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1.
Chinese Journal of Hepatology ; (12): 576-580, 2012.
Article Dans Chinois | WPRIM | ID: wpr-296846

Résumé

To investigate the effect of short hairpin RNA (shRNA)-mediated silencing of CTGF and TIMP-1 in hepatic stellate cells (HSCs) on mRNA expression of TIMP-1, CTGF, and procollagen type-I (PC I), as well as secretion of extracellular matrix (ECM) proteins. Two recombinant expression plasmids harboring shRNAs against CTGF and TIMP-1 (psiRNA-GFP-CTGF and psiRNA-GFP-TIMP-1) were transfected alone or together into TGFb1-activated HSC-T6 cells. The mRNA expression levels of CTGF, TIMP-1, and PC I were detected by fluorescence quantitative PCR (FQ-PCR). The concentrations of secreted PC type-III, hyaluronate (HA), and laminin (LN) were measured by radioimmunoassay (RIA) of culture supernatants. FQ-PCR analysis showed that CTGFshRNA and TIMP-1shRNA specifically inhibited the expression of CTGF, TIMP-1, and PC I mRNA in activated HSC-T6 cells. The concentrations of secreted PC III, HA, and LN were decreased significantly in HSC-T6 cells with shRNA-silenced CTGF or TIMP-1 (P less than 0.01 or P less than 0.05). Moreover, HSC-T6 cells with shRNA-silenced CTGF and TIMP-1 showed a more robust decrease in synthesis of PC III, HA and LN (all, P less than 0.01), as well as in mRNA expression of PC I (P less than 0.05). CTGFshRNA and TIMP-1shRNA effectively inhibit expression of the respective target genes, as well as of PC I, and decrease secretion of ECM components from HSC-T6 cells. Silencing of both CTGF and TIMP-1 produces more robust effects than either in isolation. These data suggest that CTGF and TIMP-1 may be effective targets of shRNA-based gene therapy to treat liver fibrosis.


Sujets)
Animaux , Rats , Cellules cultivées , Collagène de type I , Génétique , Métabolisme , Facteur de croissance du tissu conjonctif , Génétique , Métabolisme , Régulation négative , Matrice extracellulaire , Métabolisme , Régulation de l'expression des gènes , Extinction de l'expression des gènes , Cellules étoilées du foie , Métabolisme , Acide hyaluronique , Métabolisme , Laminine , Métabolisme , Cirrhose du foie , Métabolisme , Anatomopathologie , Réaction de polymérisation en chaîne , ARN messager , Génétique , Métabolisme , Petit ARN interférent , Génétique , Inhibiteur tissulaire de métalloprotéinase-1 , Génétique , Métabolisme , Transfection , Facteur de croissance transformant bêta , Métabolisme
2.
Chinese Journal of Experimental and Clinical Virology ; (6): 453-455, 2012.
Article Dans Chinois | WPRIM | ID: wpr-305011

Résumé

<p><b>OBJECTIVE</b>To characterize genotypic resistance within HBV RT region in chronic hepatitis B (CHB) patients with nucleos(t)ide analogue (NA) treatment.</p><p><b>METHODS</b>Serum samples of 229 CHB patients with NA treatment were obtained. Full-length HBV RT sequences were amplified, sequenced and analyzed, on the following NA resistant (NAr) mutations belonging to different NAr pathways.</p><p><b>RESULTS</b>Among 229 HBV isolates, 14.41% (33/229) and 85.59% (196/229) were genotype B and C, respectively; and the patients with HBV genotype C may be more susceptible to develope resistant mutations than patients with HBV genotype B(chi2 = 2.95, P < 0.05). NAr mutations were detected in 63 CHB patients. Mutations were not found at rtI169, rtT184, rtA194 or rtS202. RtM204 mutations were detected at the highest frequency among 63 mutants (40/63, 63.49%) and found to display 11 combination mutation patterns, in which rtM204I were associated with rtL80I/V and rtL180M, and rtM204V were associated with rtL1l80M, respectively. Conclusions There are complicated mutation patterns in the HBV RT region for chronic hepatitis B (CHB) patients with nucleos(t)ide analogue (NA) treatment. RtM204V/I mutation was the highest.</p>


Sujets)
Adolescent , Adulte , Sujet âgé , Femelle , Humains , Mâle , Adulte d'âge moyen , Jeune adulte , Antiviraux , Utilisations thérapeutiques , Virus de l'hépatite B , Génétique , Hépatite B chronique , Traitement médicamenteux , Virologie , Mutation , Nucléosides , Utilisations thérapeutiques , Nucléotides , Utilisations thérapeutiques , RNA-directed DNA polymerase , Génétique , Métabolisme , Protéines virales , Génétique , Métabolisme
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