温州医科大学学报
溫州醫科大學學報
온주의과대학학보
Journal of Wenzhou Medical University
2014年
12期
882-886
,共5页
血管内皮生长因子%内皮生长晕细胞%冻存%凋亡%信号通路
血管內皮生長因子%內皮生長暈細胞%凍存%凋亡%信號通路
혈관내피생장인자%내피생장훈세포%동존%조망%신호통로
vascular endothelium growth factor%endothelial outgrowth cells%cryopreservation%apoptosis%signaling pathway
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。
目的：探討血管內皮生長因子（VEGF）對低溫凍存內皮生長暈細胞（EOCs）凋亡率的影響及其與PI3K-Akt-Bad信號通路的關繫，研究VEGF降低低溫凍存EOCs凋亡率的可能機製。方法：採用密度梯度離心法分離人臍帶血中的單箇覈細胞，經體外擴增培養EOCs，用免疫細胞化學染色及熒光染色法鑒定細胞的內皮特性。以擴增後的第二代細胞為生物材料，將其分為W組（wortmannin榦預24 h後凍存）、W+V組（wortmannin 榦預24 h後+50μg/L VEGF凍存）、BC組（空白對照組）、V組（50μg/L VEGF凍存），于-80℃凍存24 h後複囌。用流式細胞術檢測EOCs凋亡率，Western blot法檢測p-Akt、Bad、Caspase 3的錶達量。結果：採用體外擴增培養的EOCs具有多種內皮細胞特性。低溫凍存會增加EOCs的凋亡率（為5.36%±0.27%），但50μg/L VEGF能夠降低低溫凍存和複囌過程EOCs的凋亡率（為3.36%±0.27%，P＜0.05）；經wortmannin對PI3K特異性抑製後，VEGF對EOCs的保護作用減弱，細胞的凋亡率增加（為8.34%±0.57%，P＜0.05）；加50μg/L VEGF的EOCs凍存細胞p-Akt錶達升高而Bad和Caspase 3錶達降低（均P＜0.05），經wortmannin榦預24 h後的EOCs凍存細胞p-Akt錶達降低而Bad和Caspase 3錶達升高（均P＜0.05）。結論：50μg/L VEGF能夠降低低溫凍存EOCs的凋亡率，其作用可能通過PI3K-Akt-Bad信號通路來實現。
목적：탐토혈관내피생장인자（VEGF）대저온동존내피생장훈세포（EOCs）조망솔적영향급기여PI3K-Akt-Bad신호통로적관계，연구VEGF강저저온동존EOCs조망솔적가능궤제。방법：채용밀도제도리심법분리인제대혈중적단개핵세포，경체외확증배양EOCs，용면역세포화학염색급형광염색법감정세포적내피특성。이확증후적제이대세포위생물재료，장기분위W조（wortmannin간예24 h후동존）、W+V조（wortmannin 간예24 h후+50μg/L VEGF동존）、BC조（공백대조조）、V조（50μg/L VEGF동존），우-80℃동존24 h후복소。용류식세포술검측EOCs조망솔，Western blot법검측p-Akt、Bad、Caspase 3적표체량。결과：채용체외확증배양적EOCs구유다충내피세포특성。저온동존회증가EOCs적조망솔（위5.36%±0.27%），단50μg/L VEGF능구강저저온동존화복소과정EOCs적조망솔（위3.36%±0.27%，P＜0.05）；경wortmannin대PI3K특이성억제후，VEGF대EOCs적보호작용감약，세포적조망솔증가（위8.34%±0.57%，P＜0.05）；가50μg/L VEGF적EOCs동존세포p-Akt표체승고이Bad화Caspase 3표체강저（균P＜0.05），경wortmannin간예24 h후적EOCs동존세포p-Akt표체강저이Bad화Caspase 3표체승고（균P＜0.05）。결론：50μg/L VEGF능구강저저온동존EOCs적조망솔，기작용가능통과PI3K-Akt-Bad신호통로래실현。
Objective: To investigate the impact of vascular endothelial growth factor (VEGF) on the apop-tosis 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 cryo-preservation 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 identiifed by immunocytochemical staining and lfuorescence 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 lfow 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 cryo-preservation 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 PI3K 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 PI3K 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 de-creases the apoptosis rate of cryopreserved EOCs partly via PI3K-Akt-Bad signal pathway.
