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Chinese Journal of Hepatology ; (12): 843-848, 2014.
Article in Chinese | WPRIM | ID: wpr-337094


<p><b>OBJECTIVE</b>To construct a single-chain human anti-EGFR antibody (scFv) and truncated protamine (tP) fusion protein, ScFv/tP, carrying small interfering (si)RNA directed against the heat shock protein Hsp47, a collagen-binding glycoprotein, in order to evaluate the role Hsp47 in apoptosis of hepatic stellate cells.</p><p><b>METHODS</b>A single chain of the human variable fragment was obtained by phage display and fused with the tP gene and with or without (negative control) the Hsp47 siRNA sequences. Following expression and purification of the scFv/tP fusion protein and the scFv/tPHsp47 siRNA fusion protein, internalization capabilities were tested in isolated human hepatic stellate cells and the QSG-7701 human hepatocyte cells with visualization by immunofluorescent staining. The DNA binding ability of the fusion proteins were verified by gel shift assay.Following ScFv/tP-Hsp47 siRNA fusion protein transfection into the human hepatic stellate cells, the levels of Hsp47 mRNA and protein expression were tested by RT-PCR and Western blotting; in addition, effects of siRNA-mediated silencing of Hsp47 on cell proliferation and apoptosis were analyzed by the cell counting kit (CCK)-8, flow cytometry and Western blot detection of the apoptosis marker poly (ADP-ribose) polymerase (PARP).</p><p><b>RESULTS</b>Indirect immunofluorescence revealed that the ScFv/tP fusion proteins were internalized into human hepatic stellate cells but not into the QSG-7701 cells.The ScFv/tP-Hsp47 siRNA fusion protein caused reduced expression of Hsp47 mRNA and protein expression in the human hepatic stellate cells, as well as increased the cells' apoptosis remarkably.</p><p><b>CONCLUSION</b>The ScFv/tP fusion protein can be used as a transfection reagent to deliver Hsp47 siRNA into hepatic stellate cells and to mediate apoptosis via blockade of Hsp47 expression.</p>

Apoptosis , Cell Proliferation , HSP47 Heat-Shock Proteins , Genetics , Hepatic Stellate Cells , Cell Biology , Humans , Protamines , Metabolism , RNA, Messenger , RNA, Small Interfering , ErbB Receptors , Allergy and Immunology , Single-Chain Antibodies , Transfection
Article in Chinese | WPRIM | ID: wpr-265729


<p><b>OBJECTIVE</b>To explore the relationship between the single nucleotide polymorphisms (SNPs) of rs12979860 and rs8099917 in IL28B gene and the response to interferon treatment in hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B patients.</p><p><b>METHODS</b>Peripheral blood samples were collected from 82 HBeAg-positive patients with chronic hepatitis B receiving interferon treatment, including 38 with favorable response to the treatment and 44 without response. IL28B gene was amplified from the chromosomal DNA, and rs8099917 SNP was genotyped based on PCR-RLFP and rs12979860 SNP by sequencing.</p><p><b>RESULTS</b>In the responsive patients, the distribution frequencies of TT and TG+GG genotypes and allele G in SNPrs8099917 were 81.6% (31/38), 18.4% (7/38), and 9.2% (7/76), as compared to the frequencies of 97.7% (43/44), 2.3% (1/44), and 1.1% (1/88) in nonresponsive patients, respectively. The frequencies showed significant differences between the responsive and nonresponsive patients (P=0.014 for genotypes and P=0.025 for allele G). The distribution frequencies of CT genotypes and allele T in SNPrs12979860 showed no differences between the responsive and nonresponsive patients (P<0.05).</p><p><b>CONCLUSION</b>rs8099917 SNP is probably associated with the response to interferon treatment in HBeAg-positive patients with chronic hepatitis B, and Allele G may be predictive of the treatment success.</p>

Adult , Alleles , Antiviral Agents , Therapeutic Uses , Female , Genotype , Hepatitis B e Antigens , Blood , Hepatitis B, Chronic , Genetics , Therapeutics , Humans , Interferons , Therapeutic Uses , Interleukins , Genetics , Male , Polymorphism, Single Nucleotide , Young Adult
Article in Chinese | WPRIM | ID: wpr-323685


<p><b>OBJECTIVE</b>To develop a rapid and specific method for hepatitis C virus ( HCV) genotyping using reverse dot blot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong.</p><p><b>METHODS</b>The primers and the probes targeting the 5'untranslated region (5'UTR) and core region of HCV genotypes 1b, 2a, 3a, 3b and 6a were designed, and the RT-PCR reverse dot blot hybridization (PCR-RDH) method for HCV genotyping was established. A total of 115 patients with hepatitis C were genotyped using this method, and 38 of them were also genotyped by sequencing and phylogenetic analysis to evaluate the accuracy and specificity of the method.</p><p><b>RESULTS</b>Of the 115 patients, 111 were successfully genotyped to be 1b, 2a, 3a, 3b, 6a and mix-infection of 1b/2a at frequencies of 56.8%, 8.1 %, 3.6%, 5.4%, 25.2% and 0.9% respectively, and all the 15 healthy control samples showed negative results. The accuracy and reliability of the genotyping method of PCR-RDH was confirmed in 38 cases by amplification of HCV core and NS5B regions followed by DNA sequencing and phylogenetic analysis.</p><p><b>CONCLUSION</b>This method for HCV genotyping, with high reliability and specificity, is suitable for clinical and epidemiological investigations. The prevalence of HCV genotypes 1b and 2a decreases while 1b remains the dominant genotype in Guangdong, where the prevalence of 6a significantly increases as compared with that 10 years ago.</p>

Genes, Viral , Genotype , Genotyping Techniques , Methods , Hepacivirus , Classification , Genetics , Hepatitis C , Virology , Humans , Immunoblotting , Nucleic Acid Hybridization , Reverse Transcriptase Polymerase Chain Reaction
Article in Chinese | WPRIM | ID: wpr-290023


<p><b>OBJECTIVE</b>To establish immortalized B lymphoblast cell lines (B-LCLs) from healthy anti-HBs antibody (anti-HBs)-positive volunteers and screen for human anti-HBs and the antibody-secreting cells.</p><p><b>METHODS</b>The peripheral blood mononuclear cells (PBMC) isolated from 3 healthy volunteers positive for anti-HBs with hepatitis B vaccine boost vaccination were infected with Epstein-Barr virus (EBV) and incubated in the presence of CpG DNA motifs and cyclosporin A (CyA). The anti-HBs in the culture supernatant of the immortalized B-cells was quantified by Architect anti-HBs assay with chemiluminescent microparticle technique. Immunocytochemistry was performed to identify the differentiation of the cell clones expressing anti-HBs.</p><p><b>RESULTS</b>Immortalized B-cell culture was successfully established from the cell clones secreting anti-HBs with EBV infection and CpG DNA stimulation. The titer of anti-HBs in the culture supernatant was at its peak at 3 weeks of cell culture and then decreased gradually. At 3 months of cell culture, the cells still retained the capacity of anti-HBs production as verified by the results of immunocytochemistry for CD20 and CD138.</p><p><b>CONCLUSION</b>Immortalized B-cell culture secreting anti-HBs from volunteers receiving boost hepatitis B vaccination has been successfully established by modified EBV immortalization technique.</p>

B-Lymphocytes , Allergy and Immunology , Cell Line , Cell Transformation, Viral , Hepatitis B , Hepatitis B Antibodies , Allergy and Immunology , Hepatitis B Surface Antigens , Allergy and Immunology , Hepatitis B Vaccines , Allergy and Immunology , Herpesvirus 4, Human , Allergy and Immunology , Humans , Immunization, Secondary , Vaccination