| Background and Objective:Vascular endothelial growth factor C (VEGF-C) is a new member of vascular endothelial growth factor/platelet-derived growth factor (VEGF/PDGF) family. It is the first ligand to be discovered for VEGFR-3 (Flt4), is a kind of polypeptide growth factors. VEGF-C has many biological activities binding with tyrosine kinase receptors VEGF receptor-2 (VEGFR-2) and VEGF receptor-3 (VEGFR-3). It is expressed in many human normal cells, tumor tissues, mouse embryo and avian embryo at different levels which is regulated by many elements. It plays important roles in embryonic development, differentiation of some cells and proliferations of vascular and lymphatic endothelial cells.Vascular endothelial growth factor (VEGF) is one of the important vascular endothelial growth factor, while VEGF-C which belongs to the same family with it is regarded the most important lymphatic endothelial growth factor. Overexpression of VEGF-C cDNA in the skin of transgenic mice induced lymphatic endothelial cell proliferation and hyperplasia of the lymphatic vasculature. However, there are no same effects on vascular vessels. Recombinant VEGF-C specifically stimulated lymphangiogenesis in chorioallantoic membrane. Recently studies of VEGF-C mainly focused on its expression in human tumor tissues, the relationship between it and lymphangiogenesis in tumor and that between it and carcinoma lymphatic spread. Many experimental results have suggested that the expression of VEGF-C is positively relative to the pericarcinoma lymphangiogenesis and metastasis of cancer cells by lymphatic vessels, the most important mechanism for it maybe : the increasing lymphatic vascular caused by VEGF-C make it easier for tumor cell cut through the lymphatic endothelium and metastasis. Another important research area for VEGF-C is: VEGF-C has the therapeutic potential for lymphatic insufficiency. Ithas been reported that the VEGF protein has the capacity to induce therapeutic lymphangiogenesis in acquired lymphedema rabbit ear model.Up to now, VEGF, another member of VEGF family, has been applied in gene therapies for ischemic diseases in cerebro-cardiovascular system, which has obtained great achievements. Such studies in some advanced countries have entered the first stage of clinical experiments. However, the study on application of VEGF-C gene just begins.Since 1999 our experimental team has performed a series of experiments to study the biological role of VEGF-C in carcinoma lymphatic spread and its application. To get some clues of the mechanism by which VEGF-C works in lymphangiogenesis and regulating lymphatic endothelial cells and the new method of inhibiting carcinoma lymphatic metastasis, and to further study on gene therapy for some diseases with defective lymphatic vessels, we designed and completed the following three parts experiments. Part 1: Construction of eukaryotic expression vector for human VEGF-C geneand its expression in CHO cells. Methods:1. According to human VEGF-C cDNA sequence, we designed and constructed a pair of specific primers who contained respectively digestion site of EcoR I and BamH I on the 5' end. Then RT-PCR was employed to clone VEGF-C cDNA from human breast cancer cell MCF-7. After being purified, the product of RT-PCR was ligated into a clone vector pMD18-T. The recombinant plasmid, pMD18-T first propagated in Ecoli DHS a , then extracted, purified and digested with EcoR I and BamH I, were confirmed to contain full length of VEGF-C cDNA by agarose gel analysis and DNA sequence analysis.2. The resulting EcoR I -BamH I fragment, which contained the full length of human VEGF-C cDNA, was purified and ligated into eukaryotic expression vector pcDNA3.1 (-) digested with EcoR I and BamH I with T4 DNA ligase. The pcDNA3.1(-)/VEGF-C was digested with EcoR I and BamH I to confirm it contains the VEGF-C cDNA sequence by agarose gel electrophoresis.3. The pcDNA3.1 (-)/VEGF-C plasmid was transfected into CHO cells by lipofectamine 2000.CHO cells was selected by G418. We apply western-bloting using collecte...
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