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目的:观察FOXO3a(forkhead box O3a)的活性改变对内皮祖细胞(endothelial progenitor cells,EPCs)增殖和细胞周期相关蛋白表达的影响。方法:将携带突变激活FOXO3a基因的腺病毒载体Ad-TM(triple mutant)-FOXO3a和阴性对照腺病毒载体Ad-GFP体外感染人脐血来源的EPCs。观察EPCs形态学改变,CCK-8分析转染后EPCs增殖情况,Western blot检测FOXO3a蛋白、细胞周期相关蛋白p27kip1以及CDK2的表达水平。结果:构建了的2种腺病毒相关载体被成功转染。形态学改变方面,Ad-TM-FOXO3a组EPCs细胞生长缓慢,集落不明显;Western blot和CCK-8结果显示,Ad-TM-FOXO3a转染组与阴性对照组相比,EPCs增殖被抑制,FOXO3a与p27kip1蛋白过表达,CDK2表达下调。结论:FOXO3a可能通过上调p27kip1蛋白表达,下调CDK2表达,以抑制EPCs增殖。
Objective: To observe the effect of FOXO3a (forkhead box O3a) on the proliferation and the expression of cell cycle related proteins of endothelial progenitor cells (EPCs). METHODS: Human umbilical cord blood-derived EPCs were infected in vitro with adenovirus vector Ad-TM-FOXO3a and negative control adenovirus Ad-GFP carrying the mutant FOXO3a gene. The morphological changes of EPCs were observed. The proliferation of EPCs after transfection was analyzed by CCK-8. The expression of FOXO3a, p27kip1 and CDK2 were detected by Western blot. Results: Two constructed adenoviral vectors were successfully transfected. In morphological changes, the growth of EPCs in Ad-TM-FOXO3a group was slow and the colonies were not obvious. The results of Western blot and CCK-8 showed that the proliferation of EPCs was inhibited in Ad-TM-FOXO3a transfected group compared with that in negative control group. FOXO3a With p27kip1 protein overexpression, CDK2 expression was down-regulated. Conclusion: FOXO3a may down-regulate the expression of p27kip1 and down-regulate the expression of CDK2 to inhibit the proliferation of EPCs.