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1.
Chinese Journal of Surgery ; (12): 741-745, 2011.
Artigo em Chinês | WPRIM | ID: wpr-285651

RESUMO

<p><b>OBJECTIVES</b>To study the phenomena of hepatitis B virus (HBV) integration into the tissues of hilar cholangiocarcinoma (HCCA) and to identify the integration sites in the host genome.</p><p><b>METHODS</b>Ten fresh HCCA samples were collected from the tissues by surgical ablation, 1 normal hilar bile duct sample selected as control. Cellular DNA were extracted by Wizard SV Genomic DNA Purification System. PCR-derived assay (HBV-Alu-PCR) was employed to amplify the viral-host junctions which contain the HBV sequence and the adjacent cellular flanking sequences. The PCR products were purified and subjected to sequencing by ABI-3730XL Auto DNA Analyzer. The sequence analysis of viral-host junctions was performed by DNASIS MAX 3.0 bioinformatics software. The insertion sites between viral and cellular sequences were identified through homology comparison using NCBI BLAST and MapViewer search.</p><p><b>RESULTS</b>In 10 HCCA samples, 5 were demonstrated to have HBV integration fragments with total 6 inserted sites identified. Sequence analysis from viral-host junction showed that HBV X gene inserted into host genome at random distribution with truncated fragments. HBV integration recurrently targeted the unknown region in upstream of CXXC finger protein-1 (CpG-binding protein) gene (4 cases). p53 tumor suppressor gene was also found at the integration site.</p><p><b>CONCLUSIONS</b>There is high integration rate of HBV DNA into cellular genome of HCCA. HBV integration is found frequently into or close to cancer-related genes. The findings demonstrate that HBV infection might have association with the pathogenesis of HCCA.</p>


Assuntos
Idoso , Feminino , Humanos , Masculino , Sequência de Bases , Neoplasias dos Ductos Biliares , Genética , Virologia , Colangiocarcinoma , Genética , Virologia , DNA Viral , Genética , Hepatite B , Virologia , Vírus da Hepatite B , Genética , Integração Viral
2.
Chinese Journal of Surgery ; (12): 1254-1257, 2004.
Artigo em Chinês | WPRIM | ID: wpr-345112

RESUMO

<p><b>OBJECTIVE</b>To study the effect of Hepatitis B virus X (HBx) gene transfection on expression of human telomerase reverse transcriptase (hTERT) mRNA in human bile duct carcinoma cell lines QBC939 and to elucidate the significance of cis-activation of hTERT mRNA by HBx gene on the carcinogenesis of bile duct.</p><p><b>METHODS</b>QBC939 were cultured in vitro and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using liposome-mediated gene transduction technique. Thirty six hours after transfection, EGFP expression, the indicator of successful transfection in cells, was determined. Flow cytometry was applied to determine the transfection efficiency. Cells were harvested and total RNA was extracted with TRI(ZOL) Reagent. The expression of hTERT mRNA in QBC939 was assayed by Reverse Transcription Polymerase Chain Reaction. The expression of HBx protein in QBC939 was detected by immunocytochemistry staining and western blotting.</p><p><b>RESULTS</b>The transfection efficiency was 29.6% for both HBx expression vector and vector control group. The expression of hTERT mRNA was significantly increased when transfected with HBx expression vector than that transfected with OPTI-MEM medium and vector only. The expression of HBx protein could only be found in the cells when transfected with HBx expression vector by immunocytochemistry staining and western blotting.</p><p><b>CONCLUSION</b>HBx gene transfection may up-regulate the transcriptional expression of hTERT mRNA in bile duct carcinoma cells. The cis-activation of hTERT gene by HBx gene is primary mechanism for carcinogenesis of biliary epithelia after HBV infection.</p>


Assuntos
Humanos , Neoplasias dos Ductos Biliares , Genética , Metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Metabolismo , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , RNA Mensageiro , Genética , Metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase , Metabolismo , Transativadores , Genética , Transfecção
3.
Chinese Journal of Surgery ; (12): 88-91, 2004.
Artigo em Chinês | WPRIM | ID: wpr-311143

RESUMO

<p><b>OBJECTIVE</b>To detect the expression of HBV X gene (HBx mRNA) in extrahepatic biliary tract carcinomas and the adjacent non-cancerous tissues, and to analyzed the relationship between HBV infection and incidence of biliary tract carcinomas, thereby to elucidate the possible role of HBx in the carcinogenesis of biliary tract.</p><p><b>METHODS</b>The plasmid pSPX46 was digested by appropriate restriction enzyme. HBx fragment was obtained through gel extraction kit. The digoxigenin-labeled DNA probes for HBx mRNA were prepared by a random prime technique. The expression of HBx mRNA was detected in formalin-fixed- paraffin-embedded specimens from 71 cases of biliary tract carcinomas and 39 specimens of non-cancerous tissues adjacent to cancer by in situ hybridization. The correlations between HBx mRNA expression and clinicopathological parameters were statistically analysed in 71 cases of biliary duct carcinomas.</p><p><b>RESULTS</b>Forty-three of 71 malignant specimens had detectable HBx mRNA expression with a positive rate being 61%. Only 7 of 39 specimens of non-cancerous tissues adjacent to cancer had weak HBx mRNA expression, with a positive rate being 18%, and all these positive signals were found in the hyperplastic biliary epithelium. No significant correlation was found between HBx mRNA expression and clinicopathological parameters, but a strong positive correlation was found between HBx mRNA and protein expression.</p><p><b>CONCLUSION</b>There is a high frequency of HBx mRNA expression in extrahepatic biliary tract carcinomas. HBV infection and its gene integration might play a role to certain extent in the development of biliary tract carcinomas.</p>


Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sequência de Bases , Ductos Biliares Extra-Hepáticos , Patologia , Neoplasias do Sistema Biliar , Virologia , Hepatite B , Virologia , Vírus da Hepatite B , Genética , Hibridização In Situ , Dados de Sequência Molecular , RNA Mensageiro , Genética , Transativadores , Genética
4.
Chinese Journal of Oncology ; (12): 98-101, 2004.
Artigo em Chinês | WPRIM | ID: wpr-271057

RESUMO

<p><b>OBJECTIVE</b>To detect the expression of human telomerase reverse transcriptase (hTERT) protein and mRNA in bile duct carcinomas and the adjacent tissues and to elucidate its role in bile duct carcinogenesis.</p><p><b>METHODS</b>The expression of hTERT protein and hTERT mRNA in the formalin-fixed paraffin-embedded specimens of 71 cases of bile duct cancers and 39 cases of adjacent tissues was detected by streptavidin-peroxidase immunostaining and in situ hybridization. The correlation was analysed statistically between the expression of hTERT protein and mRNA and clinicopathological parameters bile duct carcinomas.</p><p><b>RESULTS</b>The positive rate of hTERT protein expression and mRNA expression in malignant specimens was 78.9% (56/71) and 67.6% (48/71), while that in the adjacent tissues was 35.9% (14/39) and 23.1% (9/39), respectively. All the positive signals were found in the hyperplastic biliary epithelia. No significant correlation was established between hTERT expression and clinicopathological parameters.</p><p><b>CONCLUSION</b>hTERT gene transcription and protein expression is most likely involved in the proliferation and malignant transformation of bile epithelia and the malignant progression of bile duct carcinomas. The detection of hTERT expression may serve elucidating the carcinogenesis of bile duct.</p>


Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias dos Ductos Biliares , Patologia , Proteínas de Ligação a DNA , Imuno-Histoquímica , RNA Mensageiro , Telomerase , Genética
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