RESUMO
<p><b>OBJECTIVE</b>To screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs).</p><p><b>METHODS</b>Based on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR.</p><p><b>RESULT</b>Co-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration.</p><p><b>CONCLUSION</b>The expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.</p>
Assuntos
Humanos , Sequência de Bases , Carcinoma Hepatocelular , Patologia , Linhagem Celular Tumoral , Células Cultivadas , Embrião de Mamíferos , Proteínas de Fluorescência Verde , Genética , Antígenos do Núcleo do Vírus da Hepatite B , Genética , Antígenos E da Hepatite B , Genética , Vírus da Hepatite B , Genética , Rim , Biologia Celular , Neoplasias Hepáticas , Patologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro , Genética , RNA Interferente Pequeno , RNA de Transferência de Valina , Genética , RNA Viral , Genética , TransfecçãoRESUMO
<p><b>OBJECTIVE</b>To examine mitochondrial DNA mutations in mitochondrial myopathy.</p><p><b>METHODS</b>Three suspected cases of mitochondrial myopathy were examined by HE staining, histochemical staining methods and electron microscopy. The mutations in all 22 tRNA genes of mitochondrial genome were screened by polymerase chain reaction-single strand conformation polymorphism and DNA sequencing.</p><p><b>RESULTS</b>The three cases were diagnosed as mitochondrial myopathy. The examinations revealed that patient 1 had a homoplasmic A1627G mutation in tRNA-Val gene, and patient 2 had a heteroplasmic A1627G/A mutation in tRNA-Val gene, and patient 3 had two mutationsuone was homoplasmic T5554C mutation in tRNA-Trp gene, the other was heteroplasmic A10412C/A mutation in tRNA-Arg gene.</p><p><b>CONCLUSION</b>tRNA genes mutations of mtDNA might be one of the etiologies of mitochondrial myopathy.</p>