The Scaffolding Adapter Protein Gab1 Regulates BFGF-Induced Angiogenesis Through The PI3K-AKT Pathway

Objective To elucidate the role and mechanism of Grb2-associated binder 1 in basic fibroblast growth factor induced angiogenesis of vascular endothelium cells, providing experiment evidence for gene therapeutic of peripheral arterial occlusive disease.Methods Specific knockdown Gab1 of endothelial Eahy926 cells were generated by small interfere RNA-lentivirus system,and further was conformed whether the lentivirous-Gab1-si RNA could successful transfection by quantitative real-time PCR and western blot. Using western blot to detecting the optimal concentration and time of b FGF-induced angiogenesis of Eahy926 cells by analyzing the expression of p AKT and AKT. To directly demonstrate the role of Gab1 on b FGF-induced angiogenesis of Eahy926 cells, we utilizing the Matrigel tube formation model. Moreover, to illustrate the mechanism, we detected the expression of p Gab1, AKT, p AKT of downstream signaling pathway.Results ①Western blot showed the activation of AKT band and value were most remarkable stimulation with b FGF at the final concentration 30ng/ml.②Upon stimulation with 30ng/ml b FGF at the 30 minutes, the activation of AKT band and value were most remarkable as the western blot showed.③Compared with white light field, Eahy926 cell highly expressed green fluorescent protein at a multiplicity of infection of 100 on the fluorescent field. ④Quantitative real-time PCR demonstrated Gab1 m RNAs was severely impaired in Gab1 si RNA group. ⑤The total tube network length was shorted and its structure was simple and uncompleted in Gab1 si RNA group of Matrigel tube formation.⑥Western blot showed the activation of AKT band and value wereseverely impaired in Gab1 si RNA group compared to negative control group and blank control group.Conclusion ①The optimal concentration and time of Eahy926 cells stimulation with b FGF was 30ng/ml and 30 minutes respectively.②The si RNA-lentivirus could successful generated specific Gab1 knockdown of Eahy926 cells at a multiplicity of infection of 100.③The Gab1 protein was required in tube formation of Eahy926 cells.④The Gab1 protein regulated b FGF-induced angiogenesis through increase the impression of P85 and p AKT.