| Great progresses have been made in gene therapy in recent years, and virus vectors become the leading vector for gene delivery. But virus vectors have many disadvantages such as limited capacity for foreign genes, immunogenicity, difficulty in manufacture and storage, which result in the development of nonvirus vectors. In recent years, more and more researchers focus on receptor-mediated gene transfer, which have many advantages such as very low immunogenicity, relatively simple manufacturing and storage, safety and relatively targeting. Asialoglycoprotein receptor, transferrin receptor and receptors for lactoferrin, insulin, epidermal growth factor, folic acid and mannose have been used in receptor-mediated gene delivery.Tumor metastasis is the important cause for death of most cancer patients. Urokinase receptor and some members in plasminogen activation system play important roles in tumor metastasis, and they have become the main target for tumor therapy. Several technical methods affecting tumor growth and metastasis by targeting the uPA system have been explored, which are monoclonal antibodies directed to uPA or uPAR, soluble recombinant uPAR, uPA-derived peptides to block uPA/uPAR interaction and antisense gene of uPAR.To observe the effect of antisense gene of uPAR on tumor cells and investigate the possibility of receptor-mediated gene delivery targeting uPAR, we expressed antisense gene of uPAR in human lung giant caner cell lines 95D using recombinant adenovirus and ligand-conjugated poly-L-lysine-DNA complexes as vector to mediate uPAR antisense gene delivery for inhibiting invasive ability of 95D cells in vitro.Section 1 Recombinant adenovirus mediated antisense uPAR gene deliveryIn order to study the effect of uPAR antisense gene on inhibition of invasive ability of human lung giant caner cell lines 95D, 500bp fragment of uPAR cDNA between -46bp~+454bp was amplified, and recombined into plasmid pAdeno-X. The recombinant vectors were named pAdeno-X-uPAR(-) and pAdeno-X-uPAR(+) respectively. 7 days after transfecting HEK293 cell with linearized pAdeno-X-uPAR(-) and pAdeno-X-uPAR(+), the recombinant adenovirus can be obtained, which were named Ad-uPAR(-) and Ad-uPAR(+) respectively. The virus titre(pfu/ml) ofAd-uPAR(-) was 1.5 x108, and the virus titre of Ad-uPAR(+) was 0.5x108. 95D cells were infected with Ad-uPAR(-) in different multiplicity of infection (MOI). Northern blot analysis could detect the expression of antisense and sense RNA for 500bp fragment of uPAR gene. With the increase of MOI, Western blot analysis indicated that the uPAR protein level decreased, and modified Boyden's Chamber assay suggested that the invasive ability of 95D cells also decreased obviously. The results indicated that adenovirus mediated uPAR antisense gene delivery could downregulate uPAR protein level and inhibit the invasive ability of 95D human lung cancer cells.Section2 Urokinase receptor mediated reporter gene deliveryRecombinant fusion proteins that combine different functions required for cell type-specific uptake and intracellular delivery of DNA present an attractive approach for the development of nonviral vectors for targeted gene delivery. Here, we described a novel protein termed ATF-lys10 which composed of amino-terminal fragment of urokinase and ten lysines at the carboxyl terminus and facilitated cell-specific gene transfer via receptor-mediated endocytosis. Bacterially expressed ATF-lys10 proteins existed in soluble form, and had antigenicity of human urokinase. Purified ATF-lys10 specifically bound to uPAR-expressing cells and formed protein-DNA complexes with plasmid pGL3-control. These complexes after compensation of excess negative charge with poly-L-lysine, served as a specific gene delivery vector for uPAR-expressing cells. Lysosomotropic compounds such as chloroquine drastically increased ATF-lys10 mediated gene delivery, but not fusion peptide of hemagglutinin. Our results suggest that fusion protein with the properties of DNA binding and cell type targeting repres...
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