| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 92 === Angiogenesis is a term that describes the formation of new capillaries from a pre-existing vasculature and is a crucial mechanism required for a number of physiological and pathological events. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a prime regulator of angiogenesis, vasculogenesis, and vascular permeability. It is now well established that alternative exon splicing of a single VEGF gene results in at least six isoforms, having respectively 121, 165, 189, 206, 145 and 183 amino acids. To investigate the roles of different VEGF isoforms in non-small cell lung cancer tumorigenesis and angiogenesis, the four major isoforms (VEGF121, VEGF165, VEGF189 or VEGF206) were overexpressed in a female lung adenocarcinoma cell line (CL1-0). CL1-0 cells were stably transfected with constructs encoding VEGF cDNA under the control of the Tet-Off system. In the system, the tetracycline-controlled transactivator (tTA), is encoded by the pTet-Off regulator plasmid, binds the tetracycline-response element (TRE) and turn on the VEGF gene expression. Successfully transfected clones were identified using real-time quantitative RT-PCR to detect VEGF mRNA. Secretion of VEGF protein into the conditioned medium was assayed using a VEGF-specific ELISA. The possible autocrine effects of VEGF were ruled out by observations that overexpression of four VEGF isoforms did not have a significant effect on the in vitro growth rates of CL1-0 cells in monolayer culture. The condition medium derived from VEGF165-overexpressing CL1-0 cells could effectively stimulate mitogen activated phosphorylated kinase (MAPK) phosphorylation in human umbilical vein endothelial cells (HUVECs) than other VEGF isoforms. To examine the effects of VEGF on tumor growth in vivo, VEGF-overexpressing CL1-0 cells were injected subdermally into SCID mice. The results indicated tumor overexpressing VEGF165 generated dense vessel networks. Therefore, we suggest that VEGF165 is a more potent tumor angiogenesis activator than other VEGF isoforms.
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