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1.
AIM: To inhibit the expression of tyrosinase gene in murine B16 melanoma cells by antisense nucleotide. METHODS: The antisense recombinant pcDNA3.1(-)-tyr was constructed and was used to infect murine B16 melanoma cells for expression of tyr antisense nucleotide. The effect of antisense nucleotide of tyr on the expression of tyr gene was detected by determination of the activity of tyrosinase and of the production of melanin, Dopa staining and electronic microscope. RESULTS: The tyr antisense recombinant was successfully constructed and injected into murine B16 melanoma cell. The activity of tyrosinase in B16 cells infected with pcDNA3.1 (-)-tyr decreased to 0.0498±0.0036, compared to the tyrosinase activity of 0.0916±0.0132 in the control cells without treatment (P<0.01). The results of Dopa staining and electronic microscopy showed that the production of melanin also decreased significantly compared with control B16 cells. CONCLUSION: tyr antisense nucleotide significantly inhibits the expression of tyr gene in murine B16 melanoma cells.  相似文献   

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AIM: To provide experimental evidence for gene therapy of thrombophilia disease, we constructed the eukaryotic expression plasmid with human thrombomodulin (hTM) gene and observed the alteration of hTM expression on the surface of human umbilical vein endothelial cells (HUVECs) with and without the reconstructive plasmid. METHODS: The whole expressive fragment of hTM gene was amplified by PCR from human genome. Both hTM gene and pcDNA3.1(+)/neo empty vector was digested by HindⅢ and EcoRⅠ. Two digested fragments were ligated into pcDNA3.1/hTM with T4DNA ligase. After identifying, the reconstructive plasmid transfected into HUVECs using lipofectin. The hTM antigen on the HUVECs was detected by immunohistochemistry. RESULTS: The hTM reconstructive plasmid was confirmed by double endonuclease redigesting and sequencing. About 10% HUVECs were transfected by pcDNA3.1/hTM plasmid with lipofectin and the high-level hTM was detected on the transfected cells. CONCLUSION: We constructed the pcDNA3.1/hTM plasmid successfully, and it could be expressed on the HUVECs.  相似文献   

4.
AIM: To study the effect of human FAT10 on the apopotosis of HEK293 cells using flag-tagged human FAT10 protein.METHODS: The fragment of FAT10 gene was cloned into the pcDNA3-flag vector,which was identified by PCR,enzyme digestion and sequencing.The reconstructed plasmids were transfected into HEK293 cells.The expression of introduced FAT10 in the normal cultured and starved cells was detected respectively by Western blotting.XTT assay and DNA ladder method were used to analyze the effect of FAT10 on the apoptosis of starved HEK293 cells.RESULTS: The reconstructed plasmids were highly expressed in HEK293 cells with different expression mode at the mormal cultured and starved state.The livability of starved FAT10 overexpressed HEK293 cells was significantly lower than that of normal cultured cells.DNA ladder was observed in the starved FAT10 overexpressed cells,but not in the normal cultured cells.CONCLUSION: The eukaryotic expressed plasmids of flag-tagged FAT10 were constructed successfully,and highly expressed in HEK293.Overexpressed FAT10 enhances the apoptosis in the starved HEK293 cells.  相似文献   

5.
AIM:To study the roles of isotropic diffusion weighted imaging(DWI)and apparent diffusion coeficient(ADC)mapping in diagnosing early cerebral infarction.METHODS:21 patients with cerebral in farction (8hyperacute,13 acute)were imaged with both convent ional MRI and single-shot echo-planar isotropic diffusion weighted imaging.Among them 12 pat ients had CT scanning simultaneously within 24 hours after onset.The positive rate of early in farction was comparted on CT,T2WI and DEI.The change of the infarct lesion in DWI and T2WI was also analysed.The av erage ADC,relat ive ADC(rADC)and the ADC from center to periphery of the lesion were calculated.RESULTS:8 hypera cute cerebral ischemic regions were revealed at DWI and ADC mapping,but CT and conventional MR were not.Hyperacute and acute infarcts appeared as areas of hyperintensity on DWI,and their average ADC was significantly depressed comparted with homologous contralateral tissue(0.698±0.104)×10-3mm2/s vs(0.990±0.161)×10-3mm2/s(P<0.01).ADC value in 21 hypera cute had gradient sign.CONCLUSION:Isotropic diffusion weighted imaging and ADC mapping have greater senstitivity for acute and hyperacute cerebral infarction than conventional MRI and CT,and may be used to defined the core and penumbra of ischemic lesion.  相似文献   

6.
AIM: To construct a hAR and GFP fusion gene vector and to observe the AR-GFP gene expression in Hek293 cells. METHODS: A recombined vector pcDNA3.1/myc-HisA-AR-GFP (pH-AG) was constructed by gene engineering technique. The recombined vector was transfected into Hek293 cells using calcium phosphate. RESULTS: AR-GFP fusion protein was successfully expressed in Hek293 cells without biologic activity, which was confirmed by fluorescence microscopy and Western blotting. CONCLUSION: The Hek293 cells transfected by AR-GFP fusion gene can express its protein successfully. However, it is not a cellular model for ARI screening.   相似文献   

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AIM: To prepare the vaccine of DCs(pcDNA3-hCEA) and observ the immunity effect of the DCs(pcDNA3-hCEA) inoculating on CT26(hCEA+) loaded in BALB/c mice. METHODS: DCs were generated from bone marrow in the presence of rmGM-CSF and rmIL-4. A new recombinant plasmid, pcDNA3-hCEA, reformed with inserting a 2.4 kb human CEA cDNA into pcDNA3. DC vaccine was prepared by transfection with pcDNA3-hCEA using lipofectamine. CEA mRNA expressed in DCs(pcDNA3-hCEA) was confirmed by RT-PCR, CEA expression level was detected with RIA method, and CEA specific CTL was induced in vitro. After vaccination of DCs(pcDNA3-hCEA), the survival time of the BALB/c mice challenged with critical loading CT26(hCEA+) was observed. RESULTS: G418 test showed that about14% DCs were transfected with pcDNA3-hCEA. And CEA mRNA and protein could be detected respectively by RT-PCR and RIA in the genetically modified DCs. Furthermore, the DCs coud be targeted by specific CTL, the survival time of the mice challenged with CT26(hCEA+) was prolonged1-4 weeks. CONCLUSION: These results demonstrate that specific antitumor immune responses could be induced efficiently by vaccination of DCs(pcDNA3-hCEA), which is transfected eukaryotic expression vector encoding tumor antigen gene.  相似文献   

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AIM: To investigated the characterization and biological function of P53 binding element in the regulation of NOD8 gene. METHODS: Using the method of bioinformatics, we found a completely preserved P53 binding site in NOD8 core promoter both in humans and chimpanzees. NOD8 gene was amplified by PCR from human cDNA and correctly connected into the vector p NOD8 (760 bp)-EGFP-C2/ mp NOD8 (750 bp)-EGFP-C2. The constructed plasmids p NOD8 (760 bp)-EGFP- NOD8 and mp NOD8 (750 bp)-EGFP- NOD8 were transiently transfected into the cell line HEK293 by JetPeiTM and treated with pifithrin alpha (PFT-α,an inhibitor of P53) at different concentrations for 24 h. The mRNA and protein expression levels of NOD8 were detected by RT-PCR and Western blotting. In addition, chromatin immunoprecipitation (ChIP) assay was performed to determine the binding of P53 to the NOD8 promoter after recombinant plasmid p NOD8 (760 bp)-EGFP was transfected into HEK293 cells. RESULTS: The plasmids p NOD8 (760 bp) -EGFP- NOD8 and mp NOD8 (750 bp)-EGFP- NOD8 were successfully constructed and conformed by restriction digestion and sequence analysis. The results of ChIP confirmed that P53 bound to the promoter of NOD8 . The mRNA expression of NOD8 in the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 was stronger than that in the cells transfected with control vector pEGFP-C2 (P<0.05). Furthermore, the mRNA expression of NOD8 was reduced in HEK293 cells transfectnt with the mutant plasmid mp NOD8 (750 bp)-EGFP- NOD8 compared with the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 . Meanwhile, PFT-α inhibited the mRNA expression of NOD8 in the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 in a concentration-dependent manner, and PFT-α at concentration of 90 μmol/L was the most effective in inhibiting NOD8 mRNA expression (P<0.01). As expected, the protein expression of NOD8 in pNOD8 (760 bp)-EGFP- NOD8 group significantly increased compared with that in pNOD8 -C2 group, the protein expression of NOD8 in mp NOD8 (750 bp)-EGFP- NOD8 group or pNOD8 (760 bp)-EGFP- NOD8 + PFT-α group was dramatically decreased compared with that in p NOD8 (760 bp) -EGFP- NOD8 group. CONCLUSION: The results suggest that the P53 binding site is critical for the regulation of NOD8 gene and there is positive feedback regulation between P53 binding site and NOD8 , which may maintain efficient balance between defense and self-inflicted injury in response to the invasion of pathogen.  相似文献   

9.
AIM: To construct the recombination plasmid pcDNA3.1-hERβ with the human estrogen receptor 2 (ESR2) full length cDNA and transfect it into hormone-independent prostate cancer PC-3M cell line, and to study the effects of ESR2 on proliferation in transfected cells. METHODS: The complete cDNA of ESR2 was obtained from human ovary tissue by RT-PCR technique and cloned into eukaryotic expression vector pcDNA3.1 by using gene recombination technique to construct the pcDNA3.1-hERβ recombination plasmid. The plasmid was detected by endonuclease digestion and DNA sequencing and was transfected into PC-3M cells. MTT and FCAS assay were used to test the effects of ESR2 on the ability of proliferation in PC-3M cells. RT-PCR and Western blotting were used to detect the expressions of cyclinD1 and P21Cip1. RESULTS: The results of sequencing and endonuclease digestion demonstrated that the construction of pcDNA3.1-hERβ recombination plasmid was successful. The sequence analysis suggested that the ESR2 sequence detected by PCR was identical to that published in GenBank, and the product of endonuclease was as long as the complete human ESR2 gene. 48 h after transfected the pcDNA3.1-hERβ into PC-3M cells, the expression of ESR2 mRNA and protein levels increased significantly detected by RT-PCR and Western blotting. Compared to the cells transfected with vector as control, the PC-3M cells transfected with pcDNA3.1-hERβ showed that cell population decreased and proliferation activity degraded. FCAS showed that the cells in G0/G1 stage increased and in S stage or G2/M stage decreased. RT-PCR and Western blotting showed that the expression of cyclinD1 gene reduced and expression of P21Cip1 increased. CONCLUSION: The recombination of plasmid pcDNA3.1-hERβ is constructed and transfected into the PC-3M cells successfully. The activity of cell proliferation is inhibited after pcDNA3 transfection.1-hERβ. It is possible that ESR2 inhibits cell proliferation by the expression of proliferation related genes cyclinD1 and P21Cip1.  相似文献   

10.
XIAO Jian-min  FU Hui 《园艺学报》2013,29(10):1788-1792
AIM：To investigate the effects of carvedilol and other β-blockers on ryanodine receptor 2(RyR2)-mediated spontaneous calcium oscillation.
METHODS：HEK293 cells, which steadily and inducibly expressed RyR2, were generated using the Flp-In T-REx Core Kit. Single rat ventricular myocyte was isolated by the method of collagenase. The extracellular Ca2+ concentration was increased step by step to induce calcium oscillation. Single cell calcium imaging was used to test the effects of carvedilol and other β-blockers on RyR2-mediated spontaneous cal-cium oscillation in cardiac cells and HEK293 cells. RESULTS：Carvedilol at concentration of 30 μmol/L obviously suppressed the spontaneous cal-cium oscillation in cardiac cells and HEK293 cells expressing RyR2. The inhibitory rate was (6530±230)% and (6908±530)%, respectively. Metoprolol and other β-blockers had no effects on spontaneous calcium oscillation in cardiac cells and HEK293 cells. CONCLUSION：Carvedilol is the only β-blocker that suppresses RyR2-mediated spontaneous calcium oscillation. This may be one of the mechanisms that carvedilol is better than other β-blockers in reducing the mortality of heart failure.  相似文献   

11.
AIM: To isolate a gene encoding mouse ING4, construct pcDNA3.0-ING4 recombinant eukaryotic expression plasmid and investigate its effects on HeLa cells in vitro. METHODS: The mouse ING4cDNA was amplified by RT-PCR from mouse liver. The eukaryotic expression vector pcDNA3.0-ING4 was constructed by DNA recombination technique. The recombinant plasmid pcDNA3.0-ING4 was identified by PCR, restriction enzyme digestion and DNA sequence analysis, then was transfected into HeLa cells by lipofectamine. The expression was determined by RT-PCR. Apoptosis was detected by fluorescence microscope with Hoechst33258 staining and laser scanning confocal microscope. Cell cycle distribution was measured with flow cytometry. RESULTS: RT-PCR product was about 750 bp specific fragment. Analysis by restricting enzyme digestion and PCR of pcDNA3.0-ING4 recombiant plasmid showed that results were about 750 bp, DNA sequencing revealed that ING4 cloning were successful. With Hoechst fluorescence staining, we found that the percentage of apoptotic rate in HeLa cells transfected with pcDNA3.0- ING4 (21.25%) was higher than that in HeLa cells transfected with pcDNA3.0 (8.91%,P<0.01). Apoptosis was also detected by laser scanning confocal microscope. Cell cycle analysis reavealed the cell number in S phase of HeLa cells transfected with pcDNA3.0- ING4 increased. CONCLUSION: The gene encoding mouse ING4 and construction of pcDNA3.0- ING4 eukaryotic expression vector were successfully obtained, ING4 could enhance apoptosis in HeLa cells.  相似文献   

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14.
AIM:To study the potential of using antisense RNA mediated by expression vector to suppress MIF expression. METHODS:MIF gene was sub-cloned into plasmid pcDNA3 to construct MIF antisense RNA expression vector, pcDNA3-antiMIF, which was identified by restriction enzyme digestion and DNA sequencing. By using lipofectamine 2000, plasmid pcDNA3 and pcDNA3-antiMIF were transfected into MIF expression cells, 293-MIF, separately. 60 h later, the 293-MIF cells were collected and used to determine the MIF mRNA expression by real-time quantitive PCR. Plasmid pcDNA3-antiMIF was transformed into HUVECs, named HUVECs-antiMIF, to express MIF antisense RNA. HUVECs-antiMIF was screened by sulfate G418 and identified by PCR and RT-PCR analysis. Then the MIF expression vector, pSecTag-MIF, was transfected into HUVECs-antiMIF, and the MIF mRNA expression in HUVECs-antiMIF was determined by quantitative PCR. RESULTS:Restriction enzyme digestion and DNA sequencing analysis showed that MIF antisense RNA expression vector, pcDNA3-antiMIF, was constructed correctly. The results of quantitative PCR showed that MIF mRNA expression was suppressed by MIF antisense RNA at level about 32% (P<0.05) in 293-MIF cells. The HUVECs-antiMIF, which expressed MIF antisense RNA, was obtained and identified by PCR and RT-PCR assay. The results of quantitative PCR revealed that MIF mRNA expression was also down-regulated by about 40% (P<0.05) in HUVECs-antiMIF cells. CONCLUSION:MIF expression was suppressed efficiently by MIF antisense RNA mediated by expression vector, and the HUVECs-antiMIF was established to express MIF antisense RNA.  相似文献   

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17.
AIM:To investigate the changes of biological characteristics of GFP transgenic mouse bone marrow mesenchymal stem cells (MSCs), which were transfected with human β-nerve growth factor (β-NGF) gene in a recombinant eukaryotic expression vector. METHODS:MSCs obtained from GFP transgenic mice were isolated, cultured and purified by the whole bone marrow adherence methods. Human β-NGF gene was transfected into the MSCs by a recombinant eukaryotic expression vector. The β-NGF expression in the MSCs was detected by the method of immunocytochemistry. Hippocampal neurons from neonatal mice were cultured with culture supernatant of the MSCs transfected with pcDNA3-β-NGF and the biological characteristics of the MSCs were investigated 3 d after culture under inverted phase-contrast microscope. RESULTS:The β-NGF positive rate of MSCs in pcDNA3-β-NGF transfection group ［(37.12±2.14)%］ was significantly higher than that in MSCs control group ［(2.36±0.62)%］ and blank control group ［(1.43±0.76)%］.The neurite length of neonatal mouse hippocampal neurons cultured with culture supernatant from pcDNA3-β-NGF-transfected MSCs ［(31±3)μm］ was significantly longer than that in negative control group ［(23±4)μm］, suggesting that MSCs transfected with β-NGF gene maintained better biological characteristics. CONCLUSION: The constructed recombinant eukaryotic expression vector of human β-NGF gene can be transfected into MSCs efficiently and NGF can be effectively expressed in MSCs. MSCs transfected with β-NGF gene are capable of stable expression and secretion of β-NGF, and maintenance of better biological characteristics.  相似文献   

18.
HE Wei  ZOU Ping  ZHANG Min 《园艺学报》2005,21(6):1182-1186
AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.  相似文献   

19.
AIM: To investigate the effect of G/A mutation in rs5418 site of solute carrier family 2,facilitated glucose transporter,member 4 protein(SLC2A4) promoter region on gene expression. METHODS: The core promoter region of SLC2A4 gene was amplified by PCR. Mutant and wild-type recombinant expression vectors containing promoter of SLC2A4 gene were constructed by recombinant gene technique and the strategy of site-directed mutagenesis. Recombinant vectors were transfected into HEK293T cells by lipofectamine and the expression activity of the reporter gene in the recombinant expression vectors with different alleles was detected by a dual-luciferase reporter assay system. RESULTS: The 716-bp SLC2A4 promoter was amplified and the recombinant expression vectors pGL3-SLC2A4-prom(A) and pGL3-SLC2A4-prom(G) were successfully constructed. The luciferase reporter vector containing SLC2A4 promoter with rs5418-A alleles produced significantly higher relative luciferase activity (19.49±4.41) than that with rs5418-G allele (13.04±4.45; P<0.05). CONCLUSION: The G→A variation of rs5418 site in SLC2A4 promoter region increases the expression of SLC2A4 gene,thereby affecting the gene function.  相似文献   

20.
AIM: To investigate the effect of Polo-like kinase-1 (Plk1) depletion on cell cycle progression and cell growth in lung cancer cells.METHODS: A recombinant plasmid containing antisense RNA targeting Plk1 (pcDNA3-Plk1) was transfected into A549 cells by lipofectine. RT-PCR and Western blotting were used to examine Plk1 gene expression. Cell proliferation was evaluated by cell counting and BrdU labeling. Cell cycle distribution and apoptosis were examined by flow cytometry. Inhibition rate (IR) of vinorebline (NVB) was determined by MTT assay. RESULTS: After transfected with pcDNA3-Plk1 into A549 cells, the expression levels of Plk1 mRNA and protein were greatly decreased. Abnormal morphological changes of cells and growth inhibition were observed in pcDNA3-Plk1 transfected cells. The BrdU labeling index was significantly lower than that in control group (P<0.05). Cells showed a strong G2/M arrest and apoptosis 72 h post transfection. IR of vinorebline in pcDNA3-Plk1 transfected groups was significantly higher than that in other groups. CONCLUSION: Antisense RNA targeting Plk1 is capable of suppressing Plk1 expression, and therefore, significantly inhibits cellular proliferation, induces cell cycle arrest and apoptosis. Moreover, the sensitivity of lung cancer cells to chemotherapy is increased.  相似文献   

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