PI3K/AKT/mTOR/4EBP1信号通路在体外培养血管瘤血管内皮细胞中的表达

Expression of PI3K/AKT/mTOR/4EBP1 Signaling Pathway in Hemangioma Vascular Endothelial Cell in Vitro

目的:探讨PI3K/AKT/mTOR/4EBP1信号通路在体外培养血管瘤血管内皮细胞中的表达情况。方法:血管瘤标本取自男患儿腹壁血管瘤,经过胰酶处理后进行血管瘤内皮细胞的原代培养,选择培养至对数生长期的细胞进行同步化,接着分为雷帕霉素组和阴性对照组,雷帕霉素组细胞1640培养液中加入10 nmol/L雷帕霉素。选择Western blot法分析PI3K、AKT、mTOR及4EBP1等蛋白在血管瘤内皮细胞体外培养中的水平,细胞细胞周期及凋亡的的情况选择流式细胞计数仪进行相应检查。结果:通过流式细胞仪检查经过24 h处理的两组细胞凋亡情况,结果显示阴性对照组为1.27±0.25,雷帕霉素组为4.85±0.47,两组间对比差异有统计学意义(t=5.273,P<0.05)。且经雷帕霉素干预血管瘤内皮细胞24 h后,G0/G1期血管瘤内皮细胞比例相较于阴性对照组升高显著,差异有统计学意义(t=3.964,P<0.05)。同阴性对照组相比,血管瘤血管内皮细胞经雷帕霉素作用后PI3K、AKT、mTOR及4EBP1蛋白表达水平均显著降低,差异有统计学意义(P<0.05)。结论:PI3K、AKT、mTOR及4EBP1蛋白在血管瘤内皮细胞体外培养过程中均存在一定的表达情况;选择雷帕霉素干预mTOR/4EBP1信号通路可导致细胞周期停止于G0/G1期而促进细胞凋亡。

Objective:To observe the expression and regulation of PI3K/AKT/mTOR/4EBP1 in hemangioma derived endothelial cells.Method:Hemangioma derived endothelial cells were isolated from abdominal wall hemangioma in male children.During logarithmie phase,culture medium was changed to serum-free and cell synchronization started within 48 hours.The cells were divided into rapamycin group and negative control group.1640 culture medium was added with 10 nmol/L rapamycin in rapamycin group.Then the protein levels of PI3K,AKT,mTOR,4EBP1,cell cycle distribution and apoptosis were observed.Result:Flow cytometry was used to examine the apoptosis of the two groups after 24 h treatment.The results showed that the negative control group was 1.27±0.25,and the rapamycin group was 4.85±0.47.The difference between the two groups was statistically significant(t=5.273,P<0.05).After 24 hours of intervention with rapamycin,the proportion of endothelial cells in hemangioma in G0/G1 phase was significantly higher than that in negative control group(t=3.964,P<0.05).Compared with the negative control group,the expression levels of PI3K,AKT,mTOR and 4EBP1 protein in vascular endothelial cells of hemangioma decreased significantly after treated with rapamycin,and the difference was statistically significant(P<0.05).Conclusion:PI3K/AKT/mTOR/4EBP1 pathway plays an important role in G0/G1 phase arrest and apoptosis of hernangioma vascular endothelial cells in vitro.