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Objective To construct a recombinant adenovirus vector expressing mouse SPINK5 gene,and observe its curative effect on the skin lesions in atopic dermatitis mice model. Methods By recombining DNA technology,the sequence of mouse SPINK5 gene was cloned into adeno?virus shuttle plasmid. Then it was transformed into HEK 293 cells with the adenoviral backbone plasmid to obtain the recombinant adenovirus. A mouse model of atopic dermatitis was established by system and local sensitization of Balb/c mice with ovalbumin . The effect of recombinant adeno?virus on the lesions of atopic dermatitis mice model was observed. Results The SPINK5 over?expressing adenovirus vector and atopic dermatitis mice model were successfully constructed. After 2 weeks of adenovirus?mediated SPINK5 gene intracutaneous injection,the redness and edema of lesions of AD model mice were obvious relieved. The pathological detection indicated that epidermal thickness and prickle cell layer ,inflammatory cell infiltration significant decreased accompanied with the model blank control. Conclusion The adenovirus?mediated SPINK5 gene had signifi?cant therapeutic effect to the atopic dermatitis mice model ,which provided a laboratory basis of application of SPINK5 gene product to therapy atopic dermatitis.
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Objective To construct an adenovirus vector expressing small interfering RNA ( siRNA) targeting to rat Raf-1 gene and identify its function in cardiomyocytes.Methods The siRNA containing DNA sequence targeting to Raf-1 and its negative control sequence were designed, synthesized, annealed and subcloned into adenoviral shuttle vector pAdTrack-CMV.The recombinant adenovirus vector pAd-siRaf-1 was obtained by homologous recombination with pAdTrack-siRaf-1 linearized by PmeI and pAdeasy-1 in bacteria BJ5183, then transfected into HEK293 cells to package the adenovirus.Cardiomyocytes were infected with the adenovirus pAd-siRaf-1, and the expressions of Raf-1 and NF-κB protein were detected by Western blotting.[ 3 H ]-leu incorporation was evaluated by scintillation.The surface area of cardiomyocytes was measured using a HJ2000 image analysis system.Results The adenovirus vectors were verified by enzyme digestion and DNA sequencing.Compared with the Ang II group, Raf-1 and NF-κB expression, the surface area and [ 3 H]-leu incorporation of cardiomyocytes were significantly decreased in cardiomyocytes infected with the adenovirus PAd-siRaf-1.Conclusions A recombinant adenovirus vector containing rat siRaf-1 gene is successfully constructed.It can effectively reduce Raf-1 and NF-κB expression and cardiomyocyte hypertrophy induced by Ang II.
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@#Objective To construct recombinant adenovirus vector carrying the human HSP75 gene and detect its expression in C17.2 neural stem cells. Methods HSP75 gene was amplified from plasmid carrying the human HSP75 gene and inserted to the polyclonal site of adenovirus shuttle plasmid pHBAd-MCMV-GFP. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pHBAd-MCMV-HSP75-GFP and large adenovirus helper plasmid pHBAd-BHGloxΔE1,3 Cre in mediation of LipofiterTM. The recombinant adenovirus was obtained and the viral titer was examined using the method of TCID50. The transfection and expression of HSP75 was detected by fluorescence microscope, flow cytometer and Western blotting. Results Restriction digestion, sequencing analysis and PCR amplification revealed the successful construction of recombinant shuttle plasmid and recombinant adenovirus. The titer of recombinant adenovirus was 1.0×1010 PFU/ml. Western blotting indicated HSP75 gene could be expressed effectively in neural stem cells after transfection. Conclusion The recombinant adenovirus vector carrying HSP75 gene was successfully constructed and can be expressed after transfected in C17.2 neural stem cells.
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OBJECTIVE:To explore the effects of recombinant adenovirus vector Adxsi-GFP-VP3 carrying apoptin gene VP3 on the apoptosis of human lung squamous carcinoma SK-MES-1 cell lines and human lung adenocarcinoma NCI-H1299 cell lines. METHODS:The exponential phase SK-MES-1 and NCI-H1299 cell lines were respectively divided into a recombinant adenovirus (Adxsi-GFP-VP3) group,a empty virus (Adxsi-GFP) group and a cell control (phosphate buffer) group,which were marked as group A,B and C respectively. Reverse transcription polymerase chain reaction and Western blot method were used to detect the ex-pressions of VP3 mRNA and Apoptin in the cells of groups A and B 48 and 72 h after transfection. The change in the ultrastructure of the cells in group A was observed under transmission electron microscope 72 h thereafter. MTT method was adopted to detect the cell proliferation activities of three groups 24,48,72 and 96 h thereafter and flow cytometry to determine the apoptosis rates and cell cycle changes 24,48 and 72 h thereafter. RESULTS:Compared to group B,group A demonstrated the expression of VP3 mRNA in SK-MES-1 and NCI-H1299 cell lines 48 h after transfection,and Apoptin expression and ultrastructure change for apopto-sis of SK-MES-1 and NCI-H1299 cell lines 72 h thereafter. Compared to groups B and C,group A showed lower proliferation activ-ities and higher apoptosis rates of SK-MES-1 and NCI-H1299 cell lines,which had a positive correlation with transfection time;and in the group A,there was a decrease in the proportion of the SK-MES-1 and NCI-H1299 cell lines in S phase and an increase in the proportion of those in G2/M phase,72 h after transfection. There was statistically difference (P<0.05). CONCLUSIONS:Adxsi-GFP-VP3 can effectively induce the apoptosis of SK-MES-1 and NCI-H1299 cell lines.
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Objective To investigate the functions of triple point-mutants of hypoxia-inducible factor 1α(HIF1α)in angiogenesis in bone defect region under normoxic conditions.Methods Triple point-mutations (the 402,564 and 803 amino acids)in HIF1α coding sequence (CDS)were induced.The triple mutant of HIF1α(402/564/803)was inserted into the adenovirus pAdEasy-1 system to complete viral packaging and titer measurements. The wild-type HIF1α gene and the empty adenovirus vector were packaged in the same way.For the in vitro experiment,the rabbit bone marrow mesenchymal stem cells (MSCs)were divided into four experimental groups:A,MSCs infected with viral solution containing mutant HIF1α;B,MSCs infected with viral solution containing wild-type HIF1α;C,MSCs infected with viral solution without any HIF1α;and D,MSCs without viral infection. The efficiency of infection was observed by the expression of human renilla reniformis green fluorescent protein (hrGFP).The expression levels of HIF1α mRNA and protein in infected cells in each experimental group were measured.For the in vivo experiment,the MSCs were divided into the same four groups and infected with the virus solutions from each group and cultured under normoxic conditions.At 72h after the infection,the MSCs were used as seed cells and transplanted into Apatite-wollastonite magnetic bioactive glass-ceramic (AW MGC)vector to construct artificial tissue-engineering scaffolds,and then the scaffolds were implanted into the in vivo rabbit radial bone defect model.The animals from each group were sacrificed 8 weeks after the surgery and the tissues from the implantation region were harvested for evaluation of angiogenesis.Results The 402,564 and 803 amino acids in CDS area were point mutated into alanine;three types of recombinant adenovirus were successfully constructed, packaged and characterized.The expression levels of HIF1αmRNA were significantly higher in Group A and Group B than in Group C and Group D (P 0.05 ).The HIF1α protein expression in Group A was significantly higher than that in the other three groups (P 0.05).There was prominent angiogenesis in bone defect region in Group A,but not in the other three groups.Conclusion ① Triple point-mutants of HIF1αefficiently expressed functional proteins under normoxic conditions.② Triple point-mutants of HIF1αeffectively promoted in vivo angiogenesis in bone defect region.
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Objective To construct recombinant adenovirus vector carrying the human HSP75 gene and detect its expression in C17.2 neural stem cells. Methods HSP75 gene was amplified from plasmid carrying the human HSP75 gene and inserted to the polyclonal site of adenovirus shuttle plasmid pHBAd-MCMV-GFP. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pH-BAd-MCMV-HSP75-GFP and large adenovirus helper plasmid pHBAd-BHGloxΔE1,3 Cre in mediation of LipofiterTM. The recombinant ad-enovirus was obtained and the viral titer was examined using the method of TCID50. The transfection and expression of HSP75 was detect-ed by fluorescence microscope, flow cytometer and Western blotting. Results Restriction digestion, sequencing analysis and PCR amplifica-tion revealed the successful construction of recombinant shuttle plasmid and recombinant adenovirus. The titer of recombinant adenovirus was 1.0×1010 PFU/ml. Western blotting indicated HSP75 gene could be expressed effectively in neural stem cells after transfection. Conclu-sion The recombinant adenovirus vector carrying HSP75 gene was successfully constructed and can be expressed after transfected in C17.2 neural stem cells.
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Objective To construct the recombinant adenovirus vector containing ubiquinol-cytochrome C reductase core protein 1(UQCRC1) and to investigate the protective role of UQCRC1 against hypoxia/reoxygenation injury in H9c2 cardiac myocytes . Methods UQCRC1 gene was obtained from the cDNA library by PCR ,then was double-digested with restriction endonucleases SalⅠand XbaⅠand inserted into pAd Track-CMV .The identified plasmid of pAd Track-UQCRC1 was transfected into BJ 5183 contai-ning pAdEasy-1 .After screening the positive clone ,the plasmid was transfect into 293T cells with liposome to integrate and package the recombinant adenovirus .Finally ,these adenoviruses were transfected into H9c2 cardiac myocytes .The expressions of green fluo-rescence protein(GFP) ,UQCRC1 gene and protein were observed by RT-PCR and Western blot analysis .The cell viability and the LDH release were detect .Results The recombinant adenovirus-UQCRC1 was constructed successfully .The overexpression of UQCRC1 increased the cell viability(P<0 .05) and decreased the LDH release(P<0 .05) from H9C2 cardiac myocytes after suf-fering hypoxia/reoxygenation injury .Conclusion UQCRC1 has the protective effect on hypoxia/reoxygenation injury in H9c2 car-diac myocytes ,and the construction of recombinant adenovirus vector will lay the foundation for further studying the role of UQCRC1 in cardioprotection .
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Objective To probe the effects of recombinant adenovirus containing Akt on carbon tetrachioride-induced rat liver cirrhosis and portal hypertension. Methods Cirrhosis was induced in rats by a complex method of carbon tetrachloride. Recombinant adenovirus Ad-myr-HA-Akt was produced by homologous recombination in 293 cells. Rats received Ad-myr-HA-Akt via the tail vein at the second and the sixth week respectively. The pathological changes in liver tissues were observed after Van Gieson (VG) staining. Fas antigen in rat livers were determined by immunohistochemical method. The levels of alanine minotransferase( ALT), aspartate aminotransferase ( AST), albumin( ALB ) and hydroxyproline (Hyp) were measured. Fas antigen in rat livers were determined with immunohistochemical method. Expression of Akt, p-Akt, Fas and DR5 were evaluated by Western blotting. Frozen sections of the liver, heart,lung,kidney, brain,spleen and testis were made to examine the expression of enhance green flourescent protein (EGFP) by fluorescence microscopy in EGFP group. After 8-week CCl4 treatment, portal hypertensive rats in the saline group and Ad-Akt group received saline and Ad-myr-HA-Akt via the tail vein respectively. Portal vein pressure, mean arterial pressure and heart rate were measured in all rats on Day 3. Results In comparison with other cirrhosis rats, the pathological changes in the Akt group was markedly attenuated, and the levels of ALT, AST and Hyp were significantly lowered. Western blotting showed that the protein expression of p-Akt in the Akt group was higher significantly as compared with those in the negtive control group, saline group and EGFP group. Western blot also showed that the protein expression of Fas and DR5 in the Akt group was lower significantly. EGFP expression was mainly demonstrated by fluorescence microscopy on the frozen section of liver, very little fluorescene were detected in lung and kidney and there was no detectable EGFP in the other organs. Conclusions Ad-myr-HA-Ak inhibits CCl4-induced liver cirrhosis and is a potential pharmacological target for gene therapy in liver cirrhosis.
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Objective To establish Ad-hLIF transgenic feeder cells for the expansion of umbilical cord blood CD34+ HSPC in vitro and study the SCID mice model of hematopoietic stem/progenitor cell (HSPC) transplantation. Methods The expression of objective gene in Ad-hLIF transgenic feeder cells was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting(MACS) was detected by the flow cytometry. After expanded with various combinant of cytokines and transgenic feeder layer cells for 28 d, the quantity of mono-nuclear cell (MNC) and CD34+ cells rate was detected in different time. MNC after expansion stained by CFDA SE was injected to the sublethally irradiated SCID mice. Humanize gene Alu was detected by RT-PCR and fluorescence microscope. Results The green fluorescence was observed in the transgenic cells infected with 50MOI( multiplicity of infection) Ad-hLIF, and the objective gene was confirmed by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by MACS could reach 95.60% ±2.58%, Ad-hLIF transgenic feeder cells and various cytokines system increased MNC by 356.95±0.87 fold, and maximal expansion of CD34+ cells was observed during 0-14 d of culture, then down-expansion gradually. Four weeks after transplanted in SCID mice, fluorescently-labeled humanize cells still can be observed. The existence of the humanized gene Alu was confirmed by RT-PCR. Conclusion Ad-hLIF transgenic feeder cells can effectively proliferate umbilical cord blood CD34+ HSPC in vitro and delay it differentiate, what's more, it has high transplant efficacy and haematogenesis activity.
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Subject(s)
Humans , Adenoviridae , Blotting, Northern , Cell Count , Cell Line , Cell Survival , Congenital Abnormalities , DNA , Genes, Tumor Suppressor , Genes, vif , Genetic Therapy , Lac Operon , Mouth Neoplasms , RNA, Messenger , Survival Rate , TransfectionABSTRACT
Despite advances in surgery, radiotherapy, and chemotherapy, the survival of patients with oral squamous cell carcinoma has not significantly improved over the past several decades. Gene therapy is currently under investigation and shows us new possibility of cancer curing method. This experiment was undergone to find out the cell growth inhibition effect and evidence of apoptosis by HCCS-1(human cervical cancer suppressor-1), one of the candidates of tumor suppressor gene, transducted to human oral cancer cell line. To determine the efficiency of the adenovirus as a gene delivery vector cell line was transducted with LacZ gene and analysed with X-gal staining. Northern blot was performed to confirm the transfection with HSCC-1 gene and cell viability was assessed by cell cytotoxicity assay using cell count kit(CCK). To show the evidence of apoptosis, DNA fragmentation assay and flow cytometry(FACS) were performed. We had successfully construct the recombinant HSCC-1 adenovirus(Ad5CMV-HCCS-1), and importation efficiency was 20% at 2 MOI(multiplicity of infection), 80% at 20 MOI. Northern blot analysis showed that a single 0.6kb mRNA transcript was expressed in Ad5CMV-HCCS-1 transducted cell lines. As a result of CCK, when comparing to control subjects, transducted group showed 50% growth inhibition. In DNA fragmentation assay, according to increasing of MOI, DNA volume was diminished. In FACS analysis, DNA distribution showed fragmentation.This results imply that HCCS-1gene has growth inhibition effect in human oral cancer cell lines through apoptosis induction.
Subject(s)
Humans , Adenoviridae , Apoptosis , Blotting, Northern , Carcinoma, Squamous Cell , Cell Count , Cell Line , Cell Survival , DNA , DNA Fragmentation , Drug Therapy , Genes, Tumor Suppressor , Genes, vif , Genetic Therapy , Lac Operon , Mouth Neoplasms , Radiotherapy , RNA, Messenger , Transfection , Uterine Cervical NeoplasmsABSTRACT
Objective To construct recombinant human Bcl-2 adenoviral vector. Methods Sense human Bcl-2 cDNA fragment from plasimid pcDNA3 1/bcl-2 was cloned into adenovirus shuttle vector pAdCMV. pAdCMV/bcl-2 and plasmid pJM17 were cotransfected into 293 cells with lipofectamine 2000. Replication defective adenovirus was reproduced and purified. Adenoviral virus DNA was detected with polymerase chain reaction(PCR), and plaque assay was performed to determine the titer of virus. Results Recombinant adenovirus containing bcl-2 target gene was obtained. The titer of virus was 2 0?10 10 pfu/ml. Conclusion High titer of adenovirus containing target gene was successfully obtained by recombinant adenovirus vector.
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We attempted to observe the expression of stromelysin, which regulates the extracellular matrix of trabecular meshwork and is one of the family of the matrix metalloproteinases, in the trabecular cells after transfection of replication deficient recombinant adenovirus vector containing stromelysin cDNA. Stromelysin cDNA was produced by RT-PCR with total RNA extracted from cultured human trabecular cells after induction with interleukin-1 alpha, and cloned by inserting the cDNA into the TA vector. Adenovirus vector that contains stromelysin cDNA was constructed by cotransfection of pJM17 and p delta A. CMV. PA-str into the 293 cells. The expression of stromelysin in the trabecular cells was assayed by Western blot and zymography. The sequence of stromelysin cDNA was consistent with that previously reported. The constructed adenovirus vector had stromelysin cDNA but had no E1 region. The expression of stromelysin in the trabecular cells by this vector was detected in 4 days and peaked in 7 days after transfection. In conclusion, this study showed the possibility of gene therapy in the glaucoma treatment by transfecting the trabecular cells with the replication deficient recombinant adenovirus vector containing stromelysin cDNA.