Objective To construct AWP1 (associated with protein kinase C related kinase 1) recombinant adenovirus as the tool of transferring the gene and investigate its expression and localization in human vascular endothelial cell ECV304. Methods Cloned AWP1 cDNA was inserted into the multiply clone sites (MCS) of plasmid pcDNA3 for adding flag tag, and the flag-AWP1 gene was subcloned into shuttle vector pAdTrack-CMV. After identified with restrictional enzymes, plasmid pAdTrack-flag-AWP1 was linearized by digestion with restriction endonuclease PmeⅠ, and subsequently cotransformed into E.coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1 to make homologous recombination. After linearized by PacⅠ, the homologous recombinant adenovirus plasmid transfected into 293 cells with Lipofectamine to pack recombinant adenovirus. After PCR assay of recombinant adenovirus granules, recombinant adenoviruses infected 293 cells repeatedly for obtaining the high-level adenoviruses solution. And then, the recombinant adenoviruses infected human ECV304 cells for observing the expression and localization of AWP1 under laser scanning confocal microscope (LSCM). Results PCR assay showed that recombinant adenovirus Ad-flag-AWP1 was obtained successfully; and ECV304 cells were infected high-efficiently by the homologous recombinant virus. Then, it was observed that flag-AWP1 protein expressed in ECV304 cells and distributed in the leading edges of the cell membrane. Conclusion The vectors of flag-AWP1 recombinant adenovirus are constructed, and the localization of AWP1 protein in ECV304 cells might show that AWP1 may be a potential role on the cell signal transduction.
Objective To construct recombinant adenovirus vector co-expressing human interleukin (hIL)-10 and green fluorescent protein (GFP) for study of the expression of genes of interest in vascular smooth muscle cells （VSMCs）. Methods hIL-10 cDNA was amplified from pUCm-T/hIL-10 cDNA using polymerase chain reaction (PCR), and cloned into shuttle plasmid pShuttle-IRES-hrGFP-1. Kanamycin resistance screeninged for recombinant plasmids, which were linealized with PmeⅠand transformed into BJ5183-AD-1 containing pAdEasy-1 by electroporation after determining the insert’s sequence correct by NotⅠ and XholⅠdigestion， sequencing and basic local alignment search tool (BLAST). Prepared recombinant adenovirus plasmids were transformed into XL10-Gold cells. Amplified plasmids were transfected to AD-293 cells for packaging after being linearized with PacⅠ. PCR was used to determine target gene; The titer of the recombinant adenovirus was measured. VSMCs were transfected by recombinant adenovirus and viewed under fluorescence microscope. hIL-10 concentration in transfected VSMCs supernant was measured by enzyme linked immune sorbent assay (ELISA). Results Recombinant shuttle plasmids contained interest gene. Recombinant adenovirus had 30 kb and 3 kb fragments after digestion with PacⅠ. PCR indicated that the recombinant adenovirus contained interest gene. The titer of recombinant adenovirus was 3×10１0 efu/ml. Transfected VSMCs had GFP expression and hIL-10 concentration in supernatant was 25 ng/106 cells. Conclusion The recombinant adenovirus co-expressing hIL-10 and GFP is successfully constructed and could effectively express in VSMCs, this lays the foundation for the gene therapy of vascular intimal hyperplasia.
【Abstract】Objective To construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase2 (MMP2) for use in the gene therapy to inhibit the invasiveness and migratory capacity of hepatocellular carcinoma (HCC) cell line HepG2 in vitro and in vivo models. Methods Total RNA was extracted from HCC, and then a 500 bp fragment at the 5′ end of human MMP2 cDNA was synthesized by polymerase chain reaction (PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV,with the resultant plasmid and the backbone plasmid pAdEasy-1,the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed and generated. The adenoviruses(Ad-MMP2AS) were packaged and amplified in the HEK 293 cells.Then the viral titer was checked by GFP. Results The recombinant adenovirus vector carrying antisense MMP2 was constructed successfully, the b green fluorescence was observed in HEK 293 cells under a fluorescence microscopy. The viral titer was 1×108/ml. Conclusion The recombinant adenovirus Ad-MMP2AS constructed by us could introduce the antisense MMP2 into HepG2 effectively,which would provide experimental basis for reversing the overexpression of MMP2 in HCC and for inhibiting the invasiveness and migratory capacity of HepG2 in vitro and in vivo models.
ObjectiveTo investigate target gene therapy for hepatocellular carcinoma (HCC). MethodsHerpes simplex virus thymidine kinase (HSVTK) gene was inserted into the gene of AFP enhancer/ALB promoter with adenoassociated virus (AAV) plasmid (WAV2) as a carrier, and a hybrid plasmid pWAV2/AFPALB/HYTK was constructed. Besides, plasmid pEGFP1/AFPALB was also constructed. Two kinds of plasmids were transferred into AFP positive cells HepG2 and AFP negative cells 7721, SPC and 7901.ResultsIt was found that enhance green fluorescence protein could only be seen in AFP positive cells HepG2. 710 bp DNA was amplified only in AFP positive HepG2 cells.ConclusionPlasmid pWAV2/AFPALB/HYTK for HCC demonstrates specificity in vitro.
ObjectiveTo construct the recombinant adenovirus vector carrying antisense multidrug resistanceassociated protein (MRP) and transfect the human drugresistant hepatocellular carcinoma cell line(SMMC7721/ADM). MethodsThe fragment of MRP gene encoding 5′region was cloned reversely into the shuttle plasmid pAdTrackCMV, with the resultant plasmid and the backbone plasmid pAdEasy1,the homologous recombination took place in the bacteria and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged and amplified in 293 cells. Then the cell line of SMMC7721/ADM was transfected with the resultant adenoviruses.ResultsThe recombinant adenovirus vector carrying antisense MRP was constructed successfully. The viral titer was 2.5×109 efu/ml, and more than 90% SMMC7721/ADM cells could be transfected when the multiplicity of infection（MOI） was 100. ConclusionThe recombinant adenovirus vector constructed by us could introduce the antisense MRP into the human drugresistant hepatocellular cell line effectively, which would provide experimental basis for the mechanisms and reversal methods of the multidrug resistance in human hepatocellular carcinoma.
Objective To explore the feasibility of recombinant adeno-associated virus (rAAV) as a vector for the gene therapy of liver cancer. Methods The rAAV/enhance green fluorescein protein (EGFP) recombinant was prepared by the routine method of two plasmids cotransfection.Results The experiment showed that one 10cm plate could produce 107-108 infection unit recombinant by the method of two plasmids cotransfection, and the transduction of HepG2 cell was increased with the increase of infection dosage of rAAV. About 100 multiplicity of infection (MOI) AAV vector could make all the tumor cell light. Conclusion Liver cancer cell can be efficiently transduced by rAAV, and AAV vector may be a valuable vector for the gene therapy of liver cancer.
Objective To observe the expression of adenovirus vector coding for mouse endostatin gene（Ad-mES） in lung cancer cells and its antiangiogenic activity in human umbilical vein endothelial cells（ECV304） in vitro.Methods Lewis lung cancer（LLC） cells were transfected with Ad-mES at different multiplicity of infection（MOI）.The expression of mES in LLC cells and supernatant after 48 hours was detected by immunohistochemical staining and Western blot respectively.The inhibitory effect of supernatant at different MOI on ECV304 non-stamulated and stimulated by basic fibroblast growth factor（bFGF） was measured by methyl thiazolyl tetrazolium（MTT） assay.Results After transfected for 48 hours，endostatin was identified in the cell plasma of infected LLC and negative result was founded in non-infected LLC.Western blot revealed band of endostatin in 20 kDa in culture supernatant of infected LLC and negative results in non-infected LLC.The inhibitory effects on ECV304 cell proliferation were ber at higher MOI，and the difference was significant between stimulated and non-stamulated cells by bFGF（Plt;0.05）.Conclusion Ad-mES can transfect and express endostatin effectively in LLC with biological activity
Objective To investigate the expression of FLIP in the lung of rats and the protective effect in development of acute lung injury( ALI) with the adenovirus vector carrying FLIP gene( Ad-FLIP)inhaled. Methods Forty-eight rats were randomly divided into four groups, with 12 rats in each gruop. In treatment group, ALI rats model was eatablished by LPS intraperitoneal injection and then inhaled Ad-FLIP vector. In prevention group, the animals were infected with Ad-FLIP vector before ALI model wasestablished. Two control groups of treatment and prevention received Ad-EGFP vectors respectively.Pathological changes of lung were observed under light microscope. Wet/dry weight ( W/D) of lung lobes and lung permeability index( LPI) were also measured. The mRNA and protein expressions of FLIP in lungwere investigated by RT-PCR and immunohistochemistry, respectively. Results Lung histopathological changes were alleviated, the index of W/D and LPI were significantly lower, the expressions of FILP mRNA and protein in the lung were elevated both in the treatment group and prevention group compared to thecontrol groups ( all P lt;0. 01) . Conclusion Ad-FLIP transfection can up-regulate the expression of FLIP in lung of rats, and might protect respiratory membrane and lessen pulmonary edema to prevent the development of ALI.
Objective To determine the transfection efficiency of recombinant adenovirus to endothelial progenitor cells(EPCs) and provide the base of lung cancer therapy by transfecting human herpes simplex virusthymidine kinase（HSV-TK） gene to EPCs. Methods Admove recombinant adenovirus 5F35(AD5F35) which transfected with βgalactosidase(AD5F35LacZ) to the 24 well plate cultivated with EPCs and transfect the EPCs. Stain the EPCs with LacZ kit and calculate the transfection efficiency. Results The blue stain cells were cells transfected successfully with AD5F35LacZ under the optical microscope. The transfection efficiencies of adenovirus to EPCs were different under the premise of the different multiplicity of infection(MOI). In a certain range, the transfection efficiencies rise with the MOI rise. When MOI was 400,the proportion of blue stain cell is the highest, which was 98.38%±1.25%. Conclusion Recombinant adenovirus can transfect EPCs successfully. The transfection efficiencies rise with the MOI rise. When the MOI is 400,the transfection efficiency is the highest.
ObjectiveTo evaluate the effect of human heme oxygenase 1 augmentation in vein grafts by adenoviral mediated gene transfer of heme oxygenase 1 (Ad hHO 1) on intimal hyperplasia.MethodsTwenty one Japanese white rabbits were divided into three groups: control group, Ad null control group, and Ad hHO 1 group(each group 7 rabbits). During the operation of rabbits jugular vein into carotid artery interposition grafting, harvested rabbit jugular vein segments were exposed for 30min at room temperature to heparin saline, recombinant replication deficient adenovirus encoding hHO 1(Ad hHO 1, 1× 10 9pfu/ml), and nude recombinant replication deficient adenovirus (Ad null, 1×10 9pfu/ml). Quantitative histological studies of the vein segments were performed 28 days after operation. Protein of hHO 1 was detected with method of immunohistochemical staining(S P) in 14 days and 28 days after operation.ResultsThe average intimal thickness, medial thickness and intimal to medial(I/M) ratio were calculated for each group 28 days after bypass operation. Compared to intimal thickness, I/M ratio of control group veins and Ad null group veins,Ad hHO 1 group veins decreased significantly( P lt;0.01). There was no statistically difference in medial thickness ( P gt;0 05). Strong staining of hHO 1 was detected in vein grafts wall of Ad hHO 1 group.ConclusionAd hHO 1 gene therapy may inhibit intimal hyperplasia of vein grafts in rabbits.