Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.     

Construction of recombinant plasmid pEGFP-NGB and its expression in cultured neuroglial cells

AIM: To establish recombination plasmid pEGFP-NGB and to investigate the expression of pEGFP-NGB in culture neuroglia cells. METHODS: The NGB cds was isolated by using RT-PCR method with total RNA extracted from fetal Kunming mouse brain, then the NGB cds was cloned into the eukaryotic expression vector pEGFP-C1 of EGFP reported green fluorescence protein. The expression vector of recombinant plasmid pEGFP-NGB was successfully constructed. GeneJamer transfection reagent was used to transfer recombinant plasmid pEGFP-NGB into culture neuroglial cells. The mRNA and protein expression of pEGFP-NGB in culture neuroglial cells were investigated. RESULTS: The positive clone sequencing was consistent with the sequence of Genbank. The NGB mRNA and protein expression of pEGFP-NGB in culture neuroglial cells were detected at high levels. The high expression of green fluorescence protein was observed by fluorescence microscope in culture neuroglial cells. CONCLUSION: The expression vector of recombinant plasmid pEGFP-NGB was successfully constructed and green fluorescence protein was expressed in cultured neuroglial cells.     

Construction of the vector for fusion protein gene driven by IGF-Ⅱ P3 promoter and its expression in hepatocellular carcinoma cells

AIM:To construct the shuttle plasmid vector for thymidine kinase(tk) and EGFP fusion protein gene driven by IGF-Ⅱ P3 promoter,and investigate the specific killing effect of the HSV-tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro.METHODS:Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening,and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT-PCR. Cell killing after ganciclovir(GCV) application was determined by MTT.RESULTS:Identification of pDC316-tkEGFP-P3 by enzyme digestion and sequencing analysis showed that the length,inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells,but not in HeLa cells. The results of RT-PCR showed that only two bands could be seen in the samples of pDC316-tkEGFP-P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells.CONCLUSION:The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.     

Role of B7-H6 over-expression in NK cell-mediated apoptosis of hepatocytes

AIM: To investigate the role of B7 homologue 6 (B7-H6) over-expression in natural killer (NK) cell-mediated hepatocyte apoptosis. METHODS: The full-length fragment of B7-H6 gene was amplified by PCR and subcloned into linearized eukaryotic expression vector pIRES2-EGFP to construct recombinant B7-H6 over-expression vector pIRES2-EGFP-B7-H6. The recombinant plasmid was identified by double digestion, PCR and sequencing, and was then transfected into L02 cells. The expression of EGFP was observed by fluorescence microscopy and the transfection efficiency was evaluated by flow cytometry. B7-H6 expression was confirmed by qRT-PCR and Western blot. The L02 cells transfected with pIRES2-EGFP-B7-H6 recombinant plasmid were co-cultured with NK-92 cells at different effector/target ratios, and the cytotoxicity of NK-92 cells was evaluated by CCK-8 assay.RESULTS: The strong green fluorescence in the L02 cells was observed under fluorescence microscope 48 h after transfection. The transfection efficiency reached 92.6%. The expression of B7-H6 at mRNA and protein levels was remarkably increased 48 h after transfection. The cytotoxicity of NK-92 cells against L02 cells transfected with pIRES2-EGFP-B7-H6 plasmid was significantly higher than that of the null vector transfection group (P<0.05).CONCLUSION: The recombinant eukaryotic expression vector pIRES2-EGFP-B7-H6 was constructed successfully. The cytotoxic effect of NK-92 cells against L02 cells can be enhanced by transfecting L02 cells with pIRES2-EGFP-B7-H6 plasmid.     

Construction of lentiviral vector carrying the Ang-1 gene and its expression in the rMSCs

AIM: To construct lentiviral vector carrying the angiopoietin-1 (Ang-1) gene,and make it express Ang-1 in the rat mesenchymal stem cells (rMSCs).METHODS: The cDNA encoding the CDS of Ang-1 gene was obtained from the placenta of the adult Fisher 344 rats with RT-PCR.After digestion with restrication endonuclease,the Ang-1 gene was recombined to construct the transfer plasmid PNL-Ang1-IRES2-EGFP.The three-plasmid system of lentiviral vector was consisted of PNL-Ang1-IRES2-EGFP,the packaging plasmid HELPER,and the envelope plasmid VSVG,which were co-transfected to 293T cells mediated by lipofectamin2000 to produce lentiviral particles.The rMSCs were infected by obtained lentiviral particles.The insertion of Ang-1 gene was detected by PCR,the mRNA expression of Ang-1 in rMSCs was detected with RT-PCR,the protein expression of Ang-1 was observed with immunocytochemistry and Western blotting methods.RESULTS: The result of sequencing showed that the cloned Ang-1 gene was consistent with the sequence reported in GenBank.After digestion with restrication endonuclease,the 1 512 bp fragment of Ang-1 gene and the 10.5 kb vector fragment of PNL-IRES2-EGFP were observed with gel electrophoresis.The insertion of Ang-1 gene in viral genome was confirmed.The EGFP expression was observed with the fluorescent microscope.In infected rMSCs,the mRNA and protein expressions of Ang-1 were confirmed.CONCLUSION: Lentiviral vector carrying Ang-1 gene has been successfully constructed.The infected rMSCs are able to express the Ang-1 mRNA and Ang-1 abundantly.This will facilitate the following exploratory development of Ang-1 gene-modified rMSCs.     

Mechanical stretch induces translocation of high-mobility group box 1 protein in THP-1 cells

AIM: To investigate the translocation of high-mobility group box 1 protein(HMGB1) in THP-1 cells induced by mechanical stretch. METHODS: The vector that expressed the fusion protein of HMGB1 and enhanced green fluorescent protein(EGFP) was constructed. The cDNA of HMGB1 and EGFP was subcloned into hemagglutinin(HA)-tagged vector pcDNA3-HA by two-step method. THP-1 cells were transfected with pcDNA3-HMGB1-EGFP and exposed to cyclic mechanical stretch at 20 % elongation using Flexercell 4000T cell stretching unit. The translocation of HMGB1 in THP-1 cells was observed under fluorescence microscope. RESULTS: The recombinant plasmid was verified by enzyme digestion. The green fluorescence accumulated in the nuclei of the cells, indicating that the fusion protein was highly expressed in THP-1 cells and localized in the nuclei. Eighteen hours after mechanical stretch, the green fluorescence was observed in the cytoplasm. At the same time, the green fluorescence was still localized in the nuclei of the control cells treated without mechanical stretch. CONCLUSION: The HMGB1-EGFP fusion protein is successfully and effectively expressed in THP-1 cells. Mechanical stretch induces the translocation of HMGB1 protein from nucleus to cytoplasm.     

Adenoviral-mediated high efficiency expression of enhanced green fluorescence protein gene in ex vivo expanded rat mesenchymal stem cells

AIM:To investigate the feasibility and infection efficiency of MSCs with replication-deficient adenovirus containing delivered gene, and whether enhanced green fluorescence protein (EGFP) gene track the change during rMSCs differentiating neuron-like cells. METHODS: Rat marrow mesenchymal stem cells (rMSCs) were expanded in low density in vitro. Under the control of CMV promoter, pAd-EGFP-Vector was constructed by homologous recombination in E.coil BJ 5183, and the recombinant virus was produced in HEK 293 packaging cell line. rMSCs infected with Ad-EGFP were observed and analyzed with fluorescence microscope. Infection efficiency was assessed by microscopical scoring and flow cytometrics. After withdrawing serum and exposure to β-mercaptoethanol medium, rMSCs infected with Ad-EGFP was induced to differentiate into neuron-like cells. As a control, the plasmid of pTrack-EGFP also was transfected into rMSCs to evaluate transfection efficiency. RESULTS:The results showed that Adenovirus vector (AdVec) delivered EGFP gene with high efficiency to marrow mesenchymal stem cells. Gene expression analysis showed that 36%±2 % of rMSCs infected with recombinant adenovirus expressed the transgene of EGFP at high levels. However, the transfection of plasmid pTrack-EGFP using routine method of lipofectamin mixed with plasmid DNA (pTrack-EGFP) was not easily successful and the transfection efficiency was much lower. rMSCs infected with Ad-EGFP in different passage could differentiate into typical morphology alike neural cells after withdrawing serum and exposure to β-mercaptoethanol medium. Immuno-staining with neuron-specific enolase (NSE), a neuronal marker, was strong positive, which suggested that rMSCs infected with Ad-EGFP had the potential to differentiate into neurons or neuron-like cells. CONCLUSION:The AdVec system can deliver target gene into MSCs and EGFP gene carried by AdVec can track the change during rMSCs differentiating into neuron-like cells.     

Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro

AIM:To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro.METHODS:BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo.RESULTS:We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION:Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.     

Molecular cloning of ING4 gene and ING4-induced apoptosis in HeLa cells

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.     

Construction of pNTAP-PRAK eukaryotic expression plasmid and its expression in HEK293 cells

AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification (TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK, then DH5α E.coli was transformed with the recombinant plasmid. After identified by PCR, digestion with restriction endonuclease and sequencing, the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didnt influence the localization of exogenous PRAK.     

Expression of fusion protein anti-HER2-ScFv-GFP in insect cells and binding to surface of breast cancer cells

AIM: To express a recombinant fusion protein anti-human epidermal growth factor receptor-2 single-chain variable fragment with green fluorescent protein (anti-HER2-ScFv-GFP) using the insect cells-Bac-to-Bac baculovirus expression system and to analyze the binding function of this fusion protein with HER2 on the surface of the breast cancer cells. METHODS: Human anti-HER2-ScFv gene from mice was fused with GFP gene. To obtain the recombinant plasmid pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP, we inserted it into Bac-to-Bac baculovirus expression plasmid pFAST Bac-to-Bac HT A. The identified recombinant plasmid was transferred into Escherichia coli DH10Bac to allow the generation of a recombinant bacmid. After transfected the recombinant virus bacmid into the insect cells Tn-5B1-4, the recombinant virus was collected to infect Tn-5B1-4. SDS-PAGE and Western blotting analysis were used to verify the expression product in Tn-5B1-4. The fusion protein was purified with Ni²⁺-NTA affinity chromatography. The purified fusion protein was bound to the surface of HER2-positive breast cancer cells SKBR3 and HER2-negative breast cancer cells MCF7. The binding effects on the surface of breast cancer cells were observed under laser confocal microscope. RESULTS: The fusion gene anti-HER2-ScFv-GFP was successfully constructed with the length of 1 539 bp. The green fluorescence was also observed in Tn-5B1-4 cells infected with the recombinant virus under fluorescent microscope. A 60 kD protein was examined and confirmed by SDS-PAGE and Western blotting. Under laser confocal microscope, strong green fluorescence was observed on the surface of the HER2-positive breast cancer cells SKBR3. However, no green fluorescence was observed on the surface of HER2-negative breast cancer cell MCF7. Obvious green fluorescence on the surface of HER2-positive breast cancer cell SKBR3 was also found after the cells were eluted with 1×PBS. CONCLUSION: The fusion protein anti-HER2-ScFv-GFP was successfully expressed in insect cells Tn-5B1-4, and can firmly bind to the surface of breast cancer cells SKBR3 and emit the green fluorescent light.     

Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene

AIM: To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein ( RFP ) reporter gene with the approach of in vitro recombinant ligation. METHODS: The RFP gene fragment of pTurboRFP-N was digested and ligated into pShuttle transfer vector to construct recombinant vector pShuttle-TurboRFP-N. I- Ceu I/PI- Sce I were used to double digest recombinant vector pShuttle-TurboRFP-N and backbone of vector pH5'040.pkGFP-II. The target fragment was collected and ligated, and recombinant adenovirus vector AdH5'.040.CMV.RFP-N was obtained. After linearization, the vector was transfected into AD293 cells by liposome for virus packaging. The efficiency of virus packaging and RFP expression level in AD293 cells were examined using fluorescent microscope. In addition, the biological activity and titer of the virus were tested. Human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were infected with recombinant adenovirus vector AdH5'.040.CMV.RFP-N and control adenovirus vector AdH5.CMV.EGFP respectively. The infection efficiencies of the 2 vectors to different cell lines were compared by evaluating the expression levels of RFP and enhanced green fluorescent protein (EGFP). RESULTS: The recombinant adenovirus vector AdH5'.040.CMV.RFP-N was correctly constructed and confirmed by enzyme digestion. The virus was packaged by the vector in AD293 cells and had the ability to infect the target cells. The target gene in eukaryotic cells was also expressed. The number of recombinant adenoviruses and the titer of the virus after amplification and purification were 3.6×10¹⁵ vp/L and 1×10¹³ pfu/L,respectively. The infection efficiencies of recombinant adenovirus vector Ad5'.040.CMV.RFP-N to human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were higher than those in control adenovirus vector AdH5.CMV.EGFP (P<0.05). CONCLUSION: We have constructed recombinant virus vector carrying RFP reporter gene and provide an important tool for gene therapy and gene vaccine research. The reporter gene can be highly expressed in AD293 cells and has high infection efficiency to cancer cells. RFP is a good substitution and supplement to green fluorescent protein.     

Construction of recombinant plasmid pEGFP-HSP70 and its expression in rat neural stem cells

AIM：To explore the method of constructing recombinant plasmid pEGFP-HSP70 and its expression in neural stem cells. METHODS：Total RNA was acquired from the fetal liver tissue of SD rat. cDNA complete sequence of heat-shock protein 70 (HSP70) gene was amplified by RT-PCR, and cloned into an eukaryotic expression vector containing the enhanced green fluorescent protein (EGFP) reporter gene, pEGFP-C2. Sequencing analysis was performed to confirm the recombinant plasmid pEGFP-HSP70. The technique of nucleofector transfection was used to transfect the recombinant plasmid pEGFP-HSP70 into neural stem cells. RESULTS：HSP70 cDNA sequence was correctly cloned into the eukaryotic expression vector pEGFP-C2. The recombinant plasmid pEGFP-HSP70 was constructed successfully. Compared with control group, the fluorescence intensities in pEGFP-C2 group and pEGFP-HSP70 group were significantly increased. The fluorescence intensity in pEGFP-HSP70 group after 24 h of transfection was significantly decreased compared with other time points of 7 d, 14 d and 21 d. The expression level of HSP70 significantly increased 1 d, 7 d and 14 d after transfection compared with control group. CONCLUSION：The neural stem cells can be directly used as gene action target cells. The HSP70 expression level in the stem cells is closely related to the time after transfection.     

Construction of human anti-HBc ScFv eukaryotic expression vector and expression of anti-HBc ScFv in HepG2 cells

AIM: To construct an eukaryotic expression vector of human single-chain variable fragment against hepatitis B virus core protein (anti-HBc ScFv) and detect its expression in HepG2 cells. METHODS: Anti-HBc ScFv genes were amplified from the plasmids abstracted from positive clone and inserted into pEGFP-c1 vector that contained green fluorescent protein gene. The recombinant plasmids were transfected into HepG2 cells, and resistant clones were obtained by G418 selection. The expression of the gene of fusion protein was determined by fluorescent invert microscope and ELISA. RESULTS: Recombinant plasmids were successfully constructed. The plasmid transfected HepG2 cells were obtained by G418 selection. Specific fluorescence was observed in HepG2 cells 48 hours after transfection. ELISA analysis confirmed the expression of anti-HBc ScFv in the cells. CONCLUSION: The construction of human anti-HBc ScFv eukaryotic expression vector and its expression in HepG2 cells lay the foundation for advanced research of intracellular anti-HBc ScFv.     

Subcellular localization of ZNF580-EGFP fusion protein

AIM: To construct a eukaryotic expression plasmid for enhanced green fluorescent protein (EGFP) and ZNF580 fusion protein, and study its subcellular localization in the transfected MGC803 cells. METHODS: The primers were designed according to the cDNA encoding sequence of ZNF580 full-length open reading frame (1-172aa), ZNF580 amino terminus (1-93aa) and ZNF580 carboxyl terminus (94-172aa). The three cDNA segments of PCR were cloned into pGEM-T vector. Then they were subcloned respectively into plasmid pEGFP-C1 (enhanced green fluorescent protein). The subcellular localization of the fusion protein in MGC803 cells transfected with the vector was monitored by autofluorescence microscopy. RESULTS: Restricted enzymes analysis and DNA sequencing showed that the sequences of the pEGFP-ZNF580 (1-172), pEGFP-ZNF580 (1-93) and pEGFP-ZNF580 (94-172) transgenic plasmid were correct. The fusion proteins of EGFP-ZNF580 (1-172) and pEGFP-ZNF580 (94-172) were localized in the nuclei. CONCLUSION: The recombinant eukaryotic expression vector pEGFP-ZNF580 has been successfully constructed. The nuclear localization signal is within amino acid residues 94 and 172 of ZNF580 carboxyl terminus (C2H2 zinc finger domain).     

The value of intrapericardial injection with a trans-diaphragmatic approach in the gene therapy of chronic heart failure rats

AIM: To study the feasibility, security and validity of the intrapericardial injection with a trans-diaphragmatic approach in chronic heart failure rats and its value in the study of gene therapy for heart diseases, and further investigate whether adeno-associated virual gene transfer of sarcoplasmic reticulum Ca2+-ATPase （SERCA2a） gene can improve ventricular function in chronic heart failure (HF) rats. METHODS: An animal model of heart failure was obtained by creating descending aortic constriction in rats. Recombinant adeno-associated virus, carrying enhanced green fluorescent protein (EGFP) gene and recombinant adeno-associated virus carrying SERCA2a gene, were respectively injected into pericardium of heart failure rats in different groups (group HF+EGFP and group HF+SERCA2a) by intrapericardial injection with a trans-diaphragmatic approach. After 30 days, hemodynamic parameters were measured and analyzed. Cryosection was analyzed by fluorescence microscopy to examine the expression of green fluorescent protein, and Western blotting was performed to detect the expression of SERCA2a. RESULTS: Green fluorescence was detected in cryosection of the hearts in all rats in group HF+EGFP and the expression of green fluorescent protein was ubiquitously. The expression of SERCA2a in all rats in group HF+SERCA2a was more than those in group HF and group HF+EGFP. And overexpression of SERCA2a improved the systolic and diastolic function of heart failure rats significantly and the hemodynamic parameters were similar with those of the controls. CONCLUSION: The intrapericardial injection with a trans-diaphragmatic approach suggests a simple, safe, efficient and cheap technique for the gene therapy of chronic heart failure. Gene thransfer of SERCA2a may be a new approach for the treatment of chronic heart failure.     

Effect of adipose differentiation-related protein on proliferation and apoptosis in H9c2 cells

AIM: To construct an adipose differentiation-related protein (ADRP) eukaryotic expression vector and to explore the effect of ADRP on apoptosis of H9c2 cells induced by palmitic acid (PA). METHODS: The ADRP gene obtained by the method of RT-PCR was cloned into pEGFP-C1 plasmid. The recombinant plasmid was transformed into E.coli DH5α for amplification. The recombinant plasmid was extracted from E.coli DH5α and transfected into H9c2 cells by Lipofectamine^TM2000. The stable transformants were selected by G418 screening. Expression of green fluorescent protein was observed under fluorescence microscope and the ADRP expression was identified by RT-qPCR and Western blotting analysis. The effect of PA on the proliferation of H9c2 cells was detected by MTT assay. The apoptotic percentage of H9c2 cells caused by PA was determined by flow cytometry. RESULTS: The eukaryotic expression vector pEGFP-C1-ADRP was successfully constructed. Green fluorescent was observed in the cells transfected with pEGFP-C1 or pEGFP-C1-ADRP under fluorescence microscope. RT-qPCR and Western blotting analysis showed that recombinant cells exhibited high mRNA and protein levels of ADRP. After treated with PA at different concentrations, the apoptosis rates and the proliferation inhibition of recombinant cells were both lower than those of the other two cells. CONCLUSION: The transfected H9c2 cells with stable ADRP expression were successfully established. The over-expression of ADRP prevents the cells from apoptosis and inhibition of proliferation caused by PA, indicating that ADRP plays a protective role in H9c2 cells.     

VEGF gene expression in norepinephrine/ burn serum-induced rat astrocytes

AIM: To investigate the changes of gene expression of vascular endothelial growth factor (VEGF) in norepinephrine/ burn serum-induced astrocytes. METHODS: Immunofluorescence staining was used to show the distribution of VEGF in astrocytes after 24 h using norepinephrine/burn serum stimulation. Western blotting was used to detect protein expression of VEGF. Real time PCR was used to investigate expression of VEGF mRNA. RESULTS: ① Green fluorescence of protein expression of VEGF in astrocytes was increased when treated with high dose norepinephrine (50 μmol/L). Green fluorescence of protein expression of VEGF in astrocytes was increased distinctness after burn serum stimulation. Green fluorescence protein expression of VEGF in astrocytes was increased significantly when high dose norepinephrine combined with burn serum stimulation was added. ② VEGF protein expression in burn serum stimulating group was increased, and VEGF protein expression was significantly increased when burn serum was added during moderate (20 μmol/L), high dose norepinephrine stimulation. ③ Expression of VEGF mRNA was increased in burn serum-treated astrocytes. Expression of VEGF mRNA was increased significantly when norepinephrine-stimulated astrocytes exposed to barn serum, and as norepinephrine dose increases gradually. CONCLUSION: Norepinephrine and burn serum play an important role in inducing VEGF protein expression in astrocytes, suggesting that stress reaction of postburn is an important cause in inducing brain edema by excreting VEGF in astrocytes.     

Effects of up-regulation of c-myc expression on U937 cell line

AIM:To establish a human monocytic　leukemia cell line U937 stably expressing c-myc gene and to investigate the biological characteristics of this cell line. METHODS:The recombinant plasmid MSCV-c-myc-IRES-GFP (MMIG) was constructed. MMIG and MSCV-IRES-GFP (MIG) were used to package the viruses for infecting U937 cells. Fluorescence-activated cell sorter (FACS) was used for sorting U937/GFP and U937/MYC cells. The GFP-positive cells were detected by fluorescence microscopy and FACS. The protein expression of c-Myc, survivin, X-linked inhibitor of apoptosis protein (XIAP) and Bcl-2 was detected by Western blotting. Cell proliferation was evaluated by MTT assay. Propidium iodide (PI) staining was used to determine the cell cycle distribution. Self-renewal ability was observed by colony- forming assay. RESULTS:The GFP expression in the cells infected with MIG or MMIG virus was observed under fluorescence microscope. The green fluorescent rate of the cells infected with MIG was 26.0%, while that of the cells infected with MMIG was 27.7%. The protein expression of c-Myc in MMIG-infected U937 cells was higher than that in MIG-infected cells. After sorting, the green fluorescent rates of U937/GFP and U937/MYC cells reached 98.7% and 93.7%, respectively. The protein expression of c-Myc in U937/MYC cells was higher than that in U937/GFP cells. In addition, survivin, a downstream protein of c-Myc, was up-regulated, while the protein expression of XIAP and Bcl-2 remained unchanged. Cell cycle analysis showed that the percentage of the cells in S phase increased in U937/MYC cells. Moreover, the proliferation and colony-forming ability of U937/MYC cells were also enhanced. CONCLUSION: U937/MYC cell line stably expressing c-myc gene was successfully established. c-Myc may increase cell viability via enhancing the expression of anti-apoptotic protein survivin, the cell cycle transition and the self-renewal ability.