血小板活化对肠微血管内皮细胞血管生成影响的实验研究

Effect of platelet activation on angiogenesis of intestinal microvascular endothelial cells

目的研究活化血小板诱导的血管生成对肠微血管内皮细胞(Intestinal microvascular endo-thelial cells,IMECs)上皮间质转化(epithelial-mesenchymal transition,EMT)的影响及机制。方法将IMECs细胞作为研究对象,以活化的血小板刺激,观察比较加入PI3K/mTOR抑制剂BEZ235前后,IMECs细胞迁移、管腔形成能力的变化来研究活化的血小板对IMECs血管生成的影响,观察对PI3K/AKT/mTOR信号通路磷酸化相关蛋白及HIF-1α的蛋白表达的影响,以及对纤维化相关因子E-cadherin、α-SMA、Collagen I蛋白表达变化和基因表达水平的影响。结果经培养得到的IMECs经倒置显微镜形态学特征鉴定95%以上为内皮细胞特征。静息血小板加入ADP后,FCM检测P-selectin抗体浓度明显增加(P<0.01),表明活化血小板数量明显增加。加入PI3K/mTOR抑制剂BEZ235可显著抑制活化的血小板刺激增强了IMECs的细胞迁移能力、管腔形成的能力(P<0.01)。PI3K/AKT/mTOR蛋白无明显变化(P<0.05),而磷酸化蛋白水平明显受到抑制,HIF-1α、α-SMA、CollagenI的蛋白生成及基因表达明显受到抑制(P<0.01),而E-cadherin蛋白生成及基因表达则上调(P<0.01)。结论活化血小板诱导的IMECs细胞EMT的机制可能是通过PI3K/mTOR/AKT信号通路促进血管生成实现的。

Objective To investigate the effect and mechanism of activated platelet-induced angiogenesis on EMT of IMECs.Methods The effect of activated platelets on the angiogenesis of IMECs was investigated by comparing the changes in cell migration capacity and lumen formation of IMECs with and without PI3K/mTOR inhibitor BEZ235.To observe the effects on the protein expression of phosphorylation-related proteins and HIF-1αin the PI3K/AKT/mTOR signaling pathway,and the effects on the expression changes of fibrosis-related factors E-cadherin,α-SMA,and CollagenI and gene expression levels.Results The IMECs′s proportion obtained by culture and identified as endothelial cells by morphological features of inverted microscope was over 95%.After adding ADP to resting platelets,the concentration of p-selectin antibody detected by FCM was significantly increased(P<0.01),indicating that the number of activated platelets was significantly increased.Compared with the addition of PI3K/mTOR inhibitor BEZ235,activated platelet stimulation enhanced the cell migration ability and lumen formation ability of IMECs,and inhibited PI3K,AKT,mTOR phosphorylated protein and HIF-1αprotein,the protein production and gene expression of fibrosis-related factorsα-SMA and CollagenI were significantly suppressed,while E-cadherin protein production and gene expression were up-regulated.Conclusion The mechanism of activating platelet-induced EMTs in IMECs cells may be achieved by promoting angiogenesis through the PI3K/AKT signaling pathway.