VEGF165通过MAPK信号通路调节uPA蛋白表达的实验研究-外科学(血管外科)专业论文.docxVIP

Abstract PAGE PAGE III ABSTRACT Objective: In this study,we measured the phosphorylation of ERK, p38 and JNK and the expression of urokinase-type plasminogen activator（uPA）in human umbilical vein endothelial cells (HUVECs) cultured with vascular endothelial growth factor 165 ( VEGF165) , in order to investigate the role of MAPK signal transdution pathways in the effect of VEGF165 on uPA protein expression in HUVECs. Methods: Prove the influence of VEGF165 to HUVECs’viability ： HUVECs were cultured in different densities of VEGF165（0 ng/mL, 5 ng/mL, 10 ng/mL, 30 ng/mL and 50 ng/mL）, then observed the cell proliferation by CCK-8. Validate the effect exerted by VEGF165 on uPA protein expression: Divided HUVECs into two groups, one was incubated with 10 ng/ml VEGF165 and the other one with no VEGF165 as control. Then analysed the expression of uPA protein of each group at 24 h, 48 h, and 72 h by Western blot (WB). Demonstrate the role of VEGF165 on MAPK signal pathway: Divided HUVECs into two groups, one was cultured with VEGF165 and the other was for contrast with no VEGF165.Then analysed the phosphorylation of ERK, p38 and JNK in these two groups at 0 h, 6 h, 12 h and 24 h by Western blot. Confirm the related MAPK signaling pathways to the uPA protein expression: In order to identify whether the expression of uPA was ERK signaling pathway dependent, we used PD98059, a specific blocking agent of ERK signaling pathway ． Groups were like below: control group, add VEGF165 , PD98059 pretreated for 1h, PD98059 pretreated for 1h then add VEGF165. Analysed the expression of uPA protein and phosphorylation of ERK, p38 and JNK by WB. In order to identify whether the expression of uPA protein was p38 signaling pathway dependent, we used SB203580, a specific blocking agent of P38 signaling pathway．Groups and the analyses were like above．Also in order to identify whether PAGE PAGE IV the expression of uPA protein was JNK signaling pathway