The Experiment Of Adenovirus-mediated Vascular Endothelial Growth Factor Gene Targeting Transferred To The Cardiomyocyte

Objective: Numerous experiments and clinical trials have showed gene therapy's potentiallyfavorable future for ischemic heart disease, however, though many cardiovascular diseasesare theoretically amenable to this approach, translating the fruits of basic scientific progressinto clinical benefits remains a formidable challenge. At present, higher than it's reality tothe gene therapy's appraisal because of the defects of vectorsz gene deliveryz targets formyocardial gene therapy and so on. One important issue in most gene therapeutic approachesis the desirability for more precise gene delivery to the target tissue or cell type. Non-targetgene expression limited the means of gene delivery and reduced the effect of gene therapy.Tissue-specific gene expression can enhances the efficiency of gene therapy byconcentrating the gene where it is wanted and reducing the risk of gene diffusion to other sitewhere expression may have deleterious consequences. In the present study, we establish a recombinant adenovirus vectors (Ad.mlc-hVEGF165and Ad.mlc-GFP), in which the VEGF or GFP gene are under the control of theventricle-specific myosin light chain-2 promoter. For controls, the recombinant adenovirusvector Ad.hVEGF165 and Ad.GFP were also constructed in step at the same time. Thenevaluated the gene transfer targeting with cardiomyocyte in vitro /vivo and analysis thedifference between Ad.mlc-hVEGF165 and Ad.mlc-hVEGF165 in secrete VEGF and theirfunction in promoting endothelial cell's hyperplasia in vitro.Methods:1. Construction of Replication-deficient recombinant adenovirus coding for VEGF genewith the promoter mlc-2v(Ad.mlc-hVEGF165). Adenovirus shuttle vector PDC317 come from the adenovirus shuttle vector PDC315by removing it's native promoter CMV, human vascular endothelial growth factor(hVEGF165)cDNA and the promoter mlc-2v were cloned into adenovirus shuttle vector PDC317 bystandard procedure, the recombinant adenoviral plasmid PDC-MLC-VEGF was identifiedand the correct clones containing target gene in right direction were selected. Adenovirus 5第二军医大学博士学位论文 英文摘要 胸心外科专业(WAd5) and PDC-MLC-VEGF were transferred to the adenoviral packaging cell HEK 293cell by lipofectamine 2000 mediated gene transfer method to pack the virus; The desired Advectors were purified by density gradient ultracentrifuge and titrated in 293 cells afteridentified. We harvested the adenovirus vectors of Ad.mlc-GFP,Ad.mlc-hVEGF165 andAd.mlc-GFP in step by the same way.2. the experiment of gene transfer targeting with cardiomyocyte in vitro. The neonatal rat cardiomyocyte,endothelial cell,skeletal muscle cell and smoothmuscle cell were isolated and cultured, a L-40 hepatocyte was cultured; transfection abilityand efficiency was examined at various titration by fluorescence microscope when Ad.GFPtransferred with cardiomyocyte and the other cells. These cells were all infected withAd.mlc-GFP and Ad.GFP in vitro at the same MOI, respectively, and transfer targeting forcarsiomyocyte was assessed by fluorescence microscope and flow cytometer.3. the experiment of gene transfer targeting for carsiomyocyte in vivo. Ad.mlc-GFP and Ad.GFP were injected into the SD rat's liver,kidney,spleen,myocardium and skeletal muscles,respectively, after three days animals were sacrificedand five different tissues were analyzed for report gene expression by fluorescencemicroscope,confocal microscopy and polymerase chain reaction.4. The experiment of Ad.mlc-hVEGF165 transferred with cardiomyocytes in vitro. The neonatal rat cardiomyocyte was infected with Ad.mlc-hVEGF165 or Ad.hVEGF165in vitro at same MOI, respectively. Gene transcription and protein expression were evaluatedby RT-PCR and Western blot ways, respectively, the concentration of VEGF in culturemedium was tested by ELISA, and the proliferation effect of it?...