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Objective To construct the expressing vector of shRNA/mrp1 and study its expression in vitro. Methods 64bp oligonucleotides of pSUPER and the targeted senquence of siRNA/mrp1 were synthesized and annealed to form duplex strand, then were cloned into pSUPER to construct pSUPER-shRNA/mrp1 vector. Competenced Ecoli was transfected by vector of pSUPER-shRNA/ mrp1 to screen the positive clones for sequencing and extracting plasmid. The plasmids extracted were used to transfected HepG2/mrp1 cells with a control groups by negative vectors. The expression of mrp1 mRNA and MRP1 was measured by real-time PCR and resistance of HepG2/mrp1 by flowcytometry. Results pSUPER-shRNA/mrp1 was established successfully and was sequenced to test its accuracy. Expression of mrp1 mRNA in HepG2/mrp1-si was lower than that in HepG2/mrp1 (1-fold vs 179.76-fold, P<0.001). Compared to HepG2/mrp1, the expression of MRP1 in HepG2/mrp1-si was lower (11.2% vs 97.6%, P<0. 05). The sensitivity of HepG2/mrp1-si to adiramycin was higher than that of HepG2/mrp1(45.0-fold vs 1.2-fold, P<0.01). Meanwhile, the accumulation of DNR in HepG2/mrp1-si increased significantly as compared with the control (78.58 % vs 38.44%,P<0.05).Conclusion Vector of pSUPER-shRNA/mrp1 can be constructed by the technique of enzymatic incision. The multidrug resistance of HepG2/mrp1 can be reversed by RNA interference.
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<p><b>OBJECTIVE</b>To investigate the correlation between epidermal growth factor (EGF) and overexpression of vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Expressions of EGF, VEGF and microvessel density were studied through immunohistochemical SABC method in 36 HCC specimens, their paraneoplastic liver tissues and 6 normal liver tissues with the correlation between these parameters analyzed. Recombinant human EGF was used to stimulate HepG(2) cells and semi-quantitative reverse transcription PCR was adopted to detect the expression of VEGF in HepG(2) cells.</p><p><b>RESULTS</b>The positive rates of EGF and VEGF expression in HCC tissue were 75.0% and 88.9%. There was positive correlation between EGF and VEGF expression (P < 0.01, r = 0.462). Recombinant human EGF could induce the expression of VEGF in HepG(2) cells in a dose and time dependent manner.</p><p><b>CONCLUSION</b>The expression of EGF in HCC underlies the overexpression of VEGF in HCC.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Carcinoma, Hepatocellular , Metabolism , Endothelial Growth Factors , Epidermal Growth Factor , Intercellular Signaling Peptides and Proteins , Liver Neoplasms , Metabolism , Lymphokines , Neovascularization, Pathologic , Statistics as Topic , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Objective To summrize the effects of various treatment methods,including operation,transcatheter arterial embolization(TAE),and percutaneous ethanol injection(PEI) on hepatic cavernous hemangioma(HCH),in order to select an optimal therapy strategy for HCH.MethodsClinic data of 47 patients with 58 hepatic hemangiomas treated in our hospital in recent 10 years were analyzed retrospectively.Results There were 26 males and 21 females with mean age 42?2 5 years(10～72 years old)in this series.The diameters of these lesions were 3～21 5cm(mean diameter 8 5?1 8cm).Of them,obvious symptoms appeared in 39 patients,malignancy could not excluded in 6,and the size of HCH increased rapidly in 2.Operations were performed in 40 patients,including 2 patients failed to PEI;TAE was conducted in 7cases.There was no mortality or major morbidity occurred in the operative patients,except right pleual effusion in one case and subdiaphragmatic fluid collections in another case(2/40).Neither is mortality nor morbidity seen in the TAE treated case.Conclusions Operation is an effective procedure for HCH.But for more diffuse lesions or lesions located in special region,TAE is a more favorable alterative selection.Because PEI has risk of hemorrhage and hardener injection under ultrasonography guide has uncertain outcome,so it should be chosen cautiously.
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Objective To determine whether the important apoptosis-related genes of p53 and bcl-2 participate in the regulation of apoptosis of human liver cancer cells by establishing the model of apoptosis of human liver cancer cells induced by mitomycin C (MMC). Methods The selected human liver cancer cell line HepG2 was treated with 8mg/ml MMC. The morphological changes of the cells were observed using fluorescent and electron transmission microscopy and the DNA fragments of the cells analyzed by agarose gel electrophoresis to testify cell apoptosis. The expression of p53 and bcl-2 genes was detected by flow cytometry. Results The morphological observation and biochemical examination showed that MMC induced the HepG2 cells to undergo apoptosis. After administration of MMC, the expression of p53 gene changed obviously while that of bcl-2 gene was not apparent. Conclusion p53 plays an important role in the regulation of apoptosis induced by MMC in HepG2 cells. This regulation may be an early event in apoptotic signal pathway. Meanwhile, the apoptosis of the cells is p53-dependent. However, bcl-2 gene may not participate in the control of this apoptosis.
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Objective To investigate the role of cytochrome C in the apoptosis of hepatoma cell induced by flurouracil,and to analyze the distribution of cytochrome C in cytosol and the relationship between the cytochrome C distribution and Caspase-9 activation.Methods The human hepatoma HepG2 cells were treated with flurouracil at 1?10-2mol/L and 2,4,8,16,24h respectively.The chang of cytochrome C in HepG2 apoptosis was detected by using Fluorescent Assay Kit;and proteolytic cleavage of caspase-9 and distribution of cytochrome C in cytosol or in mitochondria was analyzed by Western blot.Results Four hours after cells exposure to flurouracil,the caspase-9 activity in HepG2 cells increased gradually and reached the peak at 16h,compared with the control groups,the difference was significant(P
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Objective To investigate the expressions of vascular endothelial growth factor (VEGF) , hypoxia inducible factor 1 alpha (HIF 1?) and epidermal growth factor (EGF) in hepatocellular carcinoma (HCC) and their clinical significance. Methods The expressions of VEGF, HIF 1? and EGF in 36 cases of HCC and corresponding paraneoplastic tissues and normal liver tissues (6 cases) were studied by immunohistochemistry assay. ResultsThe expression rate of VEGF, HIF 1? and EGF in HCC tissue was 89%, 67% and 75% respectively, higher than those in paraneoplastic tissues and normal liver tissues ( P
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Objective To determine the effect of caspase 9 in the apoptosis of hepatoma cells induced by flurouracil , and to investigate the activity alteration and proteolytic cleavage of caspase 9. Methods The human hepatoma HepG2 cells were treated with flurouracil for 2,4,8,16,24h respectively. The caspase 9 activity was detected using caspase 9 Fluorescent Assay Kit.Proteolytic cleavage of caspase 9 was analyzed by Western blot.The apoptotic rates of HepG2 cells induced by flurouracil with or without caspase 9 inhibitor treatment were measured by flow cytometry. Results Four hours after being treated with flurouracil, the caspase 9 activity in HepG2 cells increased gradually and reached the peak at 16h.Compared with the control group, the difference was significant (P