BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion sit...BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence.Consequently,the replication-competent HBV vectors carrying foreign genes,including pCH-BsdR,carrying blasticidin resistance gene(399 bp),and pCH-hrGFP,carrying humanized renilla green fluorescent protein gene(720 bp),were successfully obtained.However,the replication efficiency of the former is higher but it is tedious to use,while that of the latter is poor and cannot be quantified.Hence,we need to search for a new reporter gene that is convenient and quantifiable for further research.AIM To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.METHODS We utilized the replication-competent HBV viral vectors constructed by our laboratory,combined with the secreted luciferase reporter gene,to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase(SecNluc).HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene pCHsNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression.HBV replication intermediates could be produced from this vector.Via transfection with pTRE-sNLuc and selection by hygromycin,we obtained isolated cell clones,named HBV-NLuc-35 cells,which could secrete secNLuc recombinant viruses,and were sensitive to existing anti-HBV drugs.Using differentiated HepaRG cells,it was verified that recombinant HBV possessed infectivity.CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability,and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene.More importantly,the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.展开更多
Hepatitis C virus (HCV) infection and associated liver diseases are still challenging and represent a significant health care burden around the world. Although, the treatment strategies have been improved by the devel...Hepatitis C virus (HCV) infection and associated liver diseases are still challenging and represent a significant health care burden around the world. Although, the treatment strategies have been improved by the development of novel direct-acting antivirals, but such therapeutic options are still expensive and beyond the financial range of the most infected individuals in developing or even in resource replete countries. It demands an urgent need to search novel and improved alternate treatment strategies to treat the infection. The present study was aimed to develop an in vitro stable cell culture system, persistently expressing HCV genotype 1a non-structural genes and to characterize the inhibitory effects of synthetic siRNAs (short interference RNA) directed against the most conserved regions of nonstructural genes in an in vitro cell culture model. The continuous expression of nonstructural genes for more than 30 days post transfection was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis in stable human hepatoma cell line (Huh-7). The gene expression studies revealed significantly reduced gene expression of HCV nonstructural genes (i.e., NS2, NS4A and NS5A) both at mRNA and protein levels when treated against genome specific synthetic siRNAs in stable cell lines (51%, 47% and 54% respectively, p < 0.05). Similarly, a vivid decrease in HCV viral titer was exhibited by synthetic siRNAs in an in vitro viral replicate cell culture model (58%, 48% and 50%, respectively, p < 0.05) determined by quantitative Real-Time PCR (qPCR). Our data indicate that siRNA mediated gene silencing may be considered a promising alternate treatment strategy against HCV in combination with other effective therapeutic regimens in future.展开更多
Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for d...Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embrvonic stem cell lines with clarified directly differentiation ability展开更多
Chronic HBV infection is associated with a 100-fold high risk of developing hepatocellular carcinoma. Tumor recognition is of the most importance during the immune surveillance process that prevents cancer development...Chronic HBV infection is associated with a 100-fold high risk of developing hepatocellular carcinoma. Tumor recognition is of the most importance during the immune surveillance process that prevents cancer development in humans. In the present study, the expressions of MHC class Ⅰ molecules on hepatoplastoma cell line HepG2.2.15 were investigated to indicate the possible effects of HBV on the immune recognition during HBV-associated hepatocellular carcinoma. It was found that the expressions of MHC class Ⅰ molecules HLA-ABC, HLA-E and MICA were much lower in HepG2.2.15 cells compared with HepG2 cells. The expressing HBV in human hepatoplastoma cell line significantly down-regulated the expressions of MHC class Ⅰ molecules. Additionally, it was observed that in murine chronic HBsAg carriers the expression of classical MHC-I molecule on hepatocytes was down-regulated. These results demonstrated that HBV might affect the immune recognition during HBV- associated hepatocellular carcinoma such as the recognition of CD8^＋ T, NK-CTL and NK cells and prevent the immune surveillance against tumors. However, the effects of HBV down-regulation of MHC class I molecules on the target cells in vivo should be further studied. Cellular ＆ Molecular Immunology.展开更多
Hepatitis B virus X protein(HBx),a 17-kd protein encoded by X gene of hepatitis B virus(HBV),has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer element...Hepatitis B virus X protein(HBx),a 17-kd protein encoded by X gene of hepatitis B virus(HBV),has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements.The aim of the study is to investigate the extracellular regulated protein kinases(ERKs)pathway of HBx on glomerular mesangial cell(GMC)proliferation and tumor necrosis factor-α(TNF-α)expression.The HBV X gene was amplified by polymerase chain reaction(PCR),inserted into the eukaryotic expression vector pCI-neo and confirmed by restriction endonuclease digestion and sequence analysis.PCI-neo containing HBV X gene(pCI-neo-X)was then transfected into cultured GMC line via liposome.GMC proliferation,TNF-αand its mRNA expression were compared in the condition of with or without U0126 in culture media.HBx,ERK1/2 and p-ERK1/2 expression in GMCs was assessed by Western blotting.TNF-αmRNA expression was assessed by semi-quantitative reverse transcription-PCR(RT-PCR).TNF-αlevel in supernatants was measured by ELISA.GMC proliferation was detected by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide(MTT)kit.The results showed that HBx expression was found in transfected GMCs and became prominent at 36th and 48th h after transfection whether with or without U0126 in culture media.TNF-αmRNA expression was significantly decreased in U0126 group compared with U0126-free group.TNF-αlevels in supernatants in PCI-neo-X transfection without U0126 group were(189.0�18.1)and(172.3�24.3)pg/mL at 36th and 48th h after transfec-tion,respectively.In contrast,TNF-αlevels in supernatants with U0126 were(65.6�11.6)and(84.0�24.6)pg/mL at 36th and 48th h,respectively.The TNF-αlevels in the latter groups were significantly lower than those in the former groups(P<0.05).GMCs proliferation was also lower in added U0126 group at 36th and 48th h after transfection.From above,we can conclude that HBx could induce GMC proliferation and increase TNF-αmRNA expression and its protein production.HBx upregulates TNF-αexpression and induces cell proliferation of GMC line partly through ERK1/2 signal transduction pathway.展开更多
基金Supported by the National Natural Science Foundation of China,No.81672041the National Major Science and Technology Special Project for Infectious Diseases of China,No.2012ZX10004503-012
文摘BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence.Consequently,the replication-competent HBV vectors carrying foreign genes,including pCH-BsdR,carrying blasticidin resistance gene(399 bp),and pCH-hrGFP,carrying humanized renilla green fluorescent protein gene(720 bp),were successfully obtained.However,the replication efficiency of the former is higher but it is tedious to use,while that of the latter is poor and cannot be quantified.Hence,we need to search for a new reporter gene that is convenient and quantifiable for further research.AIM To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.METHODS We utilized the replication-competent HBV viral vectors constructed by our laboratory,combined with the secreted luciferase reporter gene,to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase(SecNluc).HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene pCHsNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression.HBV replication intermediates could be produced from this vector.Via transfection with pTRE-sNLuc and selection by hygromycin,we obtained isolated cell clones,named HBV-NLuc-35 cells,which could secrete secNLuc recombinant viruses,and were sensitive to existing anti-HBV drugs.Using differentiated HepaRG cells,it was verified that recombinant HBV possessed infectivity.CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability,and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene.More importantly,the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.
文摘Hepatitis C virus (HCV) infection and associated liver diseases are still challenging and represent a significant health care burden around the world. Although, the treatment strategies have been improved by the development of novel direct-acting antivirals, but such therapeutic options are still expensive and beyond the financial range of the most infected individuals in developing or even in resource replete countries. It demands an urgent need to search novel and improved alternate treatment strategies to treat the infection. The present study was aimed to develop an in vitro stable cell culture system, persistently expressing HCV genotype 1a non-structural genes and to characterize the inhibitory effects of synthetic siRNAs (short interference RNA) directed against the most conserved regions of nonstructural genes in an in vitro cell culture model. The continuous expression of nonstructural genes for more than 30 days post transfection was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis in stable human hepatoma cell line (Huh-7). The gene expression studies revealed significantly reduced gene expression of HCV nonstructural genes (i.e., NS2, NS4A and NS5A) both at mRNA and protein levels when treated against genome specific synthetic siRNAs in stable cell lines (51%, 47% and 54% respectively, p < 0.05). Similarly, a vivid decrease in HCV viral titer was exhibited by synthetic siRNAs in an in vitro viral replicate cell culture model (58%, 48% and 50%, respectively, p < 0.05) determined by quantitative Real-Time PCR (qPCR). Our data indicate that siRNA mediated gene silencing may be considered a promising alternate treatment strategy against HCV in combination with other effective therapeutic regimens in future.
文摘Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embrvonic stem cell lines with clarified directly differentiation ability
文摘Chronic HBV infection is associated with a 100-fold high risk of developing hepatocellular carcinoma. Tumor recognition is of the most importance during the immune surveillance process that prevents cancer development in humans. In the present study, the expressions of MHC class Ⅰ molecules on hepatoplastoma cell line HepG2.2.15 were investigated to indicate the possible effects of HBV on the immune recognition during HBV-associated hepatocellular carcinoma. It was found that the expressions of MHC class Ⅰ molecules HLA-ABC, HLA-E and MICA were much lower in HepG2.2.15 cells compared with HepG2 cells. The expressing HBV in human hepatoplastoma cell line significantly down-regulated the expressions of MHC class Ⅰ molecules. Additionally, it was observed that in murine chronic HBsAg carriers the expression of classical MHC-I molecule on hepatocytes was down-regulated. These results demonstrated that HBV might affect the immune recognition during HBV- associated hepatocellular carcinoma such as the recognition of CD8^＋ T, NK-CTL and NK cells and prevent the immune surveillance against tumors. However, the effects of HBV down-regulation of MHC class I molecules on the target cells in vivo should be further studied. Cellular ＆ Molecular Immunology.
基金supported by the National Natural Science Foundation of China(Grant No.30772360)the Natural Science Foundation of Health Department of Hubei Province,China(No.JX4B48).
文摘Hepatitis B virus X protein(HBx),a 17-kd protein encoded by X gene of hepatitis B virus(HBV),has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements.The aim of the study is to investigate the extracellular regulated protein kinases(ERKs)pathway of HBx on glomerular mesangial cell(GMC)proliferation and tumor necrosis factor-α(TNF-α)expression.The HBV X gene was amplified by polymerase chain reaction(PCR),inserted into the eukaryotic expression vector pCI-neo and confirmed by restriction endonuclease digestion and sequence analysis.PCI-neo containing HBV X gene(pCI-neo-X)was then transfected into cultured GMC line via liposome.GMC proliferation,TNF-αand its mRNA expression were compared in the condition of with or without U0126 in culture media.HBx,ERK1/2 and p-ERK1/2 expression in GMCs was assessed by Western blotting.TNF-αmRNA expression was assessed by semi-quantitative reverse transcription-PCR(RT-PCR).TNF-αlevel in supernatants was measured by ELISA.GMC proliferation was detected by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide(MTT)kit.The results showed that HBx expression was found in transfected GMCs and became prominent at 36th and 48th h after transfection whether with or without U0126 in culture media.TNF-αmRNA expression was significantly decreased in U0126 group compared with U0126-free group.TNF-αlevels in supernatants in PCI-neo-X transfection without U0126 group were(189.0�18.1)and(172.3�24.3)pg/mL at 36th and 48th h after transfec-tion,respectively.In contrast,TNF-αlevels in supernatants with U0126 were(65.6�11.6)and(84.0�24.6)pg/mL at 36th and 48th h,respectively.The TNF-αlevels in the latter groups were significantly lower than those in the former groups(P<0.05).GMCs proliferation was also lower in added U0126 group at 36th and 48th h after transfection.From above,we can conclude that HBx could induce GMC proliferation and increase TNF-αmRNA expression and its protein production.HBx upregulates TNF-αexpression and induces cell proliferation of GMC line partly through ERK1/2 signal transduction pathway.
