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Factors that contribute to angiogenesis contain direct inducing factor and indirect inducing factor[1-3]. Vascular endothelial growth factor (VEGF) is a powerful vascular permeability factor. In particular, VEGF165 and VEGF121 effects are significant. VEGF165 is half soluble and half insoluble proteins in a body, and can secrete in vitro. Some are combined with cell membrane or intercellular substance. However, VEGF121 is completely soluble, does not bind with the weak acidic protein of heparin, completely secrete outside cells and floate in intercellular substance, can better exert paracrine effects, and promote angiogenesis[4-8]. Flt-1 receptor of VEGF could be found in osteoblasts; VEGF induced chemotaxis through strongly expressed Flt-1 receptor in osteoblasts. Thus, osteoblasts gathered in defect sites, and differentiated into osteogenic tissue affected by VEGF[9-25]. The present study used humanized Renilla reniformis green fluorescent protein (hrGFP-1) as report gene, constructed adenovirus expression system containing VEGF121-FLAG and hrGFP-1 gene, and observed its expression in bone marrow stromal stem cells (BMSCs) in vitro, in the hope of providing experimental evidences for gene therapy for ischemic disease in the Department of Orthopaedics.

In the process of gene therapy, report gene-GFP was used to study intensity or time of exogenous gene expression in vivo. Enhanced GFP is commonly utilized to monitor in vivo gene expression and report gene of intracellular protein localization[27-28]. This study used hrGFP-1, which is twice lighter than enhanced GFP, can be easily detected, and has a small toxicity on mammalian cells. Following Ad-+VEGF121-IRES-hrGFP-1 transfection into BMSCs, target gene and hrGFP-1 transcription were on the same mRNA; internal ribosome IRES sequencing was bound with ribosome, which completed the simultaneous translation of target gene[29-32]. Utilizing PCR technique, VEGF121 gene termination codon was removed. NotI and Xho I restriction sites were added before and after gene sequence. Gene can localize between NotI and XhoI of pShuttle CMV-IRES- hrGFP-1 multiple clone sites. FLAG antigen labeling sequence is visible prior to shuttle vector internal ribosome entry site sequencing. Following translation removal and termination codon, base sequence from VEGF121 gene order translation initiation codon to FLAG epitope gene cannot lead to frameshift mutation of FLAG epitope gene, but result in read-through of two genes, and finally confluence of VEGF121 and FLAG proteins. The isomers of VEGF contain six types, but lack of antibody of VEGF121. Thus, FLAG antigen labeling is very important for VEGF121 specificity test. As a vector of mediating VEGF gene, adenovirus is characterized by ① extensive host cells in adenovirus; infection on dividing and nondividing cells; ② high target gene expression; ③ carried gene cannot integrate into genome of host cells, without the risk of insertion mutation; ④ big adenoviral genome, whereas viral reproduction-needed cis element area is small[33-34]. Up to now, no reports have addressed that recombinant adenovirus lead to tumor or other genetic diseases. Thus, it can be utilized as a satisfactory vector for VEGF gene, and introduced into host cells to satisfy the requirement of VEGF during the treatment of ischemic disease in the Department of Orthopaedics. The present study successfully constructed recombinant adenovirus vector containing VEGF121 and hrGFP-1 gene, and verified this recombinant adenovirus expressed target gene in eukaryotic cells. Therefore, author constructed recombinant adenovirus can be employed in following studies, and lay the foundation of in vivo local application of VEGF121 gene therapy for ischemic disease.

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