RESUMEN
BACKGROUND/AIMS: The recognition of a correlation between patatin-like phospholipase domain containing-protein 3 (PNPLA3) rs738409 (C>G) and the severity of liver steatosis or fibrosis in chronic hepatitis C (CHC) has not reached a consensus. This meta-analysis sought to investigate with accuracy the association between the PNPLA3 rs738409 (C>G) polymorphism and liver steatosis and advanced fibrosis in CHC patients. METHODS: We performed a comprehensive literature search from the PubMed, Embase, Web of Science, and Google Scholar databases up to December 31, 2014. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Statistical analyses were performed using Stata 12.0 software. RESULTS: The meta-analysis revealed the severity of liver fibrosis was significantly higher in CHC patients with PNPLA3 rs738409 GG in Caucasians (versus CC+CG OR, 2.29; 95% CI, 1.57 to 3.35; p<0.05) but not Asian populations. In Caucasians, liver steatosis was also more severe in CHC patients with rs738409 GG (versus CC+CG; OR, 4.33; 95% CI, 2.59 to 7.22; p<0.05). The sensitivity analysis indicated the results of this meta-analysis were stable and no publication bias was detected. CONCLUSIONS: PNPLA3 rs738409 (C>G) was associated with the risk of both advanced liver fibrosis and steatosis in patients with CHC, especially among Caucasian populations.
Asunto(s)
Hígado Graso/genética , Hepatitis C Crónica/genética , Lipasa/genética , Cirrosis Hepática/genética , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , Pueblo Asiatico/genética , Humanos , Población Blanca/genéticaRESUMEN
OBJECTIVE: To establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate. METHODS: Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction (FSR), followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis. RESULTS: Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11, leading to the plasmid p11-neo, and p11-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained. CONCLUSIONS: A stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.
Asunto(s)
Línea Celular , Replicación del ADN , ADN Viral/biosíntesis , Virus de la Hepatitis B/genética , Hepatocitos/citología , Clonación Molecular , Vectores Genéticos , Células Hep G2 , Hepatocitos/virología , Humanos , Plásmidos , ADN Polimerasa Dirigida por ARN/genética , Replicación Viral/genéticaAsunto(s)
Hígado Graso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Adulto , Anciano , Hígado Graso/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptor Tipo I de Factor de Crecimiento Transformador beta , Adulto JovenRESUMEN
OBJECTIVE: To investigate the expression of recombinant IkappaBalphaM (AdIkappaBalphaM) in human hepatocarcinoma cell line HepG(2) and the inhibiting effect to NF-kappaB. METHODS: To test the virus titer in 293 cells and the infective efficiency virus titer in HepG(2) cells with GFP and limited dilution method, then to assay the IkappaBalphaM expression of recombinant adenovirus and the alteration after induction of TNF-alpha in 293 cells and HepG(2) by Western blotting, furthermore, to observe NF-kappaB activity in HepG(2) before and after treatment of TNF-alpha by EMSA. RESULTS: The titer of AdIkappaBalphaM is 2 x 10(8) pfu/L, MOI equals to 20. AdIkappaBalphaM could be expressed stably and efficiently in HepG(2) and will not degrade by induction of TNF-alpha; but IkappaBalpha in the uninfection cell as well as AdIkappaBalpha control was increased at first and then decreased. EMSA demonstrated that the infected cells showed no activation of NF-kappaB before and after the treatment of TNF-alpha, but the cells of uninfected and infected with AdIkappaBalpha appeared excessive activation of NF-kappaB. CONCLUSION: AdIkappaBalphaM could be amplified in 293 cell and effectively infect to target cells HepG(2), and could be expressed stably in cells, and wouldn't be degrade with treatment of TNF-alpha, also it can effectually inhibit the excessive activation of NF-kappaB in HepG(2). The result of our research indicates the theoretical value of IkappaBalphaM as a super inhibitor, inhibition activity of NF-kappaB with IkappaBalphaM super-suppressor aided with routine anti-tumor therapy would become an effective method.
Asunto(s)
Proteínas I-kappa B/metabolismo , FN-kappa B/metabolismo , Adenoviridae/genética , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas I-kappa B/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVES: To transfer 1.3-fold-overlength genome of HBV expression plasmid into HepG2 cells, and observe the dynamic changes of viral replication as well as expression in the transfected cells. METHODS: 4.1 kb fragment of HBV genome, derived from pGEM-HBV, was cloned into Hind III site of the eukaryotic expression vector pCDNA3.1 to construct the recombinant plasmid pHBV. Then HepG2 hepatoma, cells were transfected with pHBV, using Lipofectamine2000 transfection reagent. After 24, 48, 72 hours, the levels of HBsAg and HBeAg in the supernatant of HepG2 cells were determined with Abbott MEIA Kit. Intracellular viral DNA and RNA were analyzed by Southern and northern blot hybridization. In addition, viral-specific proteins (HBsAg and HBcAg) were assayed by immunofluorescence staining. RESULTS: The expression vector pCDNA3.1 was constructed successfully. The levels of HBsAg were 5.36+-0.25, 13.42+-1.24, 7.52+-0.43, and the values of HBeAg were 9.16+-0.32, 22.75+-1.49, 15.96+-1.03 after 24, 48, 72 hours, respectively. All expected HBV replicative intermediates and specific transcripts were verified by Southern and northern blot analysis. The HBsAg-positive cells peaked after 24 hours, and then dropped slowly. HBsAg positive staining scattered in the cytoplasm, whereas HBcAg lied maily in the cytoplasm apart from nuclears. CONCLUSIONS: This recombinant plasmid, which initiates viral replication efficiently in infected cells, is expected as a novel tool for investigating HBV replication in vitro.
