Effects Of Survivin Antisense Oligonucleotide On Implanted Tumor Angiogenesis In Hemangioma Of Nude Mice

Hemangioma are formed by a lot of new blood capillaries and proliferation of endothelialcells.，Which is the disease of angiogenesis dependence. The core of angiogenesis isendothelial cell proliferation and apoptosis. How to intervene the key genes of angiogenesisto control the proliferation and apoptosis of endothelial cells is the key to angiogenesispromote or inhibit. Survivin is the strongest inhibitor of apoptosis which is newlydiscovered. Some studies and preliminary study of our group found that the expression ofsurvivin, VEGF, and caspase-3are different and related in hemangioma proliferation andapoptosis. In our study,by survivin and gene silencing technology, Hemangiomaendothelial cells in vitro cultivation were planted subcutaneously of nude mice to set upan human vascular tumors animal model of nude mice, Survivin antisenseoligodeoxynucleotide packaged with liposome was intervented to explore the effect ofsurvivin to vascular tumor angiogenesis,To clarify survivin and the closure of genetechnology on the role of Mechanism of angiogenesis in Hemangioma. To find a new targetfor the promotion and inhibition of angiogenesis provide the theoretical and experimentalbasis.Part one Culture and identification of endothelial cells from IE andobservation of biological character in vitroObjective： To isolated culture、purification and identification endothelial cells in vitrofrom infantile Proliferative phase hemangiomas. Methods: The endothelial cells of12cases fresh IH were cultured by explant method，After the success of cell culture，The cellcolonies without the gross morphology of endothelial cell were detached by scraping.When the bottom of the bottle was fully covered by endothelial cells, the primary cellswere subcultured. At the same time we purified the cells using differential adhesion methodand trypsin digestion method several times. Immunohistochemical staining of anti-factor Ⅷ associated protein, anti-CD31, and anti-CD34was performed respectively toidentificate the EC origin of the fourth generation cells. Purity of endothelial cell wasdetected with CD34by Immunohistochemical staining. The growth curves of cells weredrawed by counting the number of cultured cells. Results: Vascular endothelial cells weresuccessfully cultivated from specimens in12cases of hemangioma. Cultured endothelialcells were round, oval or polygonal and cell boundaries were clear. The nucleus was in thecenter of cell. The mitotic phase and dual-core were visible in some cells. Specific “pavingstone” feature of endothelial cells could be observed after cell fusion. It is indicated thatcultured ECs were homogenously positive for factor Ⅷ associated Protein, CD31andCD34by immunohistochemical staining. The number of CD34(+) cell was67.88%士4.25%in endothelial cell by inununohistochemical staining. The phenomenon of“formation tube” was found in some endothelial cells of IH. Conclusion：The purity of ECis high in the subculture cells and the cultured cells can answer for correlative experiments.Part two Effects of surviving antisense oligonucleotideon implantedtumorangiogenesis in hemangiomas of nude miceObjective：To explore the effect of Survivin ASODN on human proliferative vasculartumors implanted tumor growth in nude mice. Methods: The cultured cell concentrationwas adjusted to1.0×108/ml，Take0.2mL to Inject into nude mice subcutaneous，Humanvascular tumors of nude mice model was established. Tumor growth to diameter1cmwere randomly divided into3groups. Survivin and ASODN survivin packaged with liposomewere injected into tumor. Detect protein expression of survivin, VEGF, caspase-3byimmunohistochemistry in the tumor after the intervention. Resules: Human vasculartumors of proliferative phase animal model of implanted tumor in nude mice weresuccessfully established, In the ASODN/lip group, Tumor volume decreased in theintervention period, However, tumor volume increased in the NODN/lip group and thecontrol group. The tumor weight reduce than the other two groups, Inhibition tumor ratewas56.57%; The expression of suvrivin, VEGF protein were significantly decreased byImmunohistochemical staining in ASODN/lip group, The Upregulation expression ofCaspase-3was strongly positive. MVD of antisense group decreased significantly compared with the other two groups. Conclusion: Survivin ASODS in vivo transfectioncan effectively inhibit implanted tumor of human vascular tumors growth rate and tumorangiogeness, Tumor changes to the regression is accelerated.