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Objective: To investigate the inhibitory effect of a recombinant adenoviral vector carrying antisense matrix metalloproteinase-2(MMP2) on the growth of hepatocellular carcinoma(HCC) in vivo., Methods: The recombinant adenoviral vector carrying antisense MMP2(Ad-MMP2(AS))which had been constructed by us in readiness was used to infect the human HCC cell line (Bel-7402). Then the invasiveness of the Bel-7402 cells was assayed in Matrigel, and the production of MMP2 in the Bel-7402 cells was detected with Western blot analysis and Gelatin zymography. After the Ad-MMP2(AS)-infected Bel-7402 cells being subcutaneously inoculated in nude mice, the production of tumors was under observation, and then Ad-MMP2(AS) was injected intratumorally into the pre-existing tumors., Results: Compared with PBS or Ad-CMV-infected cells, infection of Bel-7402 cells with Ad-MMP2(AS) significantly reduced MMP2 enzyme activity, the invasiveness resulted in 52% reduction in Matrigel assays, and the tumor volume displayed a 4.3-fold reduction in nude mice. In addition, direct intratumoral injection of Ad-MMP2(AS) into pre-existing tumors significantly impaired the further expansion of the tumor mass and resulted in a 63% reduction in tumor cell growth., Conclusion: The recombinant adenovirus with antisense MMP2 can effectively inhibit the invasiveness and growth of Bel-7402 cells in vitro and in vivo, and has a therapeutic potential for HCC.

Objective: To investigate the inhibitory effects of antisense TGF beta1 on proliferation of human bladder transitional cell carcinoma in vitro and in vivo., Methods: Human bladder carcinoma cell line EJ was transfected with pRevT beta-AS, a replication defective retroviral vector carried antisense TGF beta1 fragment. The growth of the transfected cells was observed in vitro and in vivo. TGF beta1 mRNA expression and protein expression were detected by RT-PCR and ELISA. The proliferative activity was evaluated by immunohistochemistry method. The ultrastructure of cells was observed by image analysis system and electron microscopy. Cell cycle was determined by flow cytometry., Results: The expression of TGF beta1 mRNA and protein in EJ cells was inhibited by pRevT beta-AS, G(1) to S transition was restrained in cell cycle and cell proliferation decreased in vitro. The tumorigenesis and growth of EJ cells and DNA heteroploidy were reduced by antisense TGF beta1 in vivo., Conclusion: TGF beta1 plays a role in vitro proliferation and in vivo growth of bladder transitional cell carcinoma.

Objective: To determine the effects of PCMV-FGEV transfection on the profile of SMMC-7721 hepatocellular in vitro in vivo., Methods: SMMC-7721 hepatocellular was transfected with PCMV-FGEV antisense, PCMV-VEGF sense and empty vector plasmid encapsulated by lipofectamine. The positive cell clones were selected with G418. The stable transfection and expression of VEGF in the SMMC-7721 hepatocellular were determined by in situ hybridization and immunochemical analysis. The effect of PCMV-FGEV transfection on SMMC-7721 hepatocellular proliferation was observed by MTT colorimetric assay. Flow cytometry was used to determine the effects of PCMV-FGEV transfection on cell apoptosis of SMMC-7721. The growth of transfected cells was also observed in nude mice., Results: There was reduced VEGF expression in SMMC-7721 transfected with PCMV-FGEV confirmed by in situ hybridization and immunohistochemical analysis. There was no effect of PCMV-FGEV transfection on cell proliferation and cell apoptosis of SMMC-7721 in vitro. The growth of cell with PCMV-FGEV transfected was slow in nude mice (vivo) and accompanied with obvious apoptosis. The latent time of tumor in the antisense mice group was 25.0+/-1.8 days, which was longer than that in sense and control group significantly (F=19.455, P<0.01). On the other hand, the average tumor weight in antisense group (0.96 g+/-0.28 g) was the smallest among the three groups (F=21.501, P<0.01)., Conclusions: The expression of VEGF was inhibited by PCMV-FGEV. There was no effect on cell proliferation and cell apoptosis of SMMC-7721 by transferring PCMV-FGEV gene into SMMC-7721 cells in vitro. But in vivo it can inhibit tumor growth and induce cell apoptosis.

Overexpression of urokinase-type plasminogen activator receptor (uPAR) on tumor cell surface is essential for invasion and metastasis in a variety of tumor cells. To establish a retroviral-mediated antisense RNA transfer system of uPAR gene for exploring its function on down-regulation of uPAR expression in leukemia cells, the retroviral vector LaCD87SN was constructed by inserting uPAR gene into LXSN vector in an antisense orientation. An uPAR gene antisense RNA transfer system was established by liposome-mediated transfection in combination with cross infection with retrovirus. Human leukemia cells U937 were transduced with aCD87 amphotropic retrovirus, expressing uPAR antisense RNA, and the U937/aCD87 cells was obtained by G418 selection. The integration and expression of antisense uPAR gene were analyzed by polymerase chain reaction (PCR) assay. The cell surface expression of CD87 and the activities of matrix metalloproteinase (MMP) were assayed by flow cytometry (FCM) and gelatin zymography, respectively. The results showed that the amphotropic retroviral producers Am12/aCD87, which expressed antisense RNA of uPAR gene with a titer of 6.3 x 10(5) cfu/ml in supernatants, were obtained by means of transfection and superinfection. U937/aCD87 cells were established by continuative G418 selection after retrovirus infection. In U937/aCD87 cells, the integrated provirus and the overexpression of antisense uPAR gene was confirmed. Compared with U937/NeoR cells, FCM analysis revealed that CD87 expression on U937/aCD87 cell surface was not downregulated significantly. However, MMP-9 secretion was significantly suppressed in U937/aCD87 cells. In conclusion, although the retroviral-mediated antisense RNA transfer could not efficiently suppress uPAR expression on leukemic cell surface, it may interfere the uPAR-MMP interactions.

Background & Objectives: The latent membrane protein gene (LMP) of Epstein-Barr virus (EBV) was thought to play an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). In this study, the authors investigated the effects of antisense RNA (AsRNA) on LMP for down regulating at the target gene over expression in EBV transformed lymphoid cells, and set up an antisense system to inhibit LMP expression., Methods: Constructing the single strand antisense transcription system in vitro, the authors have got large amount of AsRNA. Designing and setting up an antisense tracing system in situ (ATSIS), the authors could observe the living particles of AsRNA/sense RNA duplex dimer. With time lapse phase-contrast microscopy, the agglutination phenotype on living cells was easily detected by MTT test, the inhibition rate on EBV transformed cells was calculated., Results: LMP 1.9 fragment ligated into pGEM vector in reverse orientation have been constructed and produced a plentiful amount of AsLMPmRNA which could incorporated into both B95-8 and C1936 cell lines by endophagocytosis and formed the duplex dimer of As/Sense RNA. This particles have been visualized in situ when labelling 35S isotope by ATSIS. When AsLMPmRNA acted as agents for specific inhibition to LMP over expression, the transform phenotype of cell agglutination have been suppressed and MTT particle formatin was apparently reduced both two EBV tansformed cell lines., Conclusion: AsLMPmRNA targets at sense strand have a high effectiveness of down-regulation on EBV-LMP overexpression. This down regulating function of LMP and growth inhibition on transformed cell is demonstrated by the antisenes tracing system in situ (ATSIS). The results provide a clue to overcome the latent EBV infection in human bodies all living long time and to prevent it inducing NPC in high incidence area by antisense strategies.

Objective: To inhibit invasion and metastasis of human lung carcinoma cell line 95D by antisense RNA for urokinase receptor (uPAR)., Methods: Antisense RNA expression plasmid for uPAR was introduced into the highly metastatic human lung carcinoma cells 95D. Modified Boyden's chamber was used to detect the invasion ability, and nude mice were used to determine metastasis., Results: Two transfected clones were found to integrate the antisense expression plasmid into genomic DNA and to express antisense RNA for uPAR. Antisense RNA blocked the uPAR expression in the transfected clones. The evident reductions of the invasiveness of antisense clones were observed by comparison with control cells, 95D cells and cells transfected with pcDNA3 vector. The metastatic potential of these two antisense clones decreased significantly in comparison with the controls (P < 0.05)., Conclusions: The results suggest that antisense RNA for uPAR inhibits uPAR expression and leads to a decrease of metastatic potential of 95D cells. Antisense RNA technique may have a valuable application in anti-metastatic therapy of human lung carcinoma.

Objective: To investigate the feasibility of gene therapy for human gliomas with vascular endothelial growth factor(165) (VEGF(165)) antisense RNA., Methods: The eukaryotic expression vector of antisense VEGF(165) was constructed and identified. Then the vector was transfected into human glioma cells (SHG44). The biological characteristics and tumorigenesis of SHG44 cells before and after transfection were inspected and compared. The changes were detected by Western blot, immunohistochemistry, micrangium counting, electron microscopy and flow cytometry., Results: The eukaryotic expression vector pcDNA-AVEGF(165) was successfully constructed and transfected into SHG44 glioma cells. The characterstics of the cells were not influenced by the expression of antisense gene. The capability of tumorigenesis and angiogenesis of the transfected cells in nude mices was greatly reduced (tumour end volume, experiment group 212 mm(3); control group 7 897 mm(3); P < 0.01; Blood vessel counting: experiment group, 5.50; control group 11.22; P < 0.01 )., Conclusion: The angiogenesis and tumor growth of human gliomas are effectively inhibited by VEGF(165) antisense RNA. This experiment lays a foundation for solid tumor-specific gene therapy.

The research of gene regulation become more and more important with the advancement of the technology of molecular biology and gene study. Antisense molecules can regulate and modify gene expression. By using antisense technology, antisense RNA or artificial antisense oligonucleotides can be made to inhibit or block the transcription and translation of genes. These molecules are highly specific in gene regulation. The application of antisense technology in ophthalmology is introduced briefly in the review. This may be helpful to the studies in ophthalmic research field.