Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/análise , Betacoronavirus/isolamento & purificação , COVID-19 , Proteínas do Envelope de Coronavírus , Infecções por Coronavirus/virologia , Proteínas do Nucleocapsídeo de Coronavírus , Humanos , Limite de Detecção , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Pneumonia Viral/virologia , Poliproteínas , RNA Viral/metabolismo , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Proteínas do Envelope Viral/genética , Proteínas Virais/genéticaRESUMO
AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA) expression vector. METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method. RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control. Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively. CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).
Assuntos
Regulação Viral da Expressão Gênica , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , RNA Interferente Pequeno/genética , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/genética , Antígenos E da Hepatite B/genética , Humanos , Técnicas In Vitro , Neoplasias Hepáticas , Plasmídeos , RNA Mensageiro/genética , TransfecçãoRESUMO
AIM: To prepare the liposomes which protect antisense oligodeoxynucleotides (ASON) against nuclease degradation and delivery ASON into cytoplasmic efficiently. METHODS: A cationic derivative of cholesterol, 3 beta-[N-(N',N'-dimethylaminoethan)-carbamoyl] cholesterol (DC-Chol) was synthesized and used to prepare cationic liposome. The characteristics of liposomes/ASON complexes including size, drug loaded efficiency and structure were investigated. Cellular uptake of fluorescence labled ASON (FAM-ASON) under different condition was determined by flow cytometric analysis. Denatured polyacryamide gel electrophoresis (DPGE) was used to analyze the role of liposomes in protecting ASON. RESULTS: The mean values of preliposomes and liposomes/ASON complexes size were 185.7 and 228.2 nm, respectively. Cationic liposomes showed a high adsorption capacity for ASON. When the +/- charge ratio exceeded 2:1, more than 90% of the ASON was loaded into liposomes. Agarose gel electrophoresis showed three different existence of ASON in liposomes formulation: free, absorbed and encapsulated types. Concerning cellular uptake, DC-Chol liposomes indicated high efficient effect of increasing cellular uptake of ASON. Compared with free ASON, the total fluorescence intensity in cytoplasma was significantly enhanced. The level of increasing was largely depended on +/- charge ratio. The cellular uptake of FAM-ASON decreased in the presence of serum. The cellular total fluorescence intensity in 10% and 30% fetal bovine serum of cultured medium were only 22.3% and 15.5% as that of serum-free media, respectively. DPGE confirmed that free ASON was rapidly degraded by DNase I while ASON encapsulated into liposomes was efficiently protected. CONCLUSION: The cationic DC-Chol liposomes are shown to be promising carriers to deliver ASON into cytoplasma.
Assuntos
Colesterol/análogos & derivados , Colesterol/farmacologia , Lipossomos/farmacologia , Oligonucleotídeos Antissenso/metabolismo , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Células HeLa , Humanos , Mieloma Múltiplo/patologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/sangue , Células Tumorais CultivadasRESUMO
OBJECTIVE: To observe the expression of MAGE, GAGE and BAGE genes in human hepatocellular carcinoma (HCC) cell lines. METHODS: The expression levels of MAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE mRNAs in HCC cell lines SMMC-7721, QQY-7701, BEL-7402 were studied by reverse transcription polymerase chain reaction and were compared with those in biopsied liver tissues. RESULTS: MAGE-1 and BAGE mRNAs were expressed in SMMC-7721 cells. MAGE-3 and BAGE mRNAs were expressed in QQY-7701 cells. MAGE-1 and GAGE1-2 mRNAs were expressed in BEL-7402 cells. None of these genes was expressed in biopsied liver tissues. CONCLUSIONS: MAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE are expressed in hepatocellular carcinoma cell lines. These tumor-specific antigens can be used as molecular markers for early diagnosis and possible targets for immunotherapy of human HCC.