ABSTRACT
To explore a novel strategy for antisense gene therapy of cancer, the coding sequence of human proliferating cell nuclear antigen (PCNA) cDNA was reversely inserted into the eukaryotic vector pLXSN by molecular cloning techniques and transferred into bladder cancer EJ cells with liposome. The PCNA expression in transferred cells was dynamically detected by immunofluorescence and RT-PCR techniques. Changes of proliferation activities of cancer cells were assayed by MTT colorimetric and cloning formation methods. In the experiment, the antisense eukaryotic vector was successfully constructed and named as pLAPSN. After transfection with it for 1-7 days, PCNA protein and mRNA levels in cancer cells were blocked by 16.74%-84.21% (P < 0.05) and 23.27%-86.15% (P < 0.05) respectively. The proliferation activities of transferred cells were inhibited by 27.91%-62.07% (P < 0.01), with cloning formation abilities being decreased by 50.81% (P < 0.01). It was concluded that the in vitro proliferation activities of cancer cells could be effectively inhibited by blocking PCNA expression with antisense technique, which could serve as an ideal strategy for gene therapy of bladder cancer.
Subject(s)
Humans , Cell Division , Cell Line, Tumor , Cloning, Molecular , DNA, Complementary , DNA, Neoplasm , Metabolism , Eukaryotic Cells , Metabolism , Gene Expression , Genetic Vectors , Proliferating Cell Nuclear Antigen , Genetics , RNA, Antisense , Genetics , Transfection , Urinary Bladder Neoplasms , Genetics , MetabolismABSTRACT
Objectives:Using IE 1 gene promoter of Autograph californica nuclear polyhedrosis virus(AcNPV),a transfer vector with an immediately early gene promoter was constructed. Methods:Transfer vector pAcPIneo which contains neomycin resistance gene(neo)coding sequence downstream of IE 1-promoter was constructed and cotransfected with the wild type of AcNPV DNA into Sf9 insect cells.Recombinant virus was selected by G418 resistance since the neo gene can be expressed in Sf9 cells. Results:Northern blot hybridzation with 32 P labeled neo gene fragement as probe showed that the neogene was integrated in plogene of AcNPV genome. Conclusions:Transfer vector with an immediately early gene promoter of baculovirus was constructed successfully,the neo gene was transcribed from the immediately early phase to the very late phase in infected cells.