P13K／Akt信号传导通路在大鼠骨髓来源内皮祖细胞分化中作用的研究

目的探讨P13K／Akt在内皮祖细胞（EPC）分化中的作用。方法密度梯度离心法分离大鼠骨髓EPC，经差速法接种2次贴壁细胞：采用激光共聚焦显微镜鉴定培养5dEPC；Westemblot检测第O、3、7、10、14天EPC中AC133、vWF、P13K、Akt蛋白表达水平；并在第3、7、10天加入LY294002作用12h，用RT—PCR检测AC133、vWFmRNA水平，Westernb10t检测p—Akt水平。结果经Westemblot检测，AC133在0d表达最强，第3天有弱表达，第7、10、14天几乎无表达（P〈0．05）：vWF表达强度没有明显变化（P〉0．05）；P13K和Akt在0d表达最强，第3天表达稍弱，随培养时间的延长表达逐渐减弱：LY294002作用12h后，第7天和第10天AC133mRNA表达水平低于第3天的水平（P〈0．05）；vWF的表达没有明显变化（P〉0．05）；p—Akt蛋白表达逐渐下降，第10天与第3天相比差异有统计学意义（P〈0．05）。结论EPC分化为内皮细胞过程中．可能有P13K／Akt的参与，

Study on the effect of PDK/Akt in differentiating endothelial progenitor cell derived from rat bone marrow

Objective To study the effect of PI3K/Akt in differentiation of endothelial progenitor cells（EPC）. Methods The techniques of Ficoll density gradient centrifugation and differential-speed adherence screening were adopted to separate EPC from rat bone marrow. The EPC cultured 5 days were collected and identified with laser confocal microscopy. The protein expressions of AC133, vWF, PI3K and Akt in EPC cultured at dav O, 3, 7, 10 and 14 were detected by Western blot method. LY294002, the specific inhibitor of PI3K, was added at cultured day 3, 7 and 10 of EPC. After affected 12 hours, mRNA expressions of AC133 and vWF by RT-PCR and the protein expression of p-Akt were detected by Western blot method, respectively. Results By Western blot examination the expression level of AC133 was the strongest at day O; and weaker at day 3. It was no expression at day 7, 10 and 14 of calfieation（P 〈 0.05）. The expression of vWF did not change. The expression levels of PI3K and Akt showed the strongest at day O, and weaker at day 3. The expression gradually decreased with culture time. After affected 12 hours by LY294002, the mRNA expression level of AC133 at day 7 and day 10 of cultivation were much lower than that at day 3（P〈 0.05）. The expression level of vWF was no change（P〉 0.05）. The expression level of p-Akt decreased gradually. The difference between day 3 and day 10 of cultivation was significant（P〈 0.05）. Conclusion The PI3K/Akt may possibly participate in the differentiation process from EPC to endothelial cells.