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Objective To construct the retroviral vector inserted SV40 large T antigen gene and transfect it into rat hepatocytes, analyze the status of SV40 large T antigen gene expression in rat hepatocytes, and to establish important basis for clinical hepatocytes transplanation. Methods Retroviral vector inserted SV40 large T antigen gene was(constructed) by DNA recombinant techniques in vitro, then the combinant vector was determined with enzyme(digestion) and sequencing and was transfected into the PA317 cell lines by liposome mediation and screened(anti-G4)18 positive clones. The viral titer was determined with the NIH3T3. After transfected into separated and purified rat primary hepatocytes, the SV40 large T antigen gene expression was detected by PCR and immunohistochemical (methods). Results (1)SV40 large T antigen gene fragment was inserted into retroviral vector in sense orientation. (2)The titer of pseudovirion packed by PA317 cell lines was 1.3?10~6CFU/ml. (3)SV40LT antigen gene was(integrated) into rat primary hepatocytes and its expression in transfected cells at 24 hour was higher than 96 hour (P
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Objective To study the clinicopathological significance of the expression of glutameta decarboxylase 65(GDA65) and protein kinase C(PKC) in the central cancer tissues, cancer edge tissues, paracancerous liver tissue and non-cancer liver tissues. Methods The expression of GDA65 and PKC were detected by immunohistochemical method in 10% neutral formalin- fixed and routinely paraffin-embedded sections in 37 hepatic cancer specimen. Results The positive rate and the score of GDA65 and PKC in the cancer tissues were significantly higher than that in the paracancer tissues or non-cancer liver tissues, but the PKC expression was no difference between the central cancer tissues and the cancer edge tissues . The expression of GDA 65 was related to the pathological types, differentiated degrees, liver cirrhosis or metastasis of hepatocarcinomas. No correlation was found between the expression of PKC and the clinicopathological features of hepatocarcinomas. Conclusions The expression of GDA65 and PKC might be closely related to the carcinogenesis of hepatocarcinoma, they might be important biological markers of hepatocarcinoma.
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Objective To establish an experimental model for exploring the role of RhoC gene in the invasiveness and metastasis of hepatocellular carcinoma.Method The RhoC gene was digested with restricted enzyme Hind III and XbaI,and direct cloned to pcDNA3.1.The recombinant vector (pcDNA3.1 RhoC) and the vector alone (pcDNA3.1) were transfected into HEPG2 cells with LIPFECTAMINETMReagent.After selected with hygromycin,resistant cloneies was obtained.The transcription and translation of RhoC gene were analysed with the reverse transcription PCR and immunohistochemical stain.Results The recombinant vectort (pc DNA3.1 Rhoc) express steadily in HerpG2 cells.Conclusions The modified tumor cells(HEPG2 RhoC) could be used to study the effect of RhoC protein on the invasiveness and metastasis of hepatocellular carcinoma.
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Objective To establish a recombinant eukaryotic expression vector containing human interferon-?(IFN-?) cDNA,and investigate the expression of IFN-? gene in transfected hepatocellular carcinoma (HCC) cell lines. Methods pcDNA3-IFN-?,established by subcloning IFN-? cDNA into a mammalian expression vector pcDNA3,was mediated into HCC cell lines SMMC-7721 and QGY-7701 by lipofectamine. The transfected HCC cells were selected in RPMI1640 containing G418(400～700?g).RT-PCR analysis and ELISA assay were used for mRNA transcription and protein expression of IFN-? gene. Results IFN-? mRNA was detected only in pcDNA3-IFN-? transfected cell lines, and high levels of IFN-? protein were detected only in the supernatants of IFN-? gene-modified cell lines. Conclusions pcDNA3-IFN-? can stably expressIFN-? cDNA in the transfected HCC cell strains
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Objective To study the mRNA expression of MCT1 and MCT2 genes in human hepatocellular carcinoma (HCC) and paracarcinoma liver tissue (PCLT). Methods The semi-quantitative analysis of MCT1 and MCT2 genes mRNA expression in human HCC and PCLT was conducted by RT-PCR method and electrophoresis band opacity density (OD) comparison analysis method in 25 patients with HCC. Results The mRNA expression of MCT1 was significantly higher than MCT2 in HCC and PCLT, in HCC the mRNA expression of MCT1 and MCT2 genes were significant higher than that in PCLT. Conclusions The high expression of mRNA of MCT1 and MCT2 genes in HCC indicates that these genes may take a significant role on lactate and other monocarboxylate transmembrane transportation and on pH regulation in tumor cells.