Résumé
<p><b>OBJECTIVE</b>To study in vitro and in vivo protein expression and biological function of gene pp1158, a hepatocellular carcinoma (HCC)-related gene.</p><p><b>METHODS</b>pp1158 was expressed with fusion expression vector pET-32a in E. Coli-BL21 (DE3), and rabbit anti-pp1158 fusion protein polyclonal antibody was prepared. The biological function and differential expressions of pp1158 were studied by in vitro colony formation assay nude mouse in vivo tumor formation assay of transfected BEL7402 cell line and immunohistochemistry and Western blot. Differential expression of pp1158 in human fetal tissues were examined by Northern blot.</p><p><b>RESULTS</b>In vitro experiment showed that pp1158 inhibited colony formation rate of transfected BEL 7402 cells, with an inhibition rate of 58.3% (P < 0.01). Tumor formation assay indicated that tumor formation of pCMV-Script-1158 transfected BEL 7402 cell line was significantly inhibited (P < 0.05) as compared with that of the control group. Immunohistochemical assay showed that pp1158 was expressed in human tissue in the following sequence: normal liver >/= noncancerous liver tissue > HCC. Western blot indicated that a 60kD protein (pp1158 protein) was expressed in BEL 7402 cells transfected with pCMV-Script-pp1158 DNA, while it was detected in BEL 7402 cells transfected with pCMV-Script vector DNA. Northern blot showed pp1158 was expressed in the placenta at a very high level, heart, liver, muscle, pancreas and lung but expressed poorly in the brain and kidney.</p><p><b>CONCLUSION</b>pp1158 is a new candidate tumor suppressor gene of HCC.</p>
Sujets)
Animaux , Femelle , Humains , Mâle , Souris , Protéine-4 similaire à l'angiopoïétine , Angiopoïétines , Technique de Northern , Technique de Western , Carcinome hépatocellulaire , Génétique , Métabolisme , Anatomopathologie , Régulation de l'expression des gènes tumoraux , Gènes suppresseurs de tumeur , Protéines et peptides de signalisation intercellulaire , Tumeurs du foie , Génétique , Métabolisme , Anatomopathologie , Souris de lignée BALB C , Données de séquences moléculaires , Protéines tumorales , Génétique , Métabolisme , Transplantation tumorale , Tumeurs expérimentales , Génétique , Métabolisme , Anatomopathologie , Protéines , Génétique , ARN messager , Génétique , Métabolisme , Transplantation hétérologue , Cellules cancéreuses en cultureRésumé
<p><b>BACKGROUND</b>To study the structure and function of 2.2 kb spliced variant of HBV genome from liver tissues of hepatocellular carcinoma patients.</p><p><b>METHODS</b>HBV genomes were amplified by using PCR from paired hepatocellular carcinoma tissues and peritumor tissues. The 3.2 kb full-length HBV genome and 2.2 kb spliced variant were separately cloned and sequenced. Hep G2 cells were co-transfected with full-length HBV DNA and 2.2 kb spliced variant, and after transfection, HBV DNAs from intracellular core particles were harvested and specific primers were used in PCR to evaluate the interactions between spliced variant and full-length counterpart in replication.</p><p><b>RESULTS</b>Semi-quantification by scanning density showed that 2.2 kb spliced variant was present in all tumor and peri-tumor samples studied. Sequence analyes revealed that the 5 terminus packaging signal for pregenomic and X and PreC/C genes were retained. When full-length HBV DNA was co-transfected with 2.2 kb, the replication signal of 3.2 kb HBV genome was increased 3-7 times.</p><p><b>CONCLUSIONS</b>The 2.2 kb HBV spliced variant was present in liver tissues, and relative content was higher in tumor tissues than that in the peri-tumor tissues. This spliced variant could enhance the replication of full-length HBV genome, which suggested the possible role of the variant in the pathogenesis of development of hepatocellular carcinoma.</p>
Sujets)
Adulte , Sujet âgé , Femelle , Humains , Mâle , Adulte d'âge moyen , Carcinome hépatocellulaire , Virologie , ADN viral , Variation génétique , Génome viral , Virus de l'hépatite B , Génétique , Foie , Virologie , Tumeurs du foie , Virologie , ARN viral , Analyse de séquence d'ADNRésumé
Purpose To introduce a method to isolate cDNA clones using bacteriophage P1-derived artificial chromosome (PAC) or bacterial artificial chromosome (BAC) as probe for hybridization and try to find some novel genes related to hepatocellular carcinoma. Methods PAC 579 (D17S926 locus) and BAC 1529 (D17S1529 locus) in the deletion region of chromosome 17p13.3 in human hepatocellular carcinoma were chosen to screening the human liver cDNA library as probe for hybridization. The isolated positive cDNA clones were partially sequenced, then analyzed by computer comparison and Southern blot. Results After three cycles of screening, 78 and 8 candidate positive cDNA clones were isolated using PAC 579 and BAC 1529 probes respectively. Further analysis indicated 18 cDNA clones isolated by PAC 579 probe and 5 cDNA isolated by BAC 1529 probe were potential novel genes related to hepatocellular carcinoma. Conclusions The isolation of cDNA clones using PAC and BAC probes is effective and practical.
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Purpose:To investigate the effects of tumor suppressor gene p53 on the activity of telomerase/hTERT in human HCC-9204 cell line.Methods:Lipofection-mediated gene transfection method was used to transfect wild-type p53(wt-p53) gene into HCC 9204 cell which expresses telomerse /hTERT and carries mutant-type p53 gene. The expression of p53 was confirmed by Western blot. Both the telomerase activity and hTERT mRNA expression were detected by PCR-ELISA,TRAP-silver staining,in-situ hybridization and RT-PCR methods. The apoptotic appearance was examined by FCM.Results:Higher telomerase activity and hTERT mRNA level were detected in HCC-9204,and they were markedly inhibited after transfection with wt-p53. Meanwhile,decreasing level of bcl-2 protein and appearance of apoptosis were also shown in the transfected cells.Conclusions:Over expression of the exogenous wt-p53 gene does suppress both telomrease activity and hTERT mRNA expression in HCC cell line. There is a p53-dependent regulatory pathway for activation and expression of telomerase/hTERT in HCC.
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Retroviral vectors are the most used gene delivery vehicle in human gene therapy.When the therapeutic gene is packagedfrom producer cell line, one has to determined whether thereplication-competent retrovirus(RCR) is being generated. In this study, two methods, namely the marker rescue assay and RT/PCR, were employed to detect RCR in the cells. While the marker rescue assay can detect RCR with a limit at 6?102CFU/ml, RT/PCR can be used to detect RCR at as low as lCFU/103ml. Combining the specificity of the marker rescue assay and the sensitivity of RT/PCR, both assays together should serve as an adequate test for detecting the generation of RCR in retroviral producer cell lines.
Résumé
0. 05). Conclusion: The HCAPl can be expressed in both normal peripheral white blood cells and the leukemic cells, and there is no difference in the protein levels between them.