Construction of recombinant adenovirus vector of Uhrf1 gene and its role in DNA damage repair of cardiomyocytes / 中国病理生理杂志

AIM:To construct a recombinant adenovirus vector carrying the mouse ubiquitin-like with plant homeodomain and RING finger domains 1(Uhrf1)gene,validate the expression of Uhrf1 in neonatal mouse cardiomyo-cytes and explored its role in hydrogen peroxide(H2O2)-induced DNA damage.METHODS:The mouse Uhrf1 gene cod-ing sequence was amplified by polymerase chain reaction(PCR),digested,and inserted into the pADM-CMV-C-FH vec-tor to create the recombinant adenoviral plasmid ADM-Uhrf1.Following transfection into HEK293T cells,we generated re-combinant adenoviral particles,amplified,purified,and determined the titer.Neonatal mouse cardiomyocytes were infect-ed at an multiplicity of infection(MOI)of 50,UHRF1 protein expression was validated via Western blot and immunofluo-rescence staining.H2O2-induced DNA damage was explored along with adenovirus-mediated Uhrf1 overexpression to inves-tigate its role in DNA damage repair.RESULTS:ADM-Uhrf1 virus titer,determined by capsid immunofluorescence as-say,was 1.8×1013 pfu/L.Western blot confirmed a significant increase in UHRF1 protein expression(P＜0.05),with im-munofluorescence indicating predominant nuclear localization.Uhrf1 overexpression effectively inhibited the expression of the DNA damage marker,phosphorylated H2AX protein(γH2AX)(P＜0.01).CONCLUSION:We successfully con-structed a recombinant adenoviral vector carrying the mouse Uhrf1 gene,facilitating Uhrf1 overexpression in neonatal mouse cardiomyocytes.Furthermore,this overexpression effectively alleviated DNA damage in cardiomyocytes.

The latest progress on novel coronavirus vaccination in kidney transplant recipients / 器官移植

Due to long-term use of immunosuppressant, poor immune function and a higher risk of critical diseases after novel coronavirus pneumonia in kidney transplant recipients, it is of significance to deliver prophylactic vaccination for this high-risk population. Studies have shown that the immune reaction of kidney transplant recipients to novel coronavirus vaccine is significantly lower than that of healthy counterparts. Standard vaccination program in the United States, such as 2 doses of messenger RNA (mRNA) vaccine, fails to provide sufficient protection for kidney transplant recipients. Many studies have proven that increasing the frequency of vaccination for kidney transplant recipients may enhance the vaccine efficacy. Nevertheless, the role of adjusting immunosuppressive therapy in increasing vaccine efficacy remains to be elucidated. In this article, the importance, effectiveness and particularity of novel coronavirus vaccine for kidney transplant recipients and the effect of immunosuppressive therapy on the efficacy of novel coronavirus vaccine were reviewed, aiming to provide reference on the vaccination for kidney transplant recipients.

Research progress of flavivirus vaccines based on adenovirus vector / 预防医学

@#There are more than 70 species of flaviviruses, including Zika virus, Dengue virus and Japanese encephalitis virus, and more than 33 species are known to be capable of infecting humans. Only three flavivirus vaccines have been approved, and there is a lack of safe and effective vaccines for most flaviviruses. Adenovirus-vectored vaccines have high safety, low cost, and convenience to store and transport. Currently, two adenovirus-vectored Zika vaccines are under early clinical trials, and adenovirus-vectored vaccines for Dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus are still in the phase of animal experiment. In the development of adenovirus-vectored flavivirus vaccines, there are still problems of pre-existing immunity to adenovirus, the insufficient immunogenicity of adenovirus vectors and the antibody-dependent enhancement effects among flavivirus. Based on relevant publications from January 2006 to June 2023, this article reviews the current status, challenges and solutions of the research into adenovirus-vectored flavivirus vaccines, so as to provide the reference for the development of relevant vaccines.

Hyperosmotic stress and perfusion culture strategies increase the yield of recombinant adenoviral vector produced by HEK 293 cells / 生物工程学报

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×1010 IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.

Construction and identification of recombinant adenovirus vector and observation of its transfecting rabbit bone marrow mesenchymal stem cells / 中国骨伤

OBJECTIVE@#To construct and identify adenovirus vector co-expressing hBMP2 and hVEGF165 fusion protein which labeled with green fluorescence protein, and laying the foundtion of the effect of hBMP2 and hVEGF165 gene inducing BMMSCs differentiation to osteoblast and bone defect repaired in the body.@*METHODS@#BMP2 and VEGF165 gene was amplified from cDNA library by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. Ad-BMP2- VEGF165 was recombinated and propagated in HEK293 cells by co-transfecting with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ E1, 3Cre. The recombinant adenovirus was purified and virustiter was determined, and then to research GFP expression and to calculate the adenovirus transfection rate in rabbit BMMSCs.@*RESULTS@#The recombinant adenovirus vector Ad-BMP2-VEGF165 was successfully constructed by the methods of gene analyzing, colony PCR, Western blotting and observing GFP expression, and the titer of the adenovirus was 1×10@*CONCLUSION@#Recombinant adenovirus vector containing hBMP2 and hVEGF165 gene was successfully constructed and its high titer was obtained.

Construction and characterization of a recombinant vaccine encoding the nucleocapsid protein gene of avian infectious bronchitis virus / Construção e caracterização de uma vacina recombinante codificando o gene da proteina do nucleocapsideo do virus da bronquite infecciosa das galinhas

A vacinação é a forma mais utilizada para prevenir a bronquite infecciosa causada pelo vírus da bronquite infecciosa das galinhas (IBV). Contudo, as vacinas convencionais são incapazes de diferenciar aves infectadas de vacinadas. No presente trabalho foi construído, caracterizado, e avaliado como candidato vacinal, um adenovírus recombinante expressando o gene N do IBV. O gene N foi clonado em um adenovírus humano tipo 5 defectivo e transfectado para as células HEK-293A para gerar rAd5_N. Após o vetor ser obtido como esperado e a confirmação da expressão da proteína N em HEK-293ª, foi realizada inoculação pela via oculo-nasal na dose de 10 7 TCID 50 /0,1mL para imunização de galinhas livres de patógenos específicos (SPF). A resposta imunológica do Ad5_N e a proteção contra o desafio ao IBV foram avaliadas e comparadas com uma vacina viva comercial. Não foram detectados anticorpos anti-IBV em aves vacinadas com o Ad5_N. A vacina comercial induziu anticorpos detectáveis a partir do 7º dia pós-vacinal. Em aves vacinadas com o Ad5_N não houve aumento na expressão de IFNγ. Neste estudo, o rAd5_N obtido não conferiu proteção contra desafio com IBV-M41. Os resultados indicam a necessidade de avaliar adenovírus recombinantes expressando outros genes do IBV.(AU)

Recombinase-mediated in vitro rapid construction of replication-competent human adenovirus type 14 vector encoding EGFP / 中华微生物学和免疫学杂志

Objective To rapidly and efficiently construct a replication-competent human recombi-nant adenovirus type 14 vector expressing enhanced green fluorescence protein ( rAd14-EGFP) using in vitro homologous recombination. Methods The skeleton plasmid pBRAd14 was constructed using homologous re-combination in Escherichia coli ( E. coli) BJ5183 competent cells. The plamid was linearized and transfected into AD293 cells to rescue Ad14. Exnase, a recombinase, was used to construct the shuttle plasmid pSK14-EGFP in vitro using homologous recombination among four fragments. The overlapping sequence was 15-20 bp. Three exogenous fragments generated with PCR including Ad14 E3L fragment, EGFP gene and Ad14 E3R fragment were cloned into the plasmid pBluescript Ⅱ SK (-) simultaneously. Recombinant plasmid pBRAd14-EGFP was constructed by in vitro homologous recombination between 27 kb fragment of plasmid pBRAd14 obtained through double digestion and Ad14 E3L-EGFP-Ad14 E3R fragment amplified by PCR using the shuttle plasmid pSK14-EGFP as template. The plasmid pBRAd14-EGFP was linearized and trans-fected into cells to obtain the viral vector rAd14-EGFP, which was then used to immunize mice to detect the induced immune responses. Results A replication-competent E3-deleted adenovirus vector rAd14-EGFP expressing EGFP was successfully constructed. Intracellular proliferation properties and immunogenicity of the vector were no significantly differences compared with those of Ad14. Conclusions In vitro homologous recombination using the commercial recombinase Exnase can be a rapid, efficient and accurate method to construct adenoviral vector.

Recombinase-mediated in vitro rapid construction of replication-competent human adenovirus type 14 vector encoding EGFP / 中华微生物学和免疫学杂志

Objective@#To rapidly and efficiently construct a replication-competent human recombinant adenovirus type 14 vector expressing enhanced green fluorescence protein (rAd14-EGFP) using in vitro homologous recombination.@*Methods@#The skeleton plasmid pBRAd14 was constructed using homologous recombination in Escherichia coli (E.coli) BJ5183 competent cells. The plamid was linearized and transfected into AD293 cells to rescue Ad14. Exnase, a recombinase, was used to construct the shuttle plasmid pSK14-EGFP in vitro using homologous recombination among four fragments. The overlapping sequence was 15-20 bp. Three exogenous fragments generated with PCR including Ad14 E3L fragment, EGFP gene and Ad14 E3R fragment were cloned into the plasmid pBluescript Ⅱ SK(-) simultaneously. Recombinant plasmid pBRAd14-EGFP was constructed by in vitro homologous recombination between 27 kb fragment of plasmid pBRAd14 obtained through double digestion and Ad14 E3L-EGFP-Ad14 E3R fragment amplified by PCR using the shuttle plasmid pSK14-EGFP as template. The plasmid pBRAd14-EGFP was linearized and transfected into cells to obtain the viral vector rAd14-EGFP, which was then used to immunize mice to detect the induced immune responses.@*Results@#A replication-competent E3-deleted adenovirus vector rAd14-EGFP expressing EGFP was successfully constructed. Intracellular proliferation properties and immunogenicity of the vector were no significantly differences compared with those of Ad14.@*Conclusions@#In vitro homologous recombination using the commercial recombinase Exnase can be a rapid, efficient and accurate method to construct adenoviral vector.

Effects of p38 MAPK signal pathway inhibition on upper lip scar hyperplasia at different time in rabbits / 中华医学美学美容杂志

Objective To investigate the effect of p38MAPK gene silencing recombinant adeno-virus on the expression of target gene in different time and to detect the effect of p 38MAPK signal pathway on the upper lip scar hyperplasia at different time to determine the optimal scar treatment time .Methods The adenovirus vector was injected into the scar tissue in 0 week ,1 week and 2 week after cheiloplasty in rabbit .The specimens were harvested in 3 week postoperatively .Four methods in-cluding Sirius red staining ,immunohistochemical staining (IHC) ,Western blotting (WB) ,real-time PC (RT-PCR) were used to quantitatively and quantitatively detect the relative expression levels of p38MAPK and scar-related factors (col Ⅰ ,col Ⅲ ,MM P1 ,TIMP1) .Results Sirius red staining and immunohistochemical staining showed that in 1st week the expression of col Ⅲ and MMP1 in scar tis-sue was significantly higher than that in 0 week and 2 week after operation and the expression of col Ⅰand TIMP1 was significantly less than that in 0 week and 2 week after operation .The results of WB and RT-PCR were consistent with that of IHC .Conclusions After injection into the upper lip scar tis-sue with adenovirus in 1 week ,the degree of scar hyperplasia is the least .

Effects of miR-124a overexpression on expression levels of TNF-α and IL-6 and cell cycle of J774. 1 cells / 吉林大学学报(医学版)

Objective; To explore the effects of miR-124a on the expression levels of tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6) and cell cycle in the macrophage J774. 1 cells of the rheumatoid arthritis (RA) model mice, and to elucidate the mechanism of miR-124a in the pathogenesis of RA. Methods: The J774. 1 cells in logarithmic growth phage were obtained and uniformly inoculated in the petri dish with 1 × 106 mL-1, and there were 3 multiple holes in each group; transfection was carried out when the fusion degree of the cells reached 60%. The cells were divided into blank control group (without any treatment), empty vector group (transfected with adenovirus negative fluid) and miR-124a overexpression group (transfected with miR-124a adenovirus vector). The expression levels of TNF-α and IL-6 in supernatant of the cells in three groups were detected by ELISA 48 h after transfection. RT-PCR was used to detect the relative expression levels of TNF-α and IL-6 mRNA in J774. 1 cells 48 h after transfection and the changes of cell cycle were detected using flow cytometry. Results: The expression levels of TNF-α and IL-6 in supernatant of the cells in miR-124a overexpression group were lower than those in blank control group and empty vector group 48 h after transfection (P=0. 038, P=0. 042; P=0. 043, P=0. 044). The expression levels of TNF-α mRNA and IL-6 mRNA in miR-124a group were significantly lower than those in blank control group and empty vector group (P = 0. 001, P=0.002; P=0. 001, P=0. 003). The pecentage of cells in Gi phase in miR-124a overexpression group was higher than those in blank control group and empty vector group (P<0. 01); the percentages of cells in S phase and G2 phase were lower than those in blank control group and empty vector group (P<0.01). Conclusion: MiR-124a can decrease the expression levels of inflammatory cytokines after transfecting the macrophages J774. 1 cells, and inhibit the proliferation of cells. MiR-124a is a potential therapeutic target for the treatment of RA.

Effect of ERRα in silencing DKK1 and SOST recombinant adenovirus vector-transfected MG63 Cells and bone-related proteins / 医学研究生学报

Objective WNT signaling pathway plays an important role in the formation, differentiation and maturation of bone cells, it is a classical intracellular signaling pathway involved in bone metabolism. DKK1 and Sost play a negative regulatory role in regulating bone mass and osteoblast differentiation, and are negative regulators of WNT signaling pathway. Estrogen-related receptor alpha (ERRα) regulates the functional activity of osteoblasts. The aim of study was to investigate the effect of ERRα on the transfection of MG63 cells and related proteins by the WNT signaling pathway inhibitor Dickkopf (DKK)1 and sclerostin (SOST) adenovirus vectors.Methods The cultured MG63 cells were divided into blank control group, silencing DKK1 group, silencing SOST group, silencing (DKK1+SOST) group, ERRα intervention empty adenovirus group, ERRα intervention silencing DKK1 group, ERRα intervention silencing SOST group, ERRα intervention silencing (DKK1+SOST) group. MG63 cells were transfected with packaged silencing DKK1 and SOST adenovirus vectors according to different groups. The activity of MG63 cells was detected by MTT assay, the activity of ALP was detected by alkaline phosphatase kit, and the concentration of calcium ion was analyzed by flow cytometry. Western blot was used to detect the expressions of low density lipoprotein associated protein 5 (LRP5), bone morphogenetic protein 2 (BMP2), osteopontin (OPN), osteoprotegerin(OPG).Results (1) Compared with blank control group, silencing DKK1, SOST, DKK1+SOST group and ERRα overexpression in the empty adenovirus group could increase cell activity, ALP activity, and decrease calcium ion concentration and increase the expressions of LRP5, BMP2, OPN, and OPG. Differences between groups were statistically significant(P0.05).Conclusion ERRα Overexpression can increase the activity of MG63 cells, ALP activity, LRP5, BMP2, OPN, and OPG proteins, and decrease the calcium ion concentration in silencing DKK1 and SOST adenovirus-transfected cells.

Effect of adenovirus vector encoding hepatocyte growth factor gene on learning and memory ability and expression of apoptosis protein in hippocampus of rats with hypertension and hyperlipemia / 新乡医学院学报

Objective To investigate the effect of adenovirus vector encoding hepatocyte growth factor gene (Ad-HGF) on learning and memory ability and expression of bcl-2 and bax protein in hippocampal CA1 region of rats with hypertension and hyperlipemia.Methods Thirty male Sprague Dawley rats were randomly divided into control group,model group and Ad-HGF group,with 10 rats in each group.The rats in model group and Ad-HGF group were given bilateral renal artery stenosis operation under aseptic condition to establish experimental renal hypertension;one week after operation,the rats were fed with high fat diet for 16 weeks.The rats in control group were only separated the bilateral renal arteries,and then were given normal diet for 16 weeks after the operation.After modeling,the rats in Ad-HGF group were injected with Ad-HGF (10 μL) throuth cisterna magna;the rats in control group and model group were injected with the same volume of saline through cisterna magna.The learning and memory ability of rats were evaluated by Morris water maze test at 10 d after administration;the expression of bcl-2 and bax protein in hippocampus CA1 region were detected by immunohistochemistry.Results The escape latency of rats in model group was significantly longer than that in the control group,the number of cross platform and target quadrant time were significantly less than those in the control group (P ＜ 0.05);the escape latency of rats in Ad-HGF group was significantly shorter than that in the model group,the number of cross platform and target quadrant time were significantly more than those in the model group (P ＜ 0.05).Compared with the control group,the number of the bcl-2 and bax protein positive cells in hippocampal CA1 region of rats in the model group was increased,and the ratio of bcl-2/bax was decreased(P ＜0.05);compared with the model group,the number of the bcl-2 protein positive cells in hippocampal CA1 region of rats in AdHGF group was increased,but the number of bax protein positive cells was decreased,and the ratio of bcl-2/bax was increased (P ＜ 0.05).Conclusion Exogenous Ad-HGF can significantly improve the learning and memory ability of rats with hypertensive and hyperlipidemia,and it play a neuroprotective role in the brain,which may be related to the inhibition of apoptosis of hippocampal neurons.

Construction of Recombinant Adenovirus Vector Expressing Mouse SPINK5 Gene and Its Curative Effect on Skin Lesions in Atopic Dermatitis Mice / 中国医科大学学报

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.

Experimental study on construction of ultrasound microbubbles co-carrying pAd-EGFP/SDF-1α and pAd-RFP/BMP2 / 中华超声影像学杂志

Objective To construct the uItrasound microbubbIes co-carrying recombinant adenovirus containing stromal cell derived factor 1 (SDF-1α) and bone morphogenetic protein 2 (BMP2),and to study the maximum efficiency of carrying adenovirus and the optimum proportion of double gene combined with ultrasound contrast agents.Methods Microbubbles were combined separately with recombinant adenovirus co-expression of enhanced green fluorescent protein and SDF-1α(pAd-EGFP/SDF-1α) as well as red fluorescence protein and BMP2 (pAd-RFP/BMP2) via biotin-streptavidin method,and the maximum efficiency of carrying DNA in microbubbles was detected.Three microbubbles with binary vectors were prepared by blending the two above-mentioned pAd at different ratio (1 ∶1,1 ∶ 2,2 ∶ 1) into the microbubbles.The microbubbles with binary vectors were evaluated though physiochemical properties,fluorescence microscope and flow cytometry to test the carrying rate of DNA in microbubbles.Results There was no significant difference in PH,average diameter and concentrations between targeted microbubbles and control group (P ＞0.05).The carrying efficiency of DNA increased with virus loads in microbubbles,but lowered if further increasing virus amount after reaching saturation.When the proportion of binary vectors and microbubbles was 1 ∶ 1,its efficiency of carrying SDF-1α gene and BMP-2 was approximately equal,and flow cytometry demonstrated that the positive rate of microbubbles labeled by both fluorescein isothiocyanate(FITC) and rhodamine was (65.6 ± 0.5)％.However,it was (59.0 ± 2.3)％ when their proportion was the 2 ∶ 1,which was significantly lower than those when other two proportions (1 ∶ 1 and 1 ∶ 2).Under the fluorescence microscope,the targeted microbubbles were equally surrounded by bright green or red fluorescence.Conclusions Ultrasound microbubbles of double genes carrying EGFP/SDF-1a and RFP/BMP2 is made successfully via biotin-streptavidin method.The optimal proportion of combining microbubbles with double gene is 1 ∶1,which can reveal the optimum load rate and stable combination.

Cthrc1 Inhibits Proliferation of Hepatic Stellate Cells in Mice / 胃肠病学

Background:Collagen triple helix repeat containing protein-1(Cthrc1)has been reported playing an important role in liver diseases,especially in liver fibrosis,however,its effect on hepatic stellate cells proliferation is not fully clear. Aims:To investigate the effect of Cthrc1 on proliferation of hepatic stellate cells. Methods:Recombinant adenovirus vector of Cthrc1 was constructed. After injecting Ad-Cthrc1 through tail vein,mRNA and protein expressions of Cthrc1 were determined by real-time PCR and Western blotting,respectively. Liver fibrosis model was established by bile duct ligation and fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine(DDC)in mice,respectively. The liver fibrosis mice were injected with Ad-Cthrc1 or Ad-GFP through tail vein. Immunofluorescence was used to determine number of hepatic stellate cells. Results:Recombinant Cthrc1-adenovirus vector was successfully constructed. Real-time PCR and Western blotting showed that mRNA and protein expressions of Cthrc1 were increased in Ad-Cthrc1 group than in control group. HE and Masson staining demonstrated that mice model of liver fibrosis was successfully established. Immunofluorescence showed that overexpression of Cthrc1 inhibited significantly the proliferation of hepatic stellate cells. Conclusions:Recombinant adenovirus vector of Ad-Cthrc1 constructed can express stably in vivo,and inhibit the proliferation of hepatic stellate cells. Therefore,Cthrc1 may become a potential target for treatment of liver fibrosis.

Construction and effect of recombinant adenovirus vector containing siRNA targeting Raf-1 on cardiomyocyte hypertrophy in neonatal rats / 中国比较医学杂志

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.

Comparison of the efficiency of tail vein injection and intramyocardial injection of adenovirus vector in mice / 中国比较医学杂志

Objective To investigate the efficiency of target gene transfection of the heart and liver after tail vein or intramyocardial injection of adenovirus vector (GFP-Ad).Methods GFP-AD was constructed at first.A total of 20 male 8-week old C57BL/6 mice were randomly and equally divided into tail vein injection of GFP-AD group and intramyocardial injection of GFP-AD group.The mRNA levels of GFP in the heart and liver tissues were detected by Q-PCR at different time points.Fluorescence microscopy was performed to visualize the expression of GFP fluorescence.Results Compared with the tail vein injection group, the GFP mRNA level in mouse heart tissue was apparently higher in the intramyocardial injection group.In both groups, the GFP mRNA levels in liver tissue were significantly increased compared with that in the heart tissue.In the tail vein injection group, the GFP mRNA level in liver tissue reached a peak on day 7;but in the intramyocardial injection group, the mRNA level of GFP in liver tissue reached apeak on day 3.We also observed the same trend of GFP fluorescence expression in the tail vein injection group compared with that in the intramyocardial injection group.Conclusions Intramyocardial injection of adenovirus vector is suitable to achieve a higher transfection efficiency in mouse heart tissue compared with the tail vein injection method.Although both injection methods are suitable for transfection of mouse liver, the tail vein injection method is preferential for it is simple and less invasive.

"Construction and preliminary validation of a new ""Controllable"" recombinant adenovirus vector carrying human insulin gene" / 实用医学杂志

Objective To design and construct a controlled adenovirus vector in degradating by itself after induction for solving the problem of stimulating host immune and producing replication adenovirus and providing a secure exogenous gene vectors for clinical practice. Methods Based on the traditional adenovirus vector AdEasyTM system, we inserted the Cre gene which belongs to Cre-LoxP system into the downstream of Tet-On inducible expression system. Two LoxP sites were inserted into two sides of the shuttle plasmid′s right arm genome. Then, the full-length human insulin gene was inserted HindIII enzyme site. After the recombinant adenovirus infected the rat bone marrow-derived mesenchymal stem cells , fluorescent protein expression and insulin secretion were detected before or after induction by Dox. Results A new controlled recombinant adenovirus vector carrying human insulin gene was constructed successfully, and was named AdEasyN/INS. After the transfection of this new vector into QBI-293A cells and rat bone marrow mesenchymal stem cells , green fluorescent protein could be observed. After induction by Dox, both of the ratio of fluorescent cells/total cell and the levels of insulin significantly decreased. Conclusion Construction and preliminary validation of a controlledrecombinant adenovirus vector carrying human insulin gene is constructed successfully , it could infect rat bone marrow mesenchymal stem cells, and degradate by itself after Dox induction, realize the controllability of exogenous gene carrier.

Effects of Recombinant Adenovirus Carrying Apoptin Gene on the Apoptosis of Human Lung Squamous Carcinoma SK-MES-1 Cell Lines and Human Lung Adenocarcinoma NCI-H1299 Cell Lines / 中国药房

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.

Effects of adenovir us mediated AdipoQ on mice submandibular gland in Sj?gren's syndrome / 中华风湿病学杂志

Objective To observe the expression of AdipoQ in Sj?gren's syndrome (SS) mice and its role in inflammation was investigated by recombinant gene transfection study in vivo. Methods As spon-taneous SS mice model, a total of 30 NOD mice were divided into 3 groups randomly: recombinant adenovirus (rADV-AdipoQ) group, normal saline control and simple adenoviruses (control group). The submandibular gland morphology, histopathological grading and the level of serum tumor necrosis factor-α (TNF-α), AdipoQ was compared between the three groups. The expression of AdipoQ on mice submandibular gland was assessed by means of semi-quantitative reverse transcriptase polymerase chain reaction. Results The submandibular glands of the mice of the control group were destructed, with focal lymphocytic infiltration and acinus atrophy. Compared with control model groups, the serum TNF-α and salivary gland AdipoQ expression was significantly down-regulated in the rADV-AdipoQ group [(248 ±30) vs (162 ±73) ng/ml] (P<0.05). Conclusion AdipoQ gene transfected SS mice can significantly improve the morphological features of tissues and decrease the concentration of TNF-αin serum, in addition, it can effectively inhibit inflammation, decrease the degree of protein and AdipoQgene expression. So the AdipoQ may be the protective gene in SS mice.