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With the economic growth and better standards of living,the prevalence of non-alcoholic fatty liver disease (NAFLD) is expected to increase dramatically worldwide.NAFLD is a common chronic inflammation disease.NF-κB is a transcription factor that plays crucial roles in inflammation,immunity,cell proliferation and apoptosis.It can facilitate the occurrence and development of NAFLD,and the underlying mechanisms are related to insulin resistance,oxidative stress,alteration of intestinal flora,activation ofrenin angiotensin system,etc.
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OBJECTIVE@#To screen differentially expressed genes and gene pathways in L02 cell line stably expressing hepatitis C virus (HCV) Ib type nonstructural protein 4B (NS4B) mediated by lentiviral system, and to provide a basis for further research of molecular biological mechanism of NS4B gene in chronic hepatitis C and hepatocarcinogenesis.@*METHODS@#NS4B stably overexpressed L02 cell line and negative control stable L02 cell line, designated as L02-NS4B and L02-mkate2 respectively, were resurrected and amplified in vitro. The differentially expressed genes between L02-NS4B and L02-mkate2 were determined by gene expression microarray from Human Gene 1.0ST. The significant pathways of the differential genes were selected by the Fisher's exact test and χ2 test according to kyoto encyclopedia of genes and genomes (KEGG) database. The differential expression levels of 5 selected genes including protein kinase C delta binding protein (PRKCDBP), tumor protein p53 (TP53), v-akt murine thymoma viral oncogene homolog 1 (AKT1), baculoviral IAP repeat containing 3 (BIRC3) and B-cell lymphoma 2-like1 (BCL2L1) from cDNA microarray data were further verified by real-time quantitative polymerase chain reaction (real-time QPCR).@*RESULTS@#Between L02-NS4B and L02-mkate2, the genes with flurescence intensity ratio >1.2 or <0.8 were considered as differentially expressed genes. A total of 2 682 differentially expressed genes in the known 28 869 human genes were detected in L02-NS4B, 1 446 genes were upregulated and 1 236 genes were downregulated. A total of 41 involved pathways of up-regulated differential genes were identified by KEGG database, mainly including apoptosis, extracellular matrix receptor interaction, cell cycle, pathways in cancer and Toll-like receptor signaling pathway; and 20 involved pathways of down-regulated differential genes were identified, mainly including pathways in cancer, Wnt signaling pathway and cell cycle pathway. Of the 5 upregulated genes selected from cDNA microarray data, 3 genes showed the same differential expression pattern by real-time QPCR as that shown in cDNA microarray data, namely AKT1, BIRC3 and BCL2L1. The confirmation rate of real-time QPCR was 60%.@*CONCLUSION@#HCVNS4B can up-regulate or down-regulate the expression of many genes in L02 cells, thus affecting multiple signaling pathways relevant to cell apoptosis, cell cycle and carcinogenesis.
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Humans , Cell Cycle , Cell Line , Gene Expression Profiling , Hepacivirus , Genetics , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Signal Transduction , Transcriptional Activation , Viral Nonstructural Proteins , GeneticsABSTRACT
Objective To investigate clinical features and antifungal therapeutic effect of chronic severe hepatitis (CSH) patients with invasive fungal infection (IFI), and to improve the diagnosis and treatment.Methods Clinical manifestation, blood routine, imageology and mycetology characteristic, antifungal treatment perscription and therapeutic effect of 79 CSH patients with IFI were retrospectively analyzed. Antifungal therapeutic effect was compared between fluconazole and voriconazole. Results Thirteen (16.5%) patients received glucocorticoid or other immunodepressants for a relatively long time, 40 (50.6%) patients had invasive operation, and 61 (77.2 %) patients were administered 1-6 kinds of broad-spectrum antibiotics. Seventy-three patients had fever. Leucocytes and neutrophilic granulocyte increased in 96.2% of the patients. Lung (31.6%), intestinal tract (26.2%) and oral cavity (14%) infections were common. Fungus was found in 70.9% of the patients. Candida albicans (40.9%) and aspergillus (21.1%) were often seen. Halo signs and crescent signs on lung CT were relatively specific in 40% of the patients with fungal pneumonia. Voriconazole was more effective than fluconazole(71.4% vs. 39.0%, P<0.05). Twelve patients with lung aspergillus infection were administered voriconazole, 8 (66.7%) patients of whom was effective, and the other 4 (33.3%) patients died. Conclusion There are high risk factors in major CSH patients with IFI. The most common clinical manifestations of CSH patients with IFI are fever, leukocytosis, lung and intestinal tract infection. Candida albicans and aspergillus infection are common. Voriconazole is more effective than fluconazole, and can increase the survival rate of CSH patients with IFI.
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Objective To determine the changes of serum glycosylphosphatidylinositol specific phospholipase D(GPI-PLD) activity in patients of several liver disease.Method Glycosylphosphatidylinositol (GPI) anchored placental alkaline phosphatase(PLAP) prepared by ourselves was used as a substrate.After partitioning by triton-X-114,the serum GPI-PLD activity was determined quantitatively and the data was treated by microware of SPSS 10.0.Results On the basis of the percentage of GPI-anchored PLAP conversion,the sera GPI-PLD activities of total 172 patients,included 26 severe acute viral hepatitis as group A,29 liver cirrhosis as group B,32 chronic viral hepatitis as group C,55 mild acute viral hepatitis as group D,30 primary hepatocellular carcinoma as group E,were measured.As compared with 182 healthy presons as control group,the sera GPI-PLD activities of group A and B were significantly reduced;By contraries,the activities of group D and E were significantly raised.The sera GPI-PLD activities of group C compared with healthy control group were not significantly altered.However,when paired Q-test,the changes of serum GPI-PLD activity between all paired groups among this five groups were remarkable.Conclusions The determination of sera GPI-PLD activities in patients can act as a biochemistry index for diagnosis of acute viral hepatitis,liver cirrhosis and primary hepatocellular carcinoma,as well as an auxiliary index for judgment of the curative effect and prognosis of liver diseases.
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Objective To investigate gene variation and the relationship between gene variation and pathogenicity of transfusion transmitted virus(TTV).Methods The TTV DNA in the serum sample from a blood donor(BD) and a chronic non-A-G severe hepatitis(CSH) patient with TTV infection was amplified by using PCR.The purified PCR product was cloned and 10 clones from each case were sequenced.The sequences were compared among different clones and analyzed by Phylogentic tree.Results There were two different TTV strains in the BD and seven different TTV strains in the chronic non-A-G severe hepatitis patient.The TTV clones in the BD were of G1a subtype and those of the CSH were of G1a and G1b subtype.Conclusion Gene variant of TTV was much more complicated in the CSH patients than that in the BD ones.
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Objective To evaluate the in vitro inhibiting activities of interferon-alpha (INF-?) against hepatitis B virus (HBV). Methods HepG2 2 2 15 cells were treated with INF-?. At 3rd, 6th and 9th days after treatment, the supernatant was collected for HBsAg quantitative assay, and total RNA of the cells was isolated for RT-PCR and realtime-PCR assay of HBV mRNA. Results There were no significant differences in HBsAg titer and virus mRNA level at 3rd and 6th days between the experimental and control groups. At 9th day, HBsAg titer and virus mRNA level in INF-? group were significantly lower than those in control group. And 500IU/ml INF-? could get maximal inhibiting effect against HBV. Conclusion INF-? can inhibit transcription and expression of HBV gene in HepG2 2 2 15 cells, which indicated that INF-? can inhibit HBV replication directly.
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Objective To evaluate the in vitro inhibitive activities of mycophenolic acid against hepatitis B virus in vitro. Methods The different concentrations of the test drugs in DMEM were added to the well with the monolayer growth of HepG2.2.15 cells. The culture medium was refreshed with DMEM containing the appropriate drugs every 3 days. At 3rd, 6th and 9th days, the supernatant was collected for HBsAg quantitative assay, total RNA was isolated from cells for RT-PCR and realtime-PCR assays. Results It was found no significant anti-HBV effect of mycophenolic acid on concentration of 2～250 ?g/ml, and mini inhibitory efficacy of HBV replication on concentration of 1250 ?g/ml. There was significant anti-HBV effect of lamivudine on concentration of 5 ?g/ml. When combined with lamivudine mycophenolic acid can significantly potentiate anti-HBV activities of lamivudine, and it can get maximal inhibition effect on concentration of 10 ?g/ml. Conclusions Mycophenolic acid could inhibit HBV replication and potentiate anti-HBV activities of lamivudine.