目的:寻找对腺病毒灭活的有效方法,来预防大规模生产腺病毒时所致的生产细胞的污染。方法:采用紫外线照射和不同浓度过氧乙酸的灭活方法灭活腺病毒,并将处理后的病毒加入到正常人胚肾293细胞中培养,观察细胞是否出现病变作为灭活效果判断标准。结果:我们发现一定强度紫外线照射至少2 h以上才能完全灭活腺病毒,过氧乙酸达到1 %的浓度和至少作用30 min,才能将完全病毒灭活。结论:紫外线照射2 h以上能有效灭活腺病毒,我们可以用于操作台的腺病毒灭活;1%浓度的过氧乙酸作用30 min以上能有效将腺病毒灭活,我们可以运用于实验室大空间的腺病毒灭活。
Objective To study the adenovirus-mediated human bone morphogenetic protein-2 gene （Ad-hBMP-2）transferred to the intervertebral disc cells of the New Zealand rabbit in vitro. Methods The cells of New Zealand white rabbitswere isolated from their lumbar discs. The cells were grown in the monolayer and treated with an adenovirus encoding the LacZ gene (Ad-LacZ) and Ad-hBMP-2 (50,100, 150 MOI,multiplicity of infection) in the Dulbecco’s Modified Eagle Medium and the Ham’s F-12 Medium in vitro. Three days after the Ad-hBMP-2 treatment,the expression of hBMP-2 in the cells that had been infected by different dosesof MOI was determined by immunofluorescence and the Western blot analysis, and the expression was determined in the cells with the Ad-LacZ treatment in a dose of 150 MOI. Six days after the Ad-hBMP-2 treatment, mRNA was extracted for the reverse transcription polymerase chain reaction (RT-PCR) and the difference was detected between the control group and the culture group that was treated withAd-hBMP-2 in doses of 50, 100 and 150 MOI so that the expressions of aggrecan and collagen ⅡmRNA could be observed. Results The expression of hBMP-2 in the cells was gradually increased after the transfection in an increasing dose, which was observed by immunofluorescence and the Western blot analysis. At 6 days the aggrecan and collagen type Ⅱ mRNA expressions were up-regulated by Ad-hBMP-2 after the transfection at an increasing viral concentration in the dosedependent manner. Conclusion The results show that Ad-hBMP-2 can transfect the rabbit intervertebral disc cells in vitro with a high efficiency rate and the expression of hBMP-2 after theinfection is dose-dependent in the manner. AdhBMP-2 after transfection can up-regulate the expression of aggrecan and collagen Ⅱ mRNA at an increasing viral concentration.
Objective To explore the effects of overexpression of human tissue inhibitors of metalloproteinase-1 (hTIMP-1) on proliferation of human liver cancer cell line HepG2 in vitro. Methods A recombinant adenoviral vector containing full-length cDNA of hTIMP-1 was generated and transfected into HepG2. The viral titer was checked by measuring GFP, and the expression of hTIMP-1 in vitro was detected by the techniques of Western blot and semi-quantitative RT-PCR. The ultrastructure was observed by transmission electron microscope and the effects of overexpression of hTIMP-1 on proliferation of HepG2 in vitro was analyzed by MTT assay and growth curve. Results The resultant AdhTIMP-1 was successfully constructed and the expression of hTIMP-1 was detected by Western blot and RT-PCR. The growth and proliferation of HepG2, which had been transfected with AdhTIMP-1, was significantly inhibited. Conclusion The proliferation of HepG2 was markedly inhibited by recombinant adenovirus-mediated overexpression of hTIMP-1, which may pave the way for further application in liver gene therapy.
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 replication-defective adenovirus containing tk gene (ADV-tk). Methods Recombinant adenovirus of ADV-tk was constructed using homologous recombination in cells. After the interested tk gene fragment in the recombinant plasmid obtained was confirmed by PCR, the titre of purified recombinant adenovirus was detected. In vitro study, tk gene in SMMC7721 cells transfected by ADV-tk was investigated by RT-PCR. In vivo study, ADV-tk was injected intraperitoneally into BALB/c nude mice with liver cancer and apoptosis cells in tumor were observed. Results Recombinant adenovirus containing ADV-tk was proved successfully. The titre of purified recombinant adenovirus was 1.4×1010 pfu/ml. In vitro study, tk was integrated and expressed by SMMC-7721 cells. In vivo study, with the injection of ADV-tk, apoptosis cells in tumor increased. Conclusion A replication-defective adenovirus containing tk gene is successfully constructed, which may useful for further research on tumor suicide gene therapy with ADV-tk.
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.
Objective To investigate the effect of Adenovirus-mediated averse vascular endothelial growth factor165（Ad-aVEGF165）on the growth of human melanoma cells(A375) in vivo and in vitro.Methods In vitro,the 100 multiplicity of infection of Aadenovirus-mediated green fluorescent protein（Ad-GFP）and Ad-aVEGF165 were transfected into human endothelium cell of vessel 304（ECV 304） and A 375. ECV 304 cells were divided into 3 groups: A 375 group, AdGFP group and AdaVEGF 165group. A375cells were also divided into 3 groups:1640 group, Ad-GFP group and AdaVEGF165 group. Their effects were analyzed by proliferation assay, cell cycle, and VEGF expression. In vivo,A375cells were injected into the axilla of the nude mouse. When the tumor formed, they were transplanted into another 15 mice. After treatment, the tumor was excised for naked eye observation, HE observation and microvascular density(MVD) counting. Results The cell supernatant fluid of A 375 group and AdGFP group could stimulate ECV304 cell growth，butthat of AdaVEGF165 group could inhibit the growth of ECV304 cell.All the A375cells in 3 groups had the proliferation trend, showing no statistically significant difference（Pgt;0.05）. ECV 304 cell proliferation index(PI) in Ad-aVEGF165group reduced（Plt;0.05）. There was no statistically significant difference（Pgt;0.05） in the PI of A 375 cell. The A 375cell integral optical densities were 234.41±13.8 in 1640 group， 222.73±3.67 in AdGFP group and 180.84±6.34 in Ad-aVEGF165group. The tumor volume in Ad-aVEGF165 group was smaller than that in Ad-GFP group and PBS group at 2 weeks after operation, the trend became much obvious with the time delay. AdaVEGF165 brought to much tissue necrosis under HE stain. The MVD of PBS group, Ad-GFP group and Ad-aVEGF165group were 65 10/view,52±11/view and 30±6/view, respectively. Conclusion In Vitro, Ad-VEGF 165gene could inhibited ECV304 cells’ growth by weakening VEGF expression of A 375cells. In vivo, Ad-aVEGF 165could inhibit the growth of human melanoma from blockinmicrovascular.
Objective To review the current concepts of gene therapy approachesmediated by adenovirus vectors for bone trauma and bone disease. Methods The recent literature concerned gene therapy mediated by adenovirus vectors was reviewed, which provides new insights into the treatments of bone trauma and bone disease. Results Adenovirus vectors was efficient, achieved high expression after transduction, and could transfer genes to both replicating and nonreplicating cells, such as osteoblasts, osteoclasts, fibroblasts, chondrocytes, bone marrow stromal cells, etc. Gene therapy mediated by adenovirus vectors achieved affirmative results in enhancing bone union and in curing bone diseases, such as osteoporosis and rheumatoid arthritis. Conclusion Gene therapy mediatedby adenovirus offers an exciting avenue for treatment of bone trauma and bone diseases.