PI3K/Akt参与内皮祖细胞分化的体外观察

Role of PI3K/Akt in endothelial progenitor cells differentiation in vitro

背景:内皮祖细胞分化为成熟内皮细胞的分子机制尚不清楚,要通过包括PI3K/Akt信号通路在内的多个信号通路调节细胞内部多种基因的表达来完成。目的:分析PI3K/Akt信号传导通路在内皮祖细胞分化中的作用。设计、时间及地点:分组对照体外实验,于2007-09/2008-03在中国医科大学附属盛京医院病理实验室完成。材料:4～6周龄Wistar大鼠,雌雄不限。方法:采用密度梯度离心法从大鼠骨髓分离内皮祖细胞,经差速取二次贴壁细胞接种于培养瓶内。收集培养5d的内皮祖细胞,用激光共聚焦显微镜鉴定,AC133和vWF双染阳性细胞为正在分化的内皮祖细胞。主要观察指标:应用Western blot和反转录-聚合酶链反应检测培养0,3,7,10,14d的内皮祖细胞中AC133,vWF,PI3K,Akt蛋白和mRNA表达水平。结果:经Western blot和反转录-聚合酶链反应检测,AC133在培养0d表达最强,3d有弱表达,7,10,14d几乎没有表达(P<0.05);vWF在培养过程中表达强度没有明显变化(P>0.05);PI3K和Akt在培养0d表达最强,3d表达稍弱,随着培养时间的延长,表达逐渐减弱。PI3K/Akt与AC133表达趋势一致。结论:在内皮祖细胞分化为内皮细胞过程中,可能有信号传导通路PI3K/Akt的参与。

BACKGROUND： The molecule mechanism underlying endothelial progenitor cells （EPCs） differentiation into mature endothelial cells remains poorly understood. It requires for the regulation of the expression of various genes inside cells by various signal pathways, PI3K/Akt maybe included. OBJECTIVE： To investigate the role of PI3K/Akt in EPCs differentiation. DESIGN, TIME AND SETTING： A group control in vitro experiment was performed at the Pathology Laboratory in Shengjing Hospital of China Medical University from September 2007 to March 2008. MATERIALS： Wistar rats aged 4-6 weeks, of either gender. METHODS： EPCs were isolated from rat bone marrow with density gradient centrifugation. Then difference-speed adherence screening method was used to obtain the second time adherent cells, which then were inoculated in culture flask. After 5 days of culture, cells were collected to be indentified with laser confocal microscopy. The differentiating EPCs were those that have positive results of both AC133 and vWF stainings. MAIN OUTCOME MEASURES： The mRNA and the protein expression of AC133, vWF, PI3K and Akt in EPCs were detected with Western blot and reverse transcription-polymerase chain reaction （RT-PCR） after 0, 3, 7, 10 and 14 days of inoculation respectively. RESULTS： According to the RT-PCR and Western blot detection, AC133 showed the strongest expression at day 0, the weaker at day 3 and none at day 7, 10 and 14 （P 〈 0.05）; vWF presented no significant expression change throughout the whole inoculation; the expressions of PI3K and Akt changed in the same direction with AC133, i.e. they became stronger with the increasing culture time, with the strongest at day 0, and the weaker at day 3. CONCLUSION： PI3K/Akt signal pathway possibly has its role in the differentiation of EPCs into endothelial cells.