1, a scorpion venom-derived peptide, reduces the amount of extracellular HBsAg, HBeAg, and HBV DNA productions of HepG2.2.15 cells in a dose-dependent manner and inhibits HBV capsid DNA, HBV intracellular RNA replication intermediates and the HBV Core protein in the cytoplasm of HepG2.2.15 cells. Using a mouse model of HBV infection, we found that HBV replication was significantly inhibited by intravenous injection of the Mucroporin-M1 peptide. This inhibitory activity was due to a reduction in HBV promoter activity caused by a decrease in the binding of HNF4α to the precore/core promoter region. Furthermore, we confirmed that Mucroporin-M1 could selectively activate mitogen-activated protein kinases (MAPKs) and lead to the down-regulation of HNF4α expression, which explains the decreased binding of HNF4α to the HBV promoter. Moreover, when the protein phosphorylation activity of the MAPK pathway was inhibited, both HNF4α expression and HBV replication recovered. Finally, we proved that treatment with the Mucroporin-M1 peptide increased phosphorylation of the MAPK proteins in HBV-harboring mice. These results implicate Mucroporin-M1 peptide can activate the MAPK pathway and then reduce the expression of HNF4α, resulting in the inhibition of HBV replication in vitro and in vivo. Our work also opens new doors to discovering novel anti-HBV agents or sources.     

iTRAQ‐coupled 2‐D LC‐MS/MS analysis of protein profile associated with HBV‐modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.     

The influence of the signal dynamics of activated form of IKK on NF-kappaB and anti-apoptotic gene expressions: a systems biology approach

NF-kappaB activation plays a crucial role in anti-apoptotic responses in response to the apoptotic signaling during tumor necrosis factor (TNF)-alpha stimulation. TNF-alpha induces apoptosis sensitive to the hepatitis B virus (HBV) infected cells, despite sustained NF-kappaB activation. Our results indicate that the HBV infection induces sustained NF-kappaB activation, in a manner similar to the TNF-alpha stimulation. However, these effects are not merely combined. Computational simulations show that the level of form of the IKK complex activated by phosphorylation (IKK-p) affects the dynamic pattern of NF-kappaB activation during TNF-alpha stimulation in the following ways: (i) the initial level of IKK-p determines the incremental change in IKK-p at the same level of TNF-alpha stimulation, (ii) the incremental change in IKK-p determines the amplitudes of active NF-kappaB oscillation, and (iii) the steady state level of IKK-p after the incremental change determines the period of active NF-kappaB oscillation. Based on experiments, we observed that the initial level of IKK-p was upregulated and the active NF-kappaB oscillation showed smaller amplitudes for a shorter period in HepG2.2.15 cells (HBV-producing cells) during TNF-alpha stimulation, as was indicated by the computational simulations. Furthermore, we found that during TNF-alpha stimulation, NF-kappaB-regulated anti-apoptotic genes were upregulated in HepG2 cells but were downregulated in HepG2.2.15 cells. Based on the previously mentioned results, we can conclude that the IKK-p-level changes induced by HBV infection modulate the dynamic pattern of active NF-kappaB and thereby could affect NF-kappaB-regulated anti-apoptotic gene expressions. Finally, we postulate that the sensitive apoptotic response of HBV-infected cells to TNF-alpha stimulation is governed by the dynamic patterns of active NF-kappaB based on IKK-p level changes.     

Synthesis of hemslecin A derivatives: A new class of hepatitis B virus inhibitors

A series of hemslecin A derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities, namely, inhibiting the secretion of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV DNA replication on HepG 2.2.15 cells. Most of the derivatives showed enhanced anti-HBV activities, of which compounds A1–A7, B5, C and E exhibited significant activities inhibiting HBV DNA replication with IC₅₀ values of 2.8–11.6 μM, comparable to that of the positive control, tenofovir. Compounds A1–A3, A5, B5, and C displayed low cytotoxicities, which resulted in high SI values of 89.7, 55.6, 77.8, >83.4, >55.8, and >150.5, respectively.     

Proteome responses to stable hepatitis B virus transfection and following interferon alpha treatment in human liver cell line HepG2

Hepatitis B virus (HBV) infection is a worldwide health problem and may develop to liver fibrosis, cirrhosis, and even hepatocellular carcinoma. To investigate the global proteome responses of liver‐derived cells to HBV infection and IFNα treatment, 2‐DE and MS‐based analysis were performed to compare the proteome changes between HBV stably transfected cell line HepG2.2.15 and its parental cell line HepG2, as well as HepG2.2.15 before and after IFNα treatment (5000 IU/mL for 72 h). Compared to HepG2, 12 of 18 down‐regulated and 27 of 32 up‐regulated proteins were identified in HepG2.2.15. After IFNα treatment, 6 of 7 down‐regulated and 11 of 14 up‐regulated proteins were identified. Differentially expressed proteins caused by HBV infection were involved with cytoskeletal matrix, heat shock stress, kinases/signal transduction, protease/proteasome components, etc. Prohibitin showed a dose‐dependent up‐regulation during IFNα treatment and might play a potent role in anti‐HBV activities of IFNα by enhancing the crossbinding p53 expression to achieve the apoptosis of HBV infected liver cells. Down‐regulation of interferon‐stimulated gene 15 (ISG15) in HepG2.2.15 and recovery by IFNα suggested its relationship with IFNα's anti‐HBV effect.     

Synthesis and biological evaluation of nucleoside analogues than contain silatrane on the basis of the structure of acyclovir (ACV) as novel inhibitors of hepatitis B virus (HBV)

Hepatitis B virus (HBV) infection causes major public health problems worldwide. Acyclovir (ACV) is mainly used to inhibit herpes simplex virus (HSV) rather than HBV. In this study, we used the combination principle to design and synthesize nucleoside analogues that contain silatrane on the basis of the structure of ACV. We found that the compounds were effective inhibitors of HBV, both in vitro and in vivo. All of the compounds showed suppressive activity on the expression of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the HepG2.2.15 cell line with low cytotoxicity. One of compounds was studied in HBV transgenic mice model, and the test results showed its ability to reduce the levels of HBsAg, HBeAg and HBV DNA by ELASE and qPCR. Furthermore, significant improvement of T lymphocyte was observed after treatment, as evaluated by flow cytometry (FCM).