Study On TRAIL And Its Related Molecules In The Imbalance Of Apoptosis After HBV Infection

Hepatitis B virus (HBV) is a confirmed carcinogenesis related factor.It is not clear why HBV could induce the hepatocyte injury and lead to the malignant transformation of the liver tissue cells in most cases. The disorder of immune regulation and the imbalance of hepatocyte apoptosis after HBV infection is the main cause which result in the different pathological process and clinical prognosis. Investigation of the mechanism between HBV and the immune surveillance molecule will help to understand the pathogenesis of HBV and find an effective treatment for the HBV related diseases.TRAIL(TNF related apoptosis inducing ligand ), TWEAK (TNF-like weak inducer of apoptosis) are the new members of TNF family,which have inimitable apoptosis inducing characters.TRAIL, TWEAK are widely distributed and have no cytoxicity on human tissues under physiological condition.Only in pathological condition, they are activated to participate in the immune response. As potent immune surveillance and immune effective molecules,whether TRAIL and TWEAK play roles in the pathogenesis of HBV infection is not yet known.We took the lead in carrying out the study on the mechanism of TRAIL and TWEAK that may involve in the pathogenesis of HBV infection. OBJECTIVES1. To investigate whether HBV will influence TRAIL induced cell apoptosis and its mechanism.2. To explore the regulative effect of IFN-γ on TRAIL induced apoptosis in HBV transfected cell and its mechanism.3. To elucidate whether TRAIL-TRAIL receptors contribute to the increased apoptosis of peripheral blood mononuclear cell (PBMC) in chronic Hepatitis B patients and to clarify the relativity between the expression of TRAIL receptors and the clinical incidence in chronic Hepatitis B patients.4. To elucidate whether Hepatitis B virus will influence TWEAK induced cell apoptosis and the regulative effect of IFN-y on TWEAK induced apoptosis in HBV transfected cell.METHODS1. Establishment of Human hepatoma cell lines which were transfected with HBV (adr subtype) stably1.1 Construction of the HBV gene eukaryotic expression vectors pcDNA3-l.lHBV, pcDNA3-3.0HBV respectivelya the construction of pcDNA3-l.lHBV p3.6ll which contains 1.1HBV DNA was digested with pVU II .The 3907bp fragment was obtained and cloned into pcDNA3 vector digested with EcoRV. Restriction endonuclease assay was used to select and identify the positive clones.Automatic DNA sequencing was used for the anlysis of HBV sequences.b the construction of pcDNA3-3.0HBV pUC19-3.0HBV which contains 3.0HBV DNA was digested with EcoRI and Hindlll.The 9600bp fragment was obtained and cloned into pcDNA3 vector digested with EcoRI and Hindlll.Restriction endonuclease assay was used to select and identify the positive clones.1.2 Establishment of Human hepatoma cell lines which were transfected with HBV (adr subtype) stablyBEL-7402 ^ HepG2 were transfected with the pcDNA3-l.lHBV > pcDNA3-3.0HBV mediated by lipofectin respectively.Geneticin (G418) was added into the medium after 24h.Cell clones could be observered 3-4 weeks after G418 added. The expression of HBx^ HBpreS2 mRNA in cell clones were detected by RT-PCR.The expression of HBsAg> HBeAg in cell clones were calculated by ELISA.2. Study the influence of HBV transfection on TRAIL induced cell apoptosis and its mechanism82.1 Study on the effect of HBV transfection on TRAIL induced apoptosisBEL-7402/l.lHBV and its controls (empty cell control: BEL -7402; empty vector control: BEL-7402/pcDNA3)> HepG2/l.lHBV and its controls (empty cell control: HepG2; empty vector control: HepG2/pcDNA3) were treated with TRAIL (10ng/mL),24h later,the apoptosis induced by TRAIL in these cell lines were examined by TUNEL assay ^ DNA Ladder> Caspase 3 activity assay and Caspase 3 activation.2.2 Study on the mechanism of apoptosis change in BEL-7402/l.lHBV > BEL-7402/pcDNA3x BEL-7402a Extracellular level The expressions of DR4> DR5> DcRK DcR2 in BEL-7402/l.lHBV > BEL-7402/pcDNA3, BEL-7402 were assayed by RT-PCR and fiowcytometery.b Intracellular level The activation of Caspase8 >. Caspase9 in BEL-7402/l.lHBV and its controls were assayed by Westemblot. The expressions of Bax> FLIP in BEL-7402/l.lHBV and its controls were detected by Westemblot. The activity of nuclear transcription factor - Kappa B (NF-kB) in BEL-7402/l.lHBV and its controls were detected by dual luciferase report gene assay system.3. Study on the effect of IFN-y on TRAIL induced cell apoptosis in HepG2/1.1 HBV and its mechanism3.1 Apoptosis of HepG2/l.lHBV cell induced by IFN-y> TRAIL * IFN-y plus TRAIL was detected by TUNELHepG2/l.lHBV was pretreated with IFN-y (200IU/mL) ,12h later, TRAIL (lOng/mL) was added.Another 24h later, the apoptosis in HepG2/l.lHBV was detected by TUNEL.3.2 The mRNA of DR4> DR5> DcRl, DcR2 and FLIP in HepG2/l.lHBV were assayed by RT-PCR.4. Study on the expressions of TRAIL receptors in the PBMC of chronic hepatitis B patients and its relativity with the clinical incidenceThe expressions of DR4> DR5^ DcRl and DcR2 in 55 cases of chronic hepatitis B patients and 30 cases of normal people were assayed by RT-RCR andFlowcytometery. The relativity of the expression of TRAIL receptors and the clinical incidence was analysed.5. Study on the effect of HBV on TWEAK induced apoptosisThe apoptosis of BEL-7402/1.1 HBV, HepG2/l.lHBV and their corresponding controls induced by TWEAK were calculated by TUNEL.6. Study on the effect of IFN-y on TWEAK induced cell apoptosis in BEL -7402/1.1HBV and HepG2/l.lHBVApoptosis of BEL-7402/1.1HBV, HepG2/l.lHBV cell induced by IFN-y plus TRAIL, TWEAK, IFN-y were detected by TUNEL. RESULTS1. The human hepatoma cell lines transfected with HBV (adr) were established successfullyThe HBV eukaryotic expression vectors pcDNA3-l.lHBV, pcDNA3-3.0HBV were constructed successfully. They were verified by endonuclease digestion and DNA sequencing.BEL-7402 , HepG2 were transfected with the pcDNA3-l.lHBV , pcDNA3-3.0HBV mediated by lipofectin respectively.Geneticin (G418) was added into the medium after 24h.Cell clones could be observed 3-4 weeks later.They were named as BEL-7402/1.1 HBV , HepG2/l.lHBV > BEL-7402/3.0HBV and HepG2/3 .OHBVrespectively.The expression of HBx mRNA, HBpreS2 mRNA , HBeAg and HBsAg in BEL-7402/1.1HBV,HepG2/l.lHBV,BEL-7402/3.0HBV and HepG2/3.0HBV are all positive. Moreover, the expression level of HBeAg and HBsAg is higher in pcDNA3-l.lHBV transfected cell lines than pcDNA3-3.0HBV transfected cell lines(p <0.05). Maybe it was caused by the large size of pcDNA3-3.0HBV vector, which was unstable in the infected cells.So we will select pcDNA3-l.lHBV transfected cell lines as cell models in the next research work.2. HBV transfection promote TRAIL induced apoptosis and its mechanism2.1 HBV transfection promote TRAIL induced apoptosisBEL-7402/l.lHBV, HepG2/l.lHBV cells showed higher sensitivity to TRAIL induced apoptosis than their corresponding controls ( P<0.01). Moreover, apoptosis change is more obvious in BEL-7402 series than in HepG2 series. 2.2 Study on the molecular mechanism of the increased apoptosis induced by TRAIL after HBV transfectionThe molecular mechanism of the increased apoptosis induced by TRAIL in BEL-7402/1.1 HBV, BEL-7402/pcDNA3, BEL-7402 was investigated.a Extracellular level There was no significant difference in the expression of TRAIL receptors among BEL-7402/1.1 HBV, BEL-7402/pcDNA3 and BEL-7402 (p > 0.05).b Intracellular level The activation of Caspase 8 and Caspase 9 is more thoroughly in BEL-7402/l.lHBV than its controls(p<0.05). Compared with its control, the expression of Bax was upregulated in BEL-7402/1.1HBV(/K0.05).There was no significant difference in the expression of FLIP among BEL-7402/l.lHBV and its controls (/J>0.05). The activity of NF-kB, an important nuclear transcription factor, inhibiting apoptosis by initiating apoptosis inhibiting gene expression, was drastically lower on BEL-7402/l.lHBV cells(/?<0. 05).3. IFN-y promote TRAIL induced apoptosis in HepG2/l.lHBV and its mechanismIFN-y could make HepG2/l.lHBV more sensitive to TRAIL-induced apoptosis (p<0.05). This might be realized by the downregulated expression of DcRl and FLIP.4. The expression of TRAIL receptors on the PBMC in chronic hepatitic B patients and its relativity with the clinical incidenceAmong the four receptors, the expression level of DcRl in the PBMC of chronic B hepatitis patients was much lower than that of control group (p<0.05). There was high relativity among the expression of DcRl-. the clinical stage and the degree ofliver injury (including ALT and albumin).5. HBV transfection promote TWEAK induced apoptosisBEL-7402/1.1 HBV, HepG2/l .1HBV showed higher sensitivity to TWEAK induced apoptosis than their corresponding controls (p<0.05). Moreover, apoptosischange is more obvious in BEL-7402 series than in HepG2 series.6. IFN^y promote TWEAK induced apoptosis in BEL-7402/1.1HBV .HepG2/l.lHBVIFN-y could make BEL-7402/1.1HBV, HepG2/l.lHBV more sensitive to TWEAK-induced apoptosis (p<0.05).CONCLUSIONS1. HBV gene eukaryotic expression vectors pcDNA3-l.lHBV> pcDNA3-3.0HBVwere constructed successfully. Human hepatoma cell lines transfected with HBV(adr) were established successfully.2. HBV transfection could make cells sensitive to TRAIL induced apoptosis, this may be realized the following steps: 1) Up-regulated the expression of Bax; 2) Downregulated the activity of NF-kB.3. IFN-y could sensitize HepG2/l.lHBV cell to TRAIL-induced apoptosis to great extent.lt may be realized by downregulating the expression of DcR2 and FLIP.4. The expression level of DcRl on PBMC in chronic hepatitis B patients was down-regulated, which might contribute to the increased apoptosis of PBMC in chronic B hepatitis patients. The expression of DcRl can reflect the clinical stage and the degree of liver injury in chronic hepatitis B patients to some degree.5. HBV transfection could make cells sensitive to TWEAK induced apoptosis. IFN-y could sensitize HBV transfected cell to TWEAK-induced apoptosis to great extent.