基于核酶自剪切机制稳定分泌丙型肝炎病毒的细胞模型

Objective To construct a stable HCV-producing cell model for anti-HCV drug research. Methods The HCV-ribozyme recombinant plasmid pJFHl-Rbz was constructed to generate the exact 5' and 3' ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy. Results HCV core protein was detected in die screened cell clone, and the level of HCV RNA was up to 1 ×107 in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN α. Conclusions We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.     

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

AIM: To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. METHODS: HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and El antigens in cells were examined by RT-PCR and immunological techniques (flow cytom-etry and Western blot) respectively. RESULTS: The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-El). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION: HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.     

No requirement of HCV 5‘NCR for HCV-like particles assembly in insect cells

AIM: To express all three HCV structural proteins in the presence or absence of HCV 5‘‘NCR to investigate the requirement of 5‘NCR for the assembly of HCV-like particles in insect cells. METHODS: HCV structural protein encoding sequences CEIE2 and 5‘‘NCR-CEIE2 were amplified with PCR. Recombinant baculovirus were constructed with recombinant DNA techniques. HCV structural proteins expressed in insect cells were analyzed by immunofluorescence and SDS-PAGE. Immunoprecipitation experiment of insect cell lysates with anti-E2 monoclonal antibody (Mab) was carried out and the immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotting with anti-C, anti-E2 Mabs and HCV positive serum. The virus-like particles in insect cells were visualized by electron microscopy (EM). The HCV-like particles were purified by sucrose gradient centrifugation and identified by EM and immune aggregation EM. RESULTS: The recombinant baculovirus reBV/CEIE2 containing HCV C, E1, E2 genes and reBV/CS containing the same structural protein genes plus 5‘‘NCR were constructed. The insect cells infected with either reBV/CE1E2 or reBV/CS expressed HCV C, E1 and E2 proteins with amolecular weight of 20 kD, 35 kD and 66 kD respectively. The results of immunoprecipitation and the immunoblotting revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating the interaction of HCV structural proteins expressed in insect cells. Electron microscopy of insect cells infected with reBV/CE1E2 or reBV/CS demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from sera or hepatic tissues of HCV infected humans. The HCV-like particles were partially purified by sucrose gradient centrifugation, and the purified VLPs showed immuno-reactivity with anti-HCV antibodies.CONCLUSION: HCV 5‘‘NCR is not required for the assemblyof HCV-like particles in insect cells, HCV core and envelope proteins are sufficient for viral particle formation.     

Transactivating effect of hepatitis C virus core protein： A suppression subtractive hybridization study

AIM: To investigate the transactivating effect of hepatitis C virus (HCV) core protein and to screen genes transactivated by HCV core protein. METHODS: pcDNA3.1(-)-core containing full-length HCV core gene was constructed by insertion of HCV core gene into EcoRI/BarnHI site. HepG2 cells were cotransfected with pcDNA3.1(-)-core and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-gal by an enzyme-linked immunosorbent assay (ELISA) kit. HepG2 cell swere transiently transfected with pcDNA3.1(-)-core using Upofectamine reagent. Cells were collected and total mRNA was isolated. A subtracted cDNA library was generated and constructed into a pGEM-Teasy vector. The library was amplified with E. coil strain JM109. The cDNAs were sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR). RESULTS: The core mRNA and protein could be detected in HepG2 cell lysate which was transfected by the pcDNA3.1(-)-core. The activity of β-galactosidase in HepG2 cells transfected by the pcDNA3.1(-)-core was 5.4 times higher than that of HepG2 cells transfected by control plasmid. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR showed that 213 clones contained 100-1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes. The full length sequences were obtained with bioinformatics method, accepted by Genl3ank. It was suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein. CONCLUSION: The core protein of HCV has transactivating effects on SV40 early promoter/enhancer. A total of 63 clones from cDNA library were randomly chosen and sequenced. Using the BLAST program at the National Center for Biotechnology Information, six of the sequences were unknown genes. The other 57 sequences were highly similar to known genes.     

Topological and evolutional relationships between HCV core protein and hepatic lipid vesicles： Studies in vitro and in conditionally transgenic mice

AIM： To investigate a specific association between hepatic steatosis and hepatitis C virus （HCV） core.
METHODS： HeLa cells and primary mouse hepatocytes were transfected with HCV core plasmid, and conditional transgenics in which hepatic over-expression of HCV core is regulated by the tetracycline-off system, were developed. The expression of the HCV core was assessed over one to six months after withdrawal of doxycycline （dox） by immunohistochemistry （IHC） and Western blotting and by sequential liver biopsy. Hepatic steatosis was evaluated using oil red stain. 8-hydroxydeoxyguanosine （8-OHdG） stains and caspase levels were conducted to clarify hepatic oxidative stress and apoptosis rate. Serum aminotransferase was checked.
RESULTS： The transfected hepatocytes had globular cores under the lipid vesicles. In transgenic mice on control diet, core expression was robust, localized to the cytoplasmic vesicle membrane and strongly associated with microvesicular steatosis, which was gradually replaced by macrovesicular steatosis. However, both steatosis and core positive hepatocytes diminished with time. Increases in aminotransferase, caspase and 8-OHdG were associated with peak core expression.
CONCLUSION： The core protein was readily detected and morphologically associated with steatosis in individual hepatocytes both in vitro and in vivo. In vivo, oxidative stress caused by the core potentially reduced the number of core positive hepatocytes and in parallel the level of steatosis. To our knowledge, this is the first animal model that directly shows topological relationship between HCV core and hepatic lipid vesicles.     

Transactivating effect of hepatitis C virus core protein: A suppression subtractive hybridization study

AIM:To investigate the transactivating effect of hepatitis Cvirus(HCV)core protein and to screen genes transactivatedby HCV core protein.METHODS:pcDNA3.1(-)-core containing full-length HCVcore gene was constructed by insertion of HCV core geneinto EcoRI/BamHI site.HepG2 cells were cotransfected withpcDNA3.1(-)-core and pSV-lacZ.After 48 h,cells werecollected and detected for the expression of β-gal by anenzyme-linked immunosorbent assay(ELISA)kit.HepG2 cellswere transiently transfected with pcDNA3.1(-)-core usingLipofectamine reagent.Cells were collected and total mRNAwas isolated.A subtracted cDNA library was generated andconstructed into a pGEM-Teasy vector.The library wasamplified with E.coli strain JM109.The cDNAs weresequenced and analyzed in GenBank with BLAST search afterpolymerase chain reaction(PCR).RESULTS:The core mRNA and protein could be detected inHepG2 cell lysate which was transfected by the pcDNA3.1(-)-core.The activity of β-galactosidase in HepG2 cells transfectedby the pcDNA3.1(-)-core was 5.4 times higher than that ofHepG2 cells transfected by control plasmid.The subtractivelibrary of genes transactivated by HCV core protein wasconstructed successfully.The amplified library contained 233positive clones.Colony PCR showed that 213 clonescontained 100-1000 bp inserts.Sequence analysis wasperformed in 63 clones.Six of the sequences were unknowngenes.The full length sequences were obtained withbioinformatics method,accepted by GenBank.It wassuggested that six novel cDNA sequences might be targetgenes transactivated by HCV core protein.CONCLUSION:The core protein of HCV has transactivatingeffects on SV40 early promoter/enhancer.A total of 63 clonesfrom cDNA library were randomly chosen and sequenced.Using the BLAST program at the National Center forBiotechnology Information,six of the sequences wereunknown genes.The other 57 sequences were highly similarto known genes.     

Down-regulation of survivin expression by small interfering RNA induces pancreatic cancer cell apoptosis and enhances its radiosensitivity

AIM: To investigate the inhibitory effect of small interfering RNA (siRNA) on the expression of survivin in pancreatic cancer cell line PC-2 and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity. METHODS: A siRNA plasmid expression vector against survivin was constructed and transfected into PC-2 cells with LipofectamineTM 2000. The down regulation of survivin expression was detected by semi-quantitive RT-PCR and immunohistochemical SP method and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity was detected by flow cytometry. RESULTS: The sequence-specific siRNA efficiently and specifically down-regulated the expression of survivin at both mRNA and protein levels. The expression inhibition ratio was 81.25% at mRNA level detected by semi-quantitive RT-PCR and 74.24% at protein level detected by immunohistochemical method. Forty-eight hours after transfection,apoptosis was induced in 7.03% cells by siRNA and in 14.58% cells by siRNA combined with radiation. CONCLUSION: The siRNA plasmid expression vector against survivin can inhibit the expression of survivin in PC-2 cells efficiently and specifically. Inhibiting the expression of survivin can induce ipoptosis of PC-2 cells and enhance its radiosensitivity significantly. RNAi against survivin is of potential value in gene t(?)erapy of pancreatic cancer.     

Prospective study in 142 cases of hepatitis C virus infection

AIM:There is limited information on the natural history ofHCV infection in China.We investigated the outcome ofHCV infection after nine-year follow-up and the risk factorsin blood donors in China in order to provide the foundationfor prevention and therapy.METHODS:A total of 172 cases of HCV infection with anti-HCV positive and ALT abnormality were enrolled in thearchives when was screened blood in Hebei Province in1993.In them 142 blood donors were followed up till July2002.No antiviral treatment was applied to them duringthe period of infection.In the present study,anti-HCV,HCV-RNA and aminotransferase were detected and genotypingwas conducted by the method of restriction fragment lengthpolymorphism(RFLP).B-type ultrasound detection wasperformed in all the patients.Age,sex,alcohol consumptionand clinical symptoms were questioned.RESULTS:After nine years'follow-up,10.56%(15/142)of the cases were negative for anti-HCV and 16.42%(12/134)of them were negative for HCV-RNA.The genotypes 1b,2a and 1b/2a were 91.07%,6.25% and 2.68% respectively.Twelve cases(8.45%)were negative for both HCV RNAand anti-HCV.The rate of chronicity in this group was83.58%(112/134),and the rate of viral spontaneousresolution was 16.42%(22/134).The mean level of ALT,AST,γ-GT in HCV RNA positive cases was significantlyhigher than that in HCV RNA negative cases(P<0.001).The abnormal rate of ALT and/or AST in male donors wassignificantly higher than that in female donors(P=0.005).The rate of progression to liver cirrhosis from chronic hepatitisC was significantly higher in the cases of super-infectionwith HBV than that in the cases of single HCV infection.Overdose alcohol consumption promoted the progressionto chronicity.CONCLUSION:This area(Hebei Province)has a higherrate of chronicity in HCV infection,and measures shouldbe taken to prevent its progression to serious liver diseases,especially for patients super-infected with HCV and HBV.     

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.