摘要
目的:探讨血管内皮生长因子(VEGF)对低温冻存内皮生长晕细胞(EOCs)凋亡率的影响及其与PI3K-Akt-Bad信号通路的关系,研究VEGF降低低温冻存EOCs凋亡率的可能机制。方法:采用密度梯度离心法分离人脐带血中的单个核细胞,经体外扩增培养EOCs,用免疫细胞化学染色及荧光染色法鉴定细胞的内皮特性。以扩增后的第二代细胞为生物材料,将其分为W组(wortmannin干预24 h后冻存)、W+V组(wortmannin干预24 h后+50μg/L VEGF冻存)、BC组(空白对照组)、V组(50μg/L VEGF冻存),于-80℃冻存24 h后复苏。用流式细胞术检测EOCs凋亡率,Western blot法检测p-Akt、Bad、Caspase 3的表达量。结果:采用体外扩增培养的EOCs具有多种内皮细胞特性。低温冻存会增加EOCs的凋亡率(为5.36%±0.27%),但50μg/L VEGF能够降低低温冻存和复苏过程EOCs的凋亡率(为3.36%±0.27%,P<0.05);经wortmannin对PI3K特异性抑制后,VEGF对EOCs的保护作用减弱,细胞的凋亡率增加(为8.34%±0.57%,P<0.05);加50μg/L VEGF的EOCs冻存细胞p-Akt表达升高而Bad和Caspase 3表达降低(均P<0.05),经wortmannin干预24 h后的EOCs冻存细胞p-Akt表达降低而Bad和Caspase 3表达升高(均P<0.05)。结论:50μg/L VEGF能够降低低温冻存EOCs的凋亡率,其作用可能通过PI3K-Akt-Bad信号通路来实现。
Objective： To investigate the impact of vascular endothelial growth factor（VEGF） on the apoptosis rate in cryopreservation endothelial outgrowth cells（EOCs）, as well as the relationship between VEGF and the PI3K-Akt-Bad signaling pathway, to research the mechanism of VEGF reducing the apoptosis rate in cryopreservation EOCs. Methods： The mononuclear cells were harvested from umbilical cord blood, induced into EOCs and expanded in vitro. The endothelial characteristics of the EOCs were identified by immunocytochemical staining and fl uorescence staining. The second generation of EOCs were devided into group W（cryoperserved after wortmannin intervention for 24 hours）, group W＋V（cryopreserved with 50 μg/L VEGF after wortmannin intervention for 24 hours）, group BC（control group）, and group V（cryopreserved with 50 μg/L VEGF）. All these groups were resuscitated after being cryopreservated at-80 ℃ for 24 hours. Subsequently, apoptosis rates were detected by fl ow cytometry, and the expressions of p-Akt, Bad, and Caspase 3 were measured using Western-blot test. Results： The cells cultured by adherent method showed multiple endothelial characteristics. Although cryopreservation increased the apoptosis rate of EOC（5.36%±0.27%）, VEGF protected cells from deep hypothermia and thus reduced the apoptosis rate of recovery cells（3.36%±0.27%, P〈0.05）. Inhibition of PI3 K by wortmannin decreased the protection of VEGF on the EOCs and increased the cellular apoptosis rate（8.34%±0.57%, P〈0.05）. Western-blot test showed elevation of p-Akt expressions and decline of Bad and Caspase 3 expressions in EOCs cryopreserved with VEGF（P〈0.05）. Under the PI3 K inhibition by wortmannin, the expression of p-Akt was down-regulated while expressions of Bad and Caspase 3 were up-regulated（P〈0.05）. Conclusion： VEGF decreases the apoptosis rate of cryopreserved EOCs partly via PI3K-Akt-Bad signal pathway.
出处
《温州医学院学报》
CAS
2014年第12期882-886,共5页
Journal of Wenzhou Medical College
基金
温州市科技局科研基金资助项目(Y20110032)
关键词
血管内皮生长因子
内皮生长晕细胞
冻存
凋亡
信号通路
vascular endothelium growth factor
endothelial outgrowth cells
cryopreservation
apoptosis
signaling pathway
