| Hepatitis B virus (HBV) infection induces acute or fulminant hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC), which affect hundreds of millions of people worldwide. It is demonstrated by epidemiology data that more than 2,000 million people were infected by HBV, and among those about 350 million are chonic infection. About 40% chonic patients will develop HCC, with the incidence 100-200 folds higher than that of normal people. The investigation on HBV infection is an urgent and important issue aroud the world.When challenged by HBV, most people can clear the virus and obtain recovery. Other patients would undergo different status, such as fulminant hepatitis, acute hepatitis, asymptomatic carrier (AsC), chronic hepatitis, liver cirrhosis, HCC, et al. Researchers have been dedicated in construct cell models as well as animal models to mimic different status of HBV carriers. Hepatoma cell lines transfected with HBV genome or gene fragments are very helpful in basic investigations and have been widely used. It is imperative to establish ideal animal models of HBV infection, which will benefit both for the study of molecular mechanisms during HBV induced diverse prognosis and for screening novel drugs for anti-HBV therapy. Therefor, we designed series of different mouse carriers for HBV, including acute infection and hepatitis mouse models, HBV transgenic mouse model which is tolerant to the virus, and HBV transgenic mouse hepatocarcinogenesis model. These models are used to elucidate molecular mechanisms of HBV induced hepatitis and HCC, and to filtrate key molecules in this pathological process.HBV infection induced hepatocyte apoptosis is the important molecular mechanisms during hepatocyte death and liver function damage, which takes potential roles in different prognosis in patients of HBV infection. Previous investigationsdemonstrated that tumor necrosis factor (TNF)-αand FasL induced apoptosis playsimportant role in HBV related hepatocyte injury. TRAIL (TNF-relatedapoptosis-inducing ligand) is a member of TNF family, which has been cloned andnamed in 1995. TRAIL may selectively induce apoptosis in transformed or viralinfected cells but not in normal cells. Many factors, including virus or viral proteins,X-ray, and drugs, can alter cell sensitivity to TRAIL induced apoptosis. According toour in vitro data, we firstly investigated the mechanisms of HBV sensitizing TRAILinduced apoptosis in vivo. Using mouse models, we observed the influence of HBVinfection on TRAIL induced hepatocyte apoptosis. Key molecules in the apoptosispathway were screened out and reversely verified by RNA interference (RNAi)technology. To investigate the therapeutic effect of sDR5, we used an acute hepatitismouse model with hydrodynamics-based in vivo gene transfection method. Weobserved the alleviation of liver function under treatment of sDR5 and compared theefficiency with the Stronger Neo-Minophagen C (SNMC), a commonly used drug forpatients with liver diseases. This investigation will prompt that sDR5 may be apotential therapeutic candidate, with combination of current antiviral reagents, for thesurvival of patients suffered from fulminant hepatitis.HBV transgenic mouse inevitablely develop hepatoma during the 20 month of life. This character makes HBV Tg a natural model of HBV related HCC, simulating the process of hepatocarcinogenesis. We used this model to observe dynamic changes of tumorigenesis related factors by bioinformatics approach. Liver gene expression difference was revealed by gene chip. And proteomics profiling was detected by proteomics technologies including two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption inoization-time of flight mass spectrometry (MALDI-TOF-MS), and peptide mass fingerprinting (PMF). Crucial genes and proteins can be selected combining gene chip and proteomics data, which will describe the pattern of dynamic molecular changes at systemic level. The study will benefit for elucidating the mechanisms of HBV related HCC and finding novel therapeutic targets for liver cancer. OBJECTIVES:1. Establish two kinds of acute hepatitis mouse models: HBV Tg adoptively transferred with HBV specific CTLs; pcDNA3-1.1HBV transfected by hydrodynamics-based tail vein injection method. These models will provide the platform for the study of the relationship between HBV infection and TRAIL induced apoptosis.2. Observe the influence of HBV infection on TRAIL induced hepatocyte apoptosis in the mouse models. Probe into the molecular mechanisms from membrane receptor level and cytoplasmic level, as well as mitochondrial dependent and independent pathway. Key molecules will be found and verified by RNAi.3. Investigate the therapeutic effect of sDR5 using the mouse models to observe the alleviation of liver function under treatment of sDR5. Compare the efficiency of sDR5 with the Stronger Neo-Minophagen C (SNMC), a commonly used drug for patients with liver diseases.4. Use the HBV Tg as natural model of hepatocarcinogenesis to observe dynamic changes of tumorigenesis related factors. Crucial genes and proteins can be selected combining gene chip and proteomics data, which will describe the pattern of dynamic molecular changes at systemic level.METHODS:1. Establishment and evaluation of novel mouse models of HBV induced acutehepatitis1.1 Acute hepatitis model established by adoptive transferring HBV specificCTLs to HBV Tg miceTo establish a mouse model of hepatitis B by using HBV-transgenic mice, and to transfer HBV-specific CTLs induced from syngeneic BALB/c mice immunized by a eukaryotic expression vector containing HBV complete genome DNA. Forty-eight hours later, the level of serum protein and transaminase was detected with biochemical method, liver and kidney were sectioned and stained by HE to observe the pathological changes. 1.2 Acute hepatitis mouse model established by hydrodynamics-based tail vein injectionBALB/c mice were transfected with pcDNA3-HBV1.1 by hydrodynamics-based tail vein injection. Mice were sacrificed at 8, 24, 96, 168, and 240 hours after injection of plasmids for model appraisal. Serology for transaminase activity, antigenemia and antibodies were determined. Viraemia was measured by purification and quantitation of encapsidated viral DNA using quantitative fluorescence PCR kit for detection of HBV. Liver tissues fixed in formalin were sectioned and stained by HE, and HBV core protein (HBcAg) was visualized by immunohistochemical staining.2. In vivo study of HBV infection and TRAIL apoptosis pathway2.1 Identification of key molecules of TRAIL apoptosis pathwayThe research included extracellular level and intracellular level.The expression of TRAIL receptors, DR5, DcR1, and DcR2 on the membrane of hepatocytes in the acute hepatitis mouse were measured by semi-quantitative reverse transcript PCR.Western blotting to detect the expression of Bax and the activation of Caspases 3, 8 and 9.2.2 Verifying of the key molecule Bax of TRAIL apoptosis pathwaySilence the upregulated molecule Bax by RNA interference to verify the change and function of this key molecule in HBV induced TRAIL apoptosis pathway in vivo.3. Blockade of TRAIL pathway by sDR5 ameliorates HBV-induced hepatitis3.1 Expression and preparation of recombinant human sDR5Pichia Pastoris (Pp) GS115 strain, transformed with yeast expression vector containing soluble DR5, were produced in abundance and the culture supernatant was collected. ProBondTM affinity chromatography column was used to purify recombinant soluble DR5. The purity was detected by SDS—PAGE and Western Blot. The biological activity of purified sDR5 on TRAIL-induced apoptosis was tested in Hela cell line.3.2 Reversion effects of sDR5 on the apoptosis induced by TRAIL and the alleviation of hepatitis Purified sDR5 were injected intraperitoneally into the two kinds of mouse models twice, 24 or 0 hours before model induction, respectively. Liver function and hepatocyte apoptosis were measured to observe the blockade effect of sDR5. Then a serial concentration of sDR5 (2-64 mg/kg) were used to decide the optimal dosage.3.3 Comparation of the therapeutic effect of sDR5 and SNMCThe most effective dosage of sDR5 was used to compare the therapeutic effect with SNMC (10 mg/kg).4. Bioinformatics approach for the mechanisms of hepatocarcinogenesis in HBV transgenic mice4.1 Identification of hepatocarcinogenesis model of HBV Tg miceHBV transgenic mice of 6, 12 and 20-month-old and the age-matching BALB/c mice as control were sacrificed for model identification. Liver tissues were sectioned for histological observation and related parameters were measured.4.2 Microarray analyses and molecular profiling during hepatocellular carcinogenesisThe total liver RNA was extracted and hybridized on an oligonucleotide chip containing 35,000 genes to conclude a molecular portrait of dynamic changes in gene expression associated with different stages of hepatocellular carcinogenesis. Significant differentially expressed genes were analysed by bioinformatics approach.4.3 Proteomics approach of the dynamic changes during hepatocarcinogenesis,Differentially expressed proteins between HBV Tg mice and control BALB/c mice were identified by proteomics methods of 2-DE, MOLDI-TOF-MS and PMF. The complete procedure includes: 1) Preparation and quantification of protein samples; 2) 2-DE; 3) Scan and analysis of gel images; 4) MOLDI-TOF-MS; 5) SW1SS-2DPAGE search; 6) Mascot database search to confirm the data of PMF.Gene chip and proteomics data were verified by RT-PCR, real-time PCR, and Western blotting. Functions of significant molecules were studied by gene transfection or RNAi. RESULTS1. Establishment and evaluation of novel mouse models of HBV induced acute hepatitis1.1 Acute hepatitis model established by adoptive transferring HBV specificCTLs to HBV Tg miceALT and AST levels indicate that the liver function of the models was injured. In the model group, hepatic cells of transgenic mice had diffuse ballooning degeneration, cytoplasmic loosening, acidophilic changes, appearance of acidophilic body, spotty necrosis of hepatic tissue and obvious infiltration of inflammatory cells in the portal area. There was no obvious inflammation and structure damage of the kidney.1.2 Acute hepatitis mouse model established by hydrodynamics-based tail veininjectionALT/AST activity peaked at 8 hours after hydrodynamics injection and gradually decreased to normal range at 168 hours. Viral antigens showed the same trends and completely disappeared from the blood at 168 to 240 hours, coincident with the appearance of antiviral antibodies. HBcAg-positive hepatocytes were randomly distributed throughout the liver lobule with a tendency for localization in the centralobular area. The copy number of HBV DNA was highest at 8 and 24 hours, and decreased after 96 hours. Viral replication in this model was remarkable compared with that of HBV transgenic mice.2. In vivo study of HBV infection and TRAIL apoptosis pathway2.1 Identification of key molecules of TRAIL apoptosis pathway(1) Extracellular level: There was no significant difference of TRAIL receptors DR5, DcR1, and DcR2 expression between the model and control group.(2) Intracellular level: We examined the hepatic levels of Bax protein in HBV transgenic mice and non-transgenic mice. We found that Bax protein levels in the liver were significantly higher in non-treated HBV transgenic mice (without hepatitis) than non-transgenic mice and that hepatitis further increased the Bax level in HBV transgenic mice. Degradation of both procaspases-8 and -9 was significantly enhanced as the consequence of Bax upregulation. We also examined the hepatic Bax expression in mice that had developed aseptic hepatitis following injection of Con-A. We found that hepatic Bax protein level was increased by 2.1 folds in BALB/c mice 8 hours after the onset of Con-A induced hepatitis as compared to control mice not treated with Con-A.2.2 Verifying of the key molecule Bax of TRAIL apoptosis pathwayWe examined the consequence of Bax knockdown in our model of HBV-induced hepatitis. Hydrodynamic administration of a plasmid encoding Bax siRNA significantly and specifically reduced hepatic Bax levels in mice with HBV-induced hepatitis. This was accompanied by a significant reduction of ALT and AST in the blood. Histochemical analysis of the liver sections revealed less inflammation and reduced number of apoptotic cells in Bax siRNA treated group as compared to control groups. Western blot analysis of hepatic extracts revealed that Bax siRNA preferentially blocked the degradation of pro-caspases-3 and -9 but not that of pro-caspase-8, an effect similar to that of Bax siRNA in vitro.3. Blockade of TRAIL pathway by sDR5 ameliorates HBV-induced hepatitis3.1 Expression and preparation of recombinant human sDR5Pp GS115 strain, transformed with yeast expression vector containing soluble DR5, could successfully express sDR5 (26kD). The results of SDS-PAGE and Western Blot show that highly purified, recombinant human soluble DR5 was obtained. The yield is up to 20μg/ml of yeast culture. Purified soluble DR5 could effectively block the apoptosis of Hela induced by TRAIL.3.2 Reversion effects of sDR5 on the apoptosis induced by TRAIL and the alleviation of hepatitisWe used the acute hepatitis mouse model to investigate blockade effect of purified sDR5. ALT and AST levels in group sDR5-l were much lower than those of models at 24 hours. However, this therapeutic effect in group sDR5-2 did not occur until 96 hours. These results demonstrated that earlier injection of sDR5 should be more effective. Injection of 2 mg/kg sDR5 protein had no influence on transaminase levels. The reversion effect appeared at 4 mg/kg, and formed a platform at 16-64 mg/kg. We concluded that the most effective dosage of sDR5 was 16 mg/kg. 3.3 Comparation of the therapeutic effect of sDR5 and SNMCThe therapeutic effect of sDR5 with the most effective dosage (16 mg/kg) was compared with that of SNMC. Both sDR5 and SNMC were injected twice 24 hours before model induction. Either of the treatment can alleviate liver function dramatically without significant difference. Thus, sDR5 represents a potential novel therapeutic drug for patients with fulminant hepatitis.4. Bioinformatics approach for the mechanisms of hepatocarcinogenesis in HBV transgenic mice4.1 Identification of hepatocarcinogenesis model of HBV Tg miceHistological observations confirmed that HBV transgenic mice developed hepatoma within the 20 month of life, with the incidence above 95%. Positive HBV antigens can be detected in the serum and liver.4.2 Microarray analyses and molecular profiling during hepatoceilular carcinogenesisThe number of differentially expressed transcripts was 235±32, 687±72 and 711±156, in the group of 6, 12 and 20 month, respectively. These genes were involved with important cellular pathways, including cell growth regulation, apoptosis, immune response, metabolisms, stress, and cell adhesion. Several complement components such as MBL2, C4, Factor H and Factor I were moderately downregulated at 12 month. While in 20-month-old group, MBL2, C4, Factor H and Factor I were obviously downregulated; CD59a, Maspl, C2 and C4bp were also downregulated simultaneously. We focused on several downregulated genes such as Thrsp, Cypl7al, Dbp, C4, Ugtlal, Tef, Cfi, Tnfaip9, Cfh, Tdel, Mbl2, et al. We further explored the microRNA (miRNA) profiling relevant to hepatocellular carcinogenesis, for miRNAs closely associated with the process of tumorigenesis. The downregulated genes discovered in HBV transgenic mice may be regulated by let-7, miR-122a, miR-214, miR-133, miR-370, miR-34, et al.Changes of complement components expression has been verified by RT-PCR and obtained the same result with gene chip. Other differentially expressed genes and proteins, associated with carcinogenesis, immune response and apoptosis, should be detected and further investigated.4.3 Proteomics approach of the dynamic changes during hepatocarcinogenesisThe 2-DE gels were analyzed by PDQuest software and the numbers of differentially expression proteins were 15±6 upregulated and 18±6 downregulated at 6 month, 17±5 and 20±8 at 12 month, and 25±12, 23±13 at 20 month. We have selected 126 spots totally from the gels for MOLDI-TOF-MS to produce PMFs. Some of them were identified by Mascot database search and analysis.CONCLUSIONS1. Novel mouse models of HBV induced acute hepatitis and HBV related HCC were established and identified. These models can serve as ideal platform for the investigation of TRAIL induced apoptosis in vivo, the mechanisms of hepatitis and HCC, and the screening of therapeutic drugs.2. HBV infection significantly increased the sensitivity of hepatocytes to TRAIL-induced apoptosis. The increase in TRAIL sensitivity was associated with a dramatic upregulation of Bax protein expression. Knocking down Bax expression using Bax-specific siRNA blocked HBV-induced hepatitis and hepatocyte apoptosis. The degradation of caspases-3 and -9, but not that of Bid or caspase-8, was preferentially affected by Bax knockdown. These results establish that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific siRNA is effective in inhibiting HBV-induced hepatitis and hepatic cell death.3. sDR5 alleviates liver damage by blocking TRAIL-induced apoptosis of HBV-transfected hepatocytes. sDR5 injection at 16 mg/kg 24 hours before HBV transfection was the most effective. sDR5 was equally effective in protecting liver injury as the Stronger Neo-Minophagen C (SNMC). Thus, sDR5 represents a potential novel therapeutic drug for patients with fulminant hepatitis.4. The molecular profilings of hepatocarcinogenesis were revealed by the combination of gene chip and proteomics technology. Complement components play an important role during the formation of HCC in HBV transgenic mice. INNOVATIONS AND SIGNIFICANCE1. The hepatitis mouse models are convenient for handling and easy to be widely used for mechanisms study and drug screening in different labs.2. HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific siRNA is effective in inhibiting HBV-induced hepatitis and hepatic cell death. Bax siRNA represents a potential protective agent for patients with hepatitis and has a great value of generalization.3. We firstly reported that sDR5 represents a potential novel therapeutic drug for patients with acute or fulminant hepatitis. It has great value for exploitation and used in several kinds of inflammatory diseases.4. We used HBV Tg as natural model of hepatocarcinogenesis for the first time to elucidate molecular mechanisms of HBV related HCC. The molecular profilings of hepatocarcinogenesis were revealed by the combination of gene chip and proteomics technology, which could benefit for early diagnosis, prevention and novel therapy methods.
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