抗癫痫药丙戊酸影响内皮祖细胞衰老和血管拟态的机制研究

The experimental study on the action mechanism of valproic acid in endothelial progenitor cell senescence and vascular mimicry

目的研究抗癫痫药丙戊酸影响内皮祖细胞衰老和血管拟态的机制。方法从外周血中分离出内皮祖细胞(endothelial progenitor cells,EPCs)进行鉴定。通过BrdU的掺入试验和菌落形成来测量EPCs的增殖。蛋白质印迹法检测丝氨酸/苏氨酸蛋白激酶Akt的磷酸化。荧光染料注射静脉,测定血管拟态(vascular mimicry,VM)PI3K通路机制。结果分离出的EPCs体外培养后,经酸性β-半乳糖苷酶染色,内皮细胞衰老。丙戊酸剂量依赖性地阻止EPCs在培养过程中的衰老。丙戊酸促进EPCs的增殖,蛋白质印迹法检测丝氨酸/苏氨酸蛋白激酶Akt的磷酸化,丙戊酸显著提高Akt磷酸化水平,Akt是磷酸肌醇3激酶(PI3K)的下游效应蛋白。PI3K阻滞剂(wortmannin或Y294002)预处理显著降低了丙戊酸诱导的细胞衰老。丙戊酸通过PI3K通路激活膜型基质金属蛋白-1,膜型基质金属蛋白酶-1能够激活MMP-2的蛋白水解作用,后者将LN-5γ2链水解后形成的片段有助于VM发生过程中肿瘤细胞的迁移。结论丙戊酸延缓EPCs衰老的发生,这可能与PI3K/Akt通路激活有关。丙戊酸对体外EPCs衰老的抑制作用可能在一定程度上改善EPCs的功能活性,对潜在细胞的发育具有重要意义。丙戊酸通过PI3K通路激活膜型基质金属蛋白酶-1,从而可阻断拮抗VM的发生。

Objective To investigate the action mechanism of valproic acid in endothelial progenitor cell senescence and vascular mimicry in vitro.Methods The endothelial progenitor cells(EPCs)were isolated from peripheral blood and identified.The proliferation of EPCs was measured by BrdU incorporation test and colony formation assay.The phosphorylation of serine/threonine protein kinase Akt was detected by Western blotting.The mechanism of PI3K pathway in vascular mimicry(VM)was determined by intravenous injection with fluorescent dyes.Results After the EPCs were isolated and cultured in vitro,which were stained with acid-galactosidase,and the endothelial cells were aged.The valproic acid dose-dependently prevented the senescence of EPCs during the culture,and the valproic acid promoted the proliferation of EPCs,and Western Blot was used to detect the phosphorylation of serine/threonine protein kinase Akt.The valproic acid significantly increased the phosphorylation level of Akt,which was the downstream effector protein of phosphoinositol 3 kinase(PI3K).The pretreatment with PI3K blocker(wortmannin or Y294002)significantly reduced valproic acid-induced cell senescence.The valproic acid activated membrane matrix metalloproteinase-1 through the PI3K pathway.And membrane matrix metalloproteinase-1 could activate the proteolysis of MMP-2,which contributed to the migration of tumor cells during VM generation by hydrolyzing the LN-5γ2 chain.Conclusion The valproic acid delays the senescence of EPCs,which may be related to the activation of PI-3K/Akt pathway.The inhibitory effects of valproic acid on the senescence of EPCs in vitro may improve the functional activity of EPCs to a certain extent,which is of great significance to the development of potential cells.Valproic acid activates membrane-type matrix metalloproteinase-1 via PI3K pathway,thereby,blocking the occurrence of VM.