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1.
Chinese Journal of Hepatology ; (12): 44-47, 2011.
Artigo em Chinês | WPRIM | ID: wpr-290655

RESUMO

To evaluate the inhibitory effects of long antisense RNA on HBV replication in HepG2.2.15 cells. The coding region of HBV S gene was cloned into pTARGET vector in sense and antisense orientations and the recombinant plasmids were transfected into HepG2.2.15 cells which were divided into HBS2 (antisense RNA) group, HBS4 (sense RNA) group and control group. HBsAg and HBeAg in the culture supernate were detected by ELISA. The HBV DNA in the supernate was quantified by real-time PCR. After treatment, the levels of HBsAg in HepG2.2.15 cell supernatants of three groups were 0.621+/-0.027, 3.399+/-0.018 and 2.232+/-0.187 respectively; the levels of HBeAg were 0.749+/-0.019, 1.548+/-0.025 and 1.570+/-0.044 respectively and the levels of HBV DNA were 1.597+/-0.082, 3.381+/-0.297 and 3.610+/-0.063 respectively. The expressions of HBsAg and HBeAg and the HBV DNA level in HBS2 group were remarkably reduced as compared to the control (Z = -2.309, P value is less than 0.05); whereas the sense plasmid transfection (HBS4) did not affect HBeAg (Z = -0.866) and HBV DNA (Z = -1.155) levels in the culture supernate but slightly increased the HBsAg level (Z = -2.309). Antisense RNA might be a useful tool to repress HBV replication.


Assuntos
Humanos , DNA Viral , Genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Células Hep G2 , Antígenos de Superfície da Hepatite B , Antígenos E da Hepatite B , Vírus da Hepatite B , Genética , Fisiologia , Plasmídeos , Interferência de RNA , Transfecção , Replicação Viral , Genética
2.
Chinese Journal of Hepatology ; (12): 839-842, 2009.
Artigo em Chinês | WPRIM | ID: wpr-306632

RESUMO

<p><b>OBJECTIVE</b>To investigate whether there is intercellular transfer of functional P-glycoprotein(P-gp) from P-gp-positive cells to P-gp-negative cells in vitro.</p><p><b>METHODS</b>HepG2/GFP cells, a HepG2 cell line stably expressing GFP, were co-cultured with HepG2/ADM cells, an adriamycin-resistant cell line derived from HepG2 cells. The distribution of P-gp in hepatocellular carcinoma cell was observed under laser scanning confocal microscope (LSCM). Immunomagnetic beads were used to separate HepG2/GFP cells from the mixed culture. The abundance of P-gp was analyzed by western blot, and the expression of mdr1 mRNA was detected by qRT-PCR.</p><p><b>RESULTS</b>Yellow fluorescence was detected in HepG2/aqMDR cells, green fluorescence was detected in HepG2/GFP cells, red fluorescence was detected in HepG2/ADM cells by LSCM. The level of P-gp protein in HepG2/aqMDR cells was lower than that in HepG2/ADM cells, but higher than that in HepG2/GFP cells (q = 35.07, P < 0.05) and HepG2 cells (q = 36.87, P < 0.05). The expression of mdr1 mRNA in HepG2/ADM cells was higher than that in HepG2/aqMDR, HepG2 and HepG2/GFP cells, but there was no significant difference in mdr1 mRNA among HepG2/aqMDR, HepG2 and HepG2/GFP cells (F = 2.30, P > 0.05).</p><p><b>CONCLUSIONS</b>P-gp can transfer from drug resistant hepatocellular cells to sensitive hepatocellular carcinoma cells. This study suggests a novel mechanism of multidrug resistance in hepatocellular carcinoma.</p>


Assuntos
Humanos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Genética , Metabolismo , Carcinoma Hepatocelular , Genética , Metabolismo , Patologia , Técnicas de Cocultura , Métodos , Doxorrubicina , Farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Genética , Genes MDR , Proteínas de Fluorescência Verde , Células Hep G2 , Neoplasias Hepáticas , Genética , Metabolismo , Patologia , Plasmídeos , Transporte Proteico , RNA Mensageiro , Genética , Metabolismo , Transfecção
3.
Chinese Journal of Hepatology ; (12): 675-678, 2009.
Artigo em Chinês | WPRIM | ID: wpr-306704

RESUMO

<p><b>OBJECTIVE</b>Major histocompatibility complex class I C-related molecules A and B (MICA and MICB) are innate immune system ligands for the NKG2D receptor expressed by natural killer cells and activated CD8(+)T cells. Our previous study showed that 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, can induce the expression of MICB and sensitized cells to NKL-cell-mediated cytolysis. The aim of this study was to determine the expression level of MICA in HepG2 cells (an HCC cell line) and L02 cells ( a normal liver cell), and to investigate the effect of 5-aza-dC on MICA expression in HepG2 cells.</p><p><b>METHODS</b>Cells were treated with 5-aza-dC, caffeine and ATM-specific siRNA. The cell surface MICA protein on HepG2 cells and L02 cells was determined using flow cytometry. The mRNA level was detected using real time RT-PCR.</p><p><b>RESULTS</b>MICA was undetectable on the surface of L02 cells, but was highly expressed on HepG2 cells. MICA expression was upregulated in response to 5-aza-dC treatment (P less than 0.05), and the upregulation of MICA was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase (P less than 0.05).</p><p><b>CONCLUSION</b>Our data suggest that 5-aza-dC induces the expression of MICA by a DNA damage-dependent mechanism.</p>


Assuntos
Humanos , Proteínas Mutadas de Ataxia Telangiectasia , Azacitidina , Farmacologia , Cafeína , Farmacologia , Carcinoma Hepatocelular , Metabolismo , Proteínas de Ciclo Celular , Metabolismo , Linhagem Celular , Membrana Celular , Metabolismo , Dano ao DNA , Proteínas de Ligação a DNA , Metabolismo , Citometria de Fluxo , Células Hep G2 , Hepatócitos , Metabolismo , Antígenos de Histocompatibilidade Classe I , Genética , Metabolismo , Neoplasias Hepáticas , Metabolismo , Proteínas Serina-Treonina Quinases , Metabolismo , RNA Mensageiro , Genética , Metabolismo , RNA Interferente Pequeno , Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor , Metabolismo , Regulação para Cima
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