血管内皮生长因子体外诱导CD34^+造血干/祖细胞信号传导与转录活化因子的活化

Activation of Signal Transducers and Activators of Transcription Induced by Vascular Endothelial Growth Factor in CD34~ + Hematopoietic Progenitor Cells in Vitro

目的通过对血管内皮生长因子(VEGF)诱导CD34+造血干/祖细胞内信号传导与转录活化因子(STAT)活化的研究,探索VEGF作用于CD34+造血干/祖细胞的分子机制及其信号传导途径。方法利用磁活化细胞分选系统(MACS)分离并纯化脐血CD34+造血干/祖细胞,体外以重组人细胞因子VEGF(50ng/ml)持续刺激不同时间(0,15,30,45,60,90min)后,提取细胞总蛋白,用Westernblot检测CD34+造血干/祖细胞内STAT-3和STAT-5磷酸化;免疫细胞化学鉴定CD34+造血干/祖细胞表面VEGF受体-2(VEGFR2)的表达,以及VEGF刺激不同时间后CD34+造血干/祖细胞内磷酸化STAT-3和STAT-5有无发生转核;采用能与VEGFR2特异性结合的七肽ATWLPPR封闭VEGF与VEGFR2的结合,Westernblot观察STAT-3和STAT-5的磷酸化是否被相应阻断。结果Westernblot检测结果显示,CD34+细胞在VEGF刺激下,STAT-3和STAT-5均发生了磷酸化,50ng/mlVEGF作用15min后,即可检测到磷酸化的STAT-3和STAT-5的表达,并分别在30和45min时达到峰值;之后开始下降,到90min时均已检测不到,不同刺激时间组间有显著性差异(P=0.0001)。免疫细胞化学显示VEGF能诱导磷酸化STAT-3发生转核并且在作用30min时最为显著,不同刺激时间组间有显著性差异(P=0.0001),而磷酸化STAT-5在VEGF刺激的各时间组均未出现明显的转?

Objective To investigate the activation pattern of signal transducers and activators of transcription(STAT)induced by vascular endothelial growth factor(VEGF)in CD34 + hematopoietic progenitor cells,and gain an insight into the molecular mechanism and signal transduction pathway of VEGF that has an effect on CD34 + hematopoietic progenitor cells.Methods After isolated from umbilical cord blood by using a high-gradient magnetic ally activated cell sorting system(MACS),CD34 + cells were stimulated by VEGF(50ng/ml)for different time (0,15,30,45,60,90min)to detect the tyrosine phospho-rylation and nuclear translocation of STAT-3and STAT-5with Western blot and immunocytochemistry methods.The expression of VEGF receptor-2(VEGFR2)on the membrane of CD34 + progenitor cells was examined by immunocytochemistry.ATWLPPR,an effective peptide screened from phage epitope library by affinity for membrane-expressed VEGFR2and blocking the binding of VEGF to VEGFR2, was used to determi ne whether the activation of STAT pathway induced by VEGF was blocked.Results Tyrosine phosphorylation of STAT-3and STAT-5was undetectable in unstimulated CD34 + cells,but was evident at15min in response to VEGF stimulation.VEGF resulted in a rapid and transient tyrosine phosphorylation of STAT-3and STAT-5.The maximal tyrosine phosphorylation was catched at30and45min,respectively(P=0.0001),and returned to basal levels at90min.Immunocytochemistry confirmed that increased phosphorylated STAT-3was translocated into the nuclei at30min(P=0.0001),and mainly in cytoplasms again at90min after stimulation with VEGF.However,compared with unstimulated CD34 + cells,there was only increased phosphorylation of STAT-5appeared mainly in cytoplasms,but no significant nuclear translocation was found after stimulation with VEGF(P>0.05).The presence of VEGFR2was confirmed using anti-VEGFR2antibody staining by immunocytochemistry,moreover,the phosphorylation of STAT-3and STAT-5failed to be activated by the co-culture with ATWLPPR and VEGF,suggesting that activation of the STAT pathway be specifically mediated by VEGFR2in CD34 + progenitor cells.Conclusions STAT signaling pathway participates in the signal transduction of VEGF via VEGFR2in CD34 + hemopoietic progenitor cells.