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AIM:To construct antisense VEGF_(165) eukaryotic expressionvector PCDNA_3-as-VEGF_(165) and to study its expression and effecton the proliferation of hepatocarcinoma SMMC-7721 cells.METHODS:VEGF_(165) cDNA was inserted into polylinker sitesof eukaryotic expression vector PCDNA_3 to construct PCDNA_3-as-VEGF_(165).Then the vector was transferred into humanhepatocarcinoma cell strain SMMC-7721 with cationlipofectamine 2000 mediated methods to evaluate theexpression of VEGF protein and the inhibitory effect on theproliferation of hepatocarcinoma SMMC-7721 cells.RESULTS:The detection indicated the presence of VEGFcDNA in normally cultured SMMC-7721 cells by PCR.VEGFmRNA expression was notably decreased in SMMC-7721 cellsby RT-PCR after PCDNA_3-as-VEGF_(165) transfection.The expressionof VEGF protein was dramatically inhibited (142.01±7.95 vs1625.52±64.46 pg·ml~(-1),P<0.01) 2 days after transfection,which correlated with the dose of PCDNA_3-as-VEGF_(165) gene.VEGF protein was most expressed in PCDNA_3 transferredSMMC-7721 cells but few in PCDNA_3-as-VEGF_(165) transferredcells by immunohistochemical staining.The apoptotic rateof hepatocarcinoma SMMC-7721 cells was significantlypromoted (17.98±0.86% vs 4.86±0.27%,P<0.01) and thesurvival rate was notably decreased (80.99±3.20% vs93.52±3.93%,P<0.05) due to antisense VEGF_(165) by flowcytometry (FCM).The transfection of antisense VEGF_(165) generesulted in the inhibitory effect on the proliferation ofhepatocarcinoma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the death of allhepatocarcinoma cells on day 6 after transfection.CONCLUSION:It is confirmed that antisense VEGF_(165) caninhibit the expression of VEGF protein,interfere with theproliferation and induce the apoptosis of hepatocarcinomacells in our study.Antisense VEGF_(165) gene therapy may playan important role in the treatment of human hepatocardnoma.
AIM: To construct antisense VEGF_ (165) eukaryotic expression vector PCDNA_3-as-VEGF_ (165) and to study its expression and effecton the proliferation of hepatocarcinoma SMMC-7721 cells.METHODS: VEGF_ (165) cDNA was inserted into polylinker sites of eukaryotic expression vector PCDNA_3 to construct PCDNA_3-as-VEGF_ (165) .Then the vector was transferred into human hepatocarcinoma cell strain SMMC-7721 with cationlipofectamine 2000 mediated methods to evaluate theexpression of VEGF protein and the inhibitory effect on theproliferation of hepatocarcinoma SMMC-7721 cells.RESULTS: The detection indicated the presence of VEGF cDNA in normally cultured SMMC-7721 cells by PCR. VEGF mRNA expression was not likely decreased in SMMC-7721 cells by RT-PCR after PCDNA_3-as-VEGF_ (165) transfection. The expression of VEGF protein wasappeared inhibited ± 7.95 vs 1625.52 ± 64.46 pg · ml -1, P <0.01) 2 days after transfection, which correlated with the dose of PCDNA_3-as-VEGF_ (165) gene. VEGF protein was most expres sed in PCDNA_3 transferredSMMC-7721 cells but few in PCDNA_3-as-VEGF_ (165) transferred cells by immunohistochemical staining. apoptotic rate of hepatocarcinoma SMMC-7721 cells was significantly promoted (17.98 ± 0.86% vs 4.86 ± 0.27%, P <0.01) (165) by flowcytometry (FCM). The transfection of antisense VEGF_ (165) generesulted in the inhibitory effect on the proliferation of hepatocarcinoma cells by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) and the death of all hepatocarcinoma cells on day 6 after transfection. CONCLUSION: It is confirmed that antisense VEGF_ (165) caninhibit the expression of VEGF protein, interfere with the proliferation and induce the apoptosis of hepatocarcinoma cells in our study. Antisense VEGF - (165) gene therapy may play important role in the treatment of human hepatocardnoma.