Effects Of Survivin Overexpression Mediated By Liposome On Biological Behavior Of HUVECs

Infantile hemangioma is the most common benign tumor of infancy,which is a typical representative of the "angiogenesis dependent" disease,occuring in10~12%.The ratio of male to female is1:3to5. Hemangiomais made up of a large number of new blood capillaries and the proliferationof endothelial cells,and endothelial cell proliferation and apoptosis is thecore of angiogenesis. How to intervene the key genes of angiogenesis tocontrol the proliferation and apoptosis of endothelial cells,what is the keyto promote or inhibit angiogenesis.So far the molecular mechanisms ofendothelial cell proliferation and apoptosis is not entirely clear. Survivin isthe strongest inhibitor of apoptosis by far,which is newly discovered.SomeStudies and preliminary study of our group found that the expression ofsurvivin,VEGF were different and related in the different periods ofhemangioma.In our study,by ouing liposome-mediated transfectionmethod,we transfected survivn gene into the Human umbilical veinendothelial cells in vitro to upregulate the expression of survivin,in order toinvestigate the effects of the over-expression survivin on the biologicalactivity of vascular endothelial cells and to reveal the role of survivin genein angiogenesis, which to provide the theoretical and experimental basis forfinding a new target for the promotion and inhibition of angiogenesis.Part one Construction of eukaryotic expression vectorcontaining human survivin geneObjective: To construct the human survivin gene eukaryotic expressionvector with the green fluorescent protein, which is named recombinantplasmid pEGFP-N3/survivin. Method: Using RT-PCR technology, obtaine the target gene of survivin with EcoR I and Bgl II enzyme cutting site in theproliferative hemangiomas endothelial cells,after T-A cloning,then clonedinto plasmid pEGFP-N3to construct recombinant plasmidpEGFP-N3/survivin,which was identified by restriction enzymes,and DNAsequencing.Results:The target gene sequence of pEGFP-N3/survivin wereconfirmed correct by restriction enzyme digestion and sequencinganalysis.Conclusion: The human survivin gene eukaryotic expressionvector pEGFP-N3/survivin was successfully constructed, which can meetthe needs of this experiment. Objective: Liposome-mediated transfection of survivin gene into HUVECsto change the biological behavior,promote cell growth and proliferation,aiming to explore the mechanism of survivin gene involved inangiogenesis.Methods:Up-regulate survivin expression by liposomemediated recombinant plasmid pEGFP-N3/survivin transfectedHUVECs;RT-PCR assay for the detection of Survivin,VEGF mRNAexpression, immunocytochemistry and Western-blot assay for the detectionof Survivin,VEGF protein expression;MTT and cell growth curve wasdrawn to observe the proliferation ability of HUVECs.Results:1.Liposome mediated recombination plasmid pEGFP-N3/survivinsuccessfully transfected HUVECs,cytoplasm visible green fluorescenceunder inverted fluorescence microscope;2.By immunocytochemistry,RT-PCR and Western-blot detected that the Survivin, VEGF mRNA andprotein expression were upregulated in the transfected cells compared withthe normal control group and there were significant differences (p<0.05);3.MTT detection and mapping of the cell growth curve observed the growth and proliferation ability were enhanced in the transfected cells. Conclusion:1.Liposome mediated recombinant plasmid pEGFP-N3/survivinsuccessfully transfected HUVECs,could upregulate survivin geneexpression in the cells;2.survivin gene overexpression improved the growthand proliferation capacity of the cells;3.Survivin gene overexpression canchange the biological behavior of HUVECs,upregulate VEGF expression,both having relevancet;4.Survivin gene may be a key gene controllingangiogenesis,thus becoming the new target to promote and inhibitangiogenesis.