ABSTRACT
<p><b>OBJECTIVE</b>To study the anti-tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro.</p><p><b>METHODS</b>RT-PCR technique was used to amplify Flk-1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET-28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR I and Hind III. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using VIII factor-related antigen antibody. The expressed product was analyzed about its effect on endothelial cell's growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection.</p><p><b>RESULTS</b>1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET-28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell(s), growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT-PCR and SDS-PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control.</p><p><b>CONCLUSION</b>Soluble VEGFR fragment is a kind of effective gene engineer product for anti-tumor angiogenesis gene therapy and the development of anti-tumor drug.</p>
Subject(s)
Animals , Mice , Cell Line, Tumor , Cell Proliferation , Cloning, Molecular , Endothelial Cells , Cell Biology , Genetic Vectors , Melanoma, Experimental , Metabolism , Pathology , Mice, Inbred BALB C , Neoplasm Transplantation , Neovascularization, Pathologic , Pathology , RNA, Messenger , Genetics , Retroviridae , Genetics , Sarcoma 180 , Metabolism , Pathology , Transfection , Vascular Endothelial Growth Factor Receptor-2 , Genetics , PhysiologyABSTRACT
Objective To observe the alteration of astrocyte and apoptosis in neonatal hypoxic-ischemic encephalopathy(HIE).Me-thods The pathological brain specimens of 25 newborns who died of HIE were observed macroscopically and microscopically,included male 14 cases and female 11 cases.The immunochemistry staining with glial fibrillary acidic protein(GFAP)and in situ end terminal dexynucleotidy 1 transferase mediated deoxyuridine triphosphate-biotion nick and labeling(TUNEL)methods were used to observe the changes of astrocyte and the neurons apoptosis in HIE.The relationship between the change of astrocyte and the neurons apoptosis and the occurrence of HIE and course of disease were further synthetically analyzed.Results The brain specimens of 25 newborn HIE had apoptosis cells at the different level.The degree of apoptosis of cerebral neurons was severe in the cases of 24 h-6 d survival(P