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Objective To investigate the effect of anti-sense osteopontin(ANOPN)on proliferation and metastasis of esophagus cancer cells.Methods An OPN gene recombinant expression vector plasmid was constructed by RT-PCR from human umbilical vein endothelial cell gene and cloned into a mammalian expression vector pcDNA3.1(+).PcDNA3.1-ANOPN was introduced by LipofectinTM.Positive cell clones(ECA-ANOPN),vector-transfected cells ECA-vect and blank cell ECA were used as three groups.RT-PCR and immunocytochemistry assay were used to investigate the expressions of OPN mRNA and protein.The metastasis characteristics of cells were studied by Transwell method.Results The vector was constructed successfully,the sequencing result was identical with that reported in GenBank.Compared with vector-transfected cells(ECA-vect cells) and ECA cells,the growth rate of ECA-ANOPN cells was significantly slowed(P
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Objective To observe the effect of gene gun transduction of K-RAS Antisense gene on the expression of K-RAS P21 protein in human pancreatic carcinoma cells.Methods K-RAS Antisense gene was transduced into pancreatic carcinoma cells by gene gun transduction, the expression of K-RAS P21 protein on BxPC-3、AsPC-1 and MiaPaCa-2 pancreatic carcinoma cells line were examined by western blot and immunocytochemistry staining. Results The expression of K-RAS P21 protein in AsPC-1 and MiaPaCa-2 pancreatic carcinoma cells was obviously lower after the transduction of specific K-RAS Antisense gene,which has little impact on BxPC-3 pancreatic carcinoma cells. Conclusion Gene gun transduction of K-RAS Antisense gene is a potential method for treatment of pancreatic carcinoma.
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Objective To observe the inhibition of vascular endotheilal growth factor (VEGF) gene transcribition and translation with antisense VEGF165 adenovirus vector transfection. Method To transfect the synovial cells with antisense VEGF165 adenovirus and detect the gene translation level with northern-blot method. Result The Northern-blot showed that: the transcription of VEGF gene were inhibited markedly after the antisense VEGF165 gene transfection for 3 days and the inhibition efficiency was obivious in 4th day. In the 5th day, the VEGF translation signal was hardly detected. Conclusion The antisense VEGF165 adenovirus gene transfection can inhibite not only the transcription but the translation of VEGF gene in synovial cells. Thus it can inhibit the secretion of VEGF in synovial cells with high efficiency.
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Objective To explore the effect of c fos antisense gene transfection on the protection of cardiomyocytes following hypoxia and burn serum treatment. Methods Burn serum was collected from Wistar rats with 30% total body surface area(TBSA) of Ⅲ degree burns. The mixture gas containing 1% O 2 was used as hypoxia model. The c fos antisense gene recombinant was constructed by genetic recombination technique. Cardiomyocytes from neonatal Wistar rats were cultured in vitro with hypoxia and burn serum treatment. c fos antisense gene recombinant was transfected into the cultured cardiomyocytes. Expression of c fos mRNA was determined by RT PCR. Expressions of c fos protein, troponin T and ? Tubulin in cardiomyocytes were determined by Western blotting in the transfected and non transfected groups. Results RT PCR results showed that the expression of c fos mRNA increased significantly in the non transfected group. But after transfection of c fos antisense gene recombinant, the expression of c fos mRNA decreased significantly as compared with the non transfected cardiomyocytes. Western blotting results showed that the expression of c fos protein in the transfected group decreased remarkably as compared with the non transfected group, but the expressions of ? Tubulin and troponin T increased significantly in the transfected group. Conclusion Burn serum and hypoxia can cause the injury of cardiomyocytes. c fos antisense gene recombinant transfection has the protective effect on cardiomyocytes exposed to burn serum and hypoxia.
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Objective To clone human beta-2 adrenoceptor gene from human bladder smooth muscle and to construct its antisense eukaryotic expression vector. Methods The ?_2-AR full length cDNA was cloned from human detrusor cells through RT-PCR and subcloned into clone vector (pUC18).The objective gene was then cut from ClaⅠ/HindⅢ sites of pUC18 with restriction endonulcease and subcloned into pLNCX vector in trans-direction.Finally the constructed ?_2-AR gene antisense expresstion vector was identified through restriction endonuclease analysis and sequencing. Results The sequence of cloned ?_2-AR full length cDNA was certified by comparison with the database of the Genebank.The constructed antisense eukaryotic expression vector was proved to be same with designed by restriction endonuclease analysis and sequencing. Conclusions ?_2-AR full length cDNA was cloned and its antisense eukaryotic expression vector was successfully constructed.This technique establishs the foundation for the further research on drug treatment of bladder dysfunction.
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Objective To study the growth inhibition of human hepatoma cells tranfected with type Ⅱ insulin like growth factor receptor (IGF ⅡR) antisense gene. Methods We constructed the recombinant eucaryotic expression plasmids of IGF ⅡR sense and antisense genes, which were transfected into SMMC 7721 human hepatoma cell line using calcium phosphate coprecipitation, and selected in DMEM supplemented with 500 ?g/ml G418(Geneticin) for 2～3 weeks. Then the total number of colonies formed was counted. The effect of IGF ⅡR antisense gene transfection on endogenous IGF ⅡR mRNA levels was examined by Northern blot, and the growth rate and the ability of the transfected cells to form colonies in soft agar medium were also examined. Results The hepatoma cells transfected with IGF ⅡR antisense gene exhibited significant reduction in endogenous IGF ⅡR messenger RNA(mRNA) levels and loss of their ability for anchorage independent growth in soft agar. The IGF ⅡR antisense gene supressed the formation of colonies of SMMC 7721 cells resistant to G418 without alteration on cell growth curve. Conclusion The IGF ⅡR antisense gene expression effectively blocks IGF Ⅱ to IGF ⅡR autocrine and/or paracrine signal transduction pathway and reverses certain aspects of the cell malignant phenotype.
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ObjectiveTo study the effect of TGF-? 1 signal transduction blocked by antisense-Smad 4 gene on progression of experimental liver fibrosis of Kunming mice.MethodsWe transfered retrovirus-mediated antisense Smad 4cDNA into the CCl4/ethanol induced cirrhotic liver of Kunming mouse model to investigate the antisense-Smad 4 gene integration by Southern blot. The expression of Smad 4 in cirrhotic liver was observed by Northern blot, RT-PCR, and Western blot. The fibrotic degree of the livers among the three groups were compared.ResultsThe antisense-Smad 4 gene was confirmed to integrate into the liver of the retrovirus-treated mouse .In the non-treated cirrhotic liver, the expression of Smad 4 mRNA was significantly increased than normal liver, the antisense Smad 4 gene could decrease the expression of Smad 4. Compared with the non-treated liver, the fibrotic septa of the liver in the retrovirus-treated mouse were fewer,narrow, and incomplete,and their fibrotic degree of the treated-liver was reduced.ConclusionThese results indicate that antisense-Smad 4 gene can reduce the expression of Smad 4,inhibit the progression of liver fibrosis.
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Vascular endothelial growth factor (VEGF)165 cDNA were inserted into the expression plasmid pcDNA3.1 site in an antisense orientation to produce antise VEGF165 expression vector.Then electrophoresis and sequencing were carried out,the antisense vector were verified and could be used for related study of antisense gene expression.