Background Previous studies showed that overexpression of sarco-endoplasmic retieulum calcium ATPase （SERCA2a） in a variety of heart failure （HF） models was associated with greatly enhanced cardiac performance. Ho...Background Previous studies showed that overexpression of sarco-endoplasmic retieulum calcium ATPase （SERCA2a） in a variety of heart failure （HF） models was associated with greatly enhanced cardiac performance. However, it still undefined the effect of SERCA2a overexpression on the systemic inflammatory response and neuro-hormonal factors. Methods A rapid right ventricular pacing model of experimental HF was used in beagles. Then the animals underwent recombinant adeno-associated vires 1 （rAAV1） mediated gene trans- fection by direct intra-myocardium injection. HF animals were randomized to receive the SERCA2a gene, enhanced green fluorescent protein （control） gene, or equivalent phosphate buffered saline. Thirty days after gene delivery, the cardiac function was evaluated by echocardiographic testing. The protein level of SERCA2a was measured by western blotting. The proteomic analysis of left ventricular （LV） sample was determined using two-dimensional （2-D） gel el^ctrophoresis and MALDI-TOF-MS. The serum levels of the systemic inflammatory and neuro-hormonal factors were assayed using radioimmunoassay kits. Results The cardiac function improved after SERCA- 2a gene transfer due to the significantly increased SERCA2a protein level. Beagles treated with SERCA2a had significantly decreased serum levels of the inflammatory markers （interleukin-6 and tumor necrosis factor-a） and neuro-hormonal factors （brain natriuretic peptide, endothelin-1 and angiotensin II） compared with HF animals. The myocardial proteomic analysis showed that haptoglobin heavy chain, heat shock protein （alpha-crystallin-related, B6） were down-regulated, and galectin-1 was up-regulated in SERCA2a group compared with HF group, companied by up-regulated contractile proteins and NADH dehydrogenase. Conclusions These findings demonstrate that regional intramyocardial injections of rAAV 1-SERCA2a vectors may improve global LV function, correlating with reverse activation of the systemic inflammatory, excessive neuroendocrine factors and the stress-associated myocardial proteins, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenuation of stress-associated reaction.展开更多
Objective: To establish a sensitive and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p...Objective: To establish a sensitive and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H2O2 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H2O2, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP. hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.展开更多
Objective： To construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide（LPS） in vitro. Methods： pShutt...Objective： To construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide（LPS） in vitro. Methods： pShuttleHl-siCas3 containing Oligo DNA of the targeting sequences and pEGFPC1-Cas3 containing caspase-3 and EGFP sequences were constructed respectively, pShuttleH 1-siCas3 and pEGFPC 1-Cas3 were co-transfected to the 293 cells by liposomes to determine interfering efficacy by flow cytometry, pShuttleHl-siCas3 was linearized and transformed into E. coli B J5183 cells containing backbone plasmid pAdEasy-1. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siCas3. The titers of adenovirus were determined by the specific 50% tissue culture infection dosage method. After virus infected the cultured hippocampus neurons, LPS-induced apoptosis and caspase-3 mRNA expression were observed. Results： It was identified that the sequence of target gene was correctly inserted into the genome of virus. The expression of green fluorescence protein was reduced by pShuttleHl-siCas3 in 293 cells. The titer of recombinant adenovirus was 1.06×10^10pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed. Conclusion： The recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.展开更多
Objective： To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cell...Objective： To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cells. Methods： A cloning and expression vector, pEGFP-NI-lac, was constructed by inserting the prokaryotic lac promoter of pUC 19 into the eukaryotic expression vector, pEGFP-N1, between the eukaryotic PCMV promoter and enhanced green fluorescent protein （EGFP） open reading frames. To assess the function of pEGFP-NI-lac, the nucleotide sequence encoding the hepatitis C virus （HCV） core protein was cloned into the multiple cloning sites. Western blotting analysis was used to detect the expression of the HCV core protein in Escherichia coli DH5a and HepG2 cells. Results： Restriction enzyme digestion and sequence analysis indicated that pEGFP-NI-lac was successfully constructed and the HCV core gene was cloned into this vector. The Western blotting results showed that pEGFP-NI-lac promoted expression of HCV core gene in prokaryotic E. coli DH5a and eukaryotic HepG2 cells. Conclusion： The pEGFP-NI-lac vector has been successfully constructed and functions in both prokaryotic and eukaryotic cells. The EGFP reporter can be used as an insert-inactivation marker for clone selection or as an expression tag. This vector can be used for cloning and expression of genes in both prokaryotic and eukaryotic cells, making gene cloning, expression and functional studies convenient as well as time- and labor-efficient展开更多
基金grants from the National Nature Science Foundation,China
文摘Background Previous studies showed that overexpression of sarco-endoplasmic retieulum calcium ATPase （SERCA2a） in a variety of heart failure （HF） models was associated with greatly enhanced cardiac performance. However, it still undefined the effect of SERCA2a overexpression on the systemic inflammatory response and neuro-hormonal factors. Methods A rapid right ventricular pacing model of experimental HF was used in beagles. Then the animals underwent recombinant adeno-associated vires 1 （rAAV1） mediated gene trans- fection by direct intra-myocardium injection. HF animals were randomized to receive the SERCA2a gene, enhanced green fluorescent protein （control） gene, or equivalent phosphate buffered saline. Thirty days after gene delivery, the cardiac function was evaluated by echocardiographic testing. The protein level of SERCA2a was measured by western blotting. The proteomic analysis of left ventricular （LV） sample was determined using two-dimensional （2-D） gel el^ctrophoresis and MALDI-TOF-MS. The serum levels of the systemic inflammatory and neuro-hormonal factors were assayed using radioimmunoassay kits. Results The cardiac function improved after SERCA- 2a gene transfer due to the significantly increased SERCA2a protein level. Beagles treated with SERCA2a had significantly decreased serum levels of the inflammatory markers （interleukin-6 and tumor necrosis factor-a） and neuro-hormonal factors （brain natriuretic peptide, endothelin-1 and angiotensin II） compared with HF animals. The myocardial proteomic analysis showed that haptoglobin heavy chain, heat shock protein （alpha-crystallin-related, B6） were down-regulated, and galectin-1 was up-regulated in SERCA2a group compared with HF group, companied by up-regulated contractile proteins and NADH dehydrogenase. Conclusions These findings demonstrate that regional intramyocardial injections of rAAV 1-SERCA2a vectors may improve global LV function, correlating with reverse activation of the systemic inflammatory, excessive neuroendocrine factors and the stress-associated myocardial proteins, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenuation of stress-associated reaction.
文摘Objective: To establish a sensitive and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H2O2 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H2O2, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP. hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.
文摘Objective： To construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide（LPS） in vitro. Methods： pShuttleHl-siCas3 containing Oligo DNA of the targeting sequences and pEGFPC1-Cas3 containing caspase-3 and EGFP sequences were constructed respectively, pShuttleH 1-siCas3 and pEGFPC 1-Cas3 were co-transfected to the 293 cells by liposomes to determine interfering efficacy by flow cytometry, pShuttleHl-siCas3 was linearized and transformed into E. coli B J5183 cells containing backbone plasmid pAdEasy-1. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siCas3. The titers of adenovirus were determined by the specific 50% tissue culture infection dosage method. After virus infected the cultured hippocampus neurons, LPS-induced apoptosis and caspase-3 mRNA expression were observed. Results： It was identified that the sequence of target gene was correctly inserted into the genome of virus. The expression of green fluorescence protein was reduced by pShuttleHl-siCas3 in 293 cells. The titer of recombinant adenovirus was 1.06×10^10pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed. Conclusion： The recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.
基金Supported by the National High Technology Research and Development Program of China (863 Program, 2009AA02Z111)the National Natural Science Foundation of China (30872223)the Funds of the State Key Laboratory of Pathogen and Biosecurity
文摘Objective： To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cells. Methods： A cloning and expression vector, pEGFP-NI-lac, was constructed by inserting the prokaryotic lac promoter of pUC 19 into the eukaryotic expression vector, pEGFP-N1, between the eukaryotic PCMV promoter and enhanced green fluorescent protein （EGFP） open reading frames. To assess the function of pEGFP-NI-lac, the nucleotide sequence encoding the hepatitis C virus （HCV） core protein was cloned into the multiple cloning sites. Western blotting analysis was used to detect the expression of the HCV core protein in Escherichia coli DH5a and HepG2 cells. Results： Restriction enzyme digestion and sequence analysis indicated that pEGFP-NI-lac was successfully constructed and the HCV core gene was cloned into this vector. The Western blotting results showed that pEGFP-NI-lac promoted expression of HCV core gene in prokaryotic E. coli DH5a and eukaryotic HepG2 cells. Conclusion： The pEGFP-NI-lac vector has been successfully constructed and functions in both prokaryotic and eukaryotic cells. The EGFP reporter can be used as an insert-inactivation marker for clone selection or as an expression tag. This vector can be used for cloning and expression of genes in both prokaryotic and eukaryotic cells, making gene cloning, expression and functional studies convenient as well as time- and labor-efficient
