Effect Of VEGF Secreted By Hep-2 Cells On The Proliferation And Differentiation Of HUVEC Under Normoxia Or Hypoxia Condition

Objectives:Similar to most tumors, some cells in laryngocarcinoma under hypoxia condition. The durative growth and metastasis of the tumor depend on neovascular formation mostly. Terefore, the therapy target of neovessels has recently become the new hotspot of tumor biological research. This study was carried out to investigate the expression of vascular endothelial growth factor (VEGF) in human laryngeal squamous cells (Hep-2) and the cultrue supernatant, and to study the expression of vascular endothelial growth factor receptor 1 (Flt-1) and vascular endothelial growth factor receptor 2 (KDR) in human umbilical vein endothelia cell (HUVEC) when cultrued in laryngeal squamous cells cultrue supernatant, and further to explore the certain effect of these protein in metastasis of laryngocarcinoma. Our findings may provide a new experiment foundation for treatment of head and neck neoplasms.Methods:1 Hep-2 cells were cultrued in the medium of RPMI-1640 containing 10% newborn calf serum, penicilin (100U/mL) and Stmycin (100μg/mL) under normoxic condition of 37℃, 5% CO₂ and 20% O₂ or under hypoxic condition of 37℃, 5% CO₂, 2% O₂ and 93% N₂.2 When the cells grow into the exponential phase, the Hep-2 cells were diviced into 2 groups: group A (normoxia 24h) and group B (hypoxia 24h). Supernatant of each group was collected, and was applied to stimulate HUVEC growth. The expression level of Flt-1 and KDR in HUVEC were detected by immunocytochemical. The proliferation of HUVEC was measured by MTT. 3 At the exponential phase, the Hep-2 cells were also diviced into 2 groups: group A (normoxia 3h, normoxia 6h, normoxia 12h, normoxia 24h, normoxia 48h) and group B (hypoxia 3h, hypoxia 6h, hypoxia 12h, hypoxia 24h, hypoxia 48h). Cells and supernatant of each group were collected and the expression level of VEGF was measured by ELISA.Results:1 Immunocytochemical results showed that the positive particles of Flt-1 and KDR was brown and mainly located in the cytoblastema and the nucleus of HUVEC. Furthermore, the expression level of Flt-1 and KDR in Hep-2 cells under hypoxic condition (48h) was higher than that under normoxic condition (48h), and the difference was significant (P<0.05).2 MTT results: The supernatant of Hep-2 cells can stimulate the growth of HUVEC no matter under normoxic condition or hypoxic condition. Moreover, proliferation of HUVEC was higher in hypoxic group (48h, 72h, 96h, 120h) than in normoxic group (48h, 72h, 96h, 120h) (P<0.05).3 ELISA results indicated that the VEGF expression level of Hep-2 cells and their supernatant in the group of normoxia(12h, 24h, 48h) was lower than that in hypoxia(12h, 24h, 48h) and the difference was significant (P<0.05).Conclusions:1 Supernatant of Hep-2 cells can greatly raise the expression of Flt-1 and KDR in HUVEC under hypoxic condition.2 Supernatant of Hep-2 cells can significantly improve the growth of HUVEC under hypoxic condition.3 The expression of VEGF in Hep-2 cells was notably increased under hypoxic condition.