活血益气方促缺氧人脐静脉内皮细胞迁移及其相关调控基因mRNA表达的拆方研究

Dismantling Study on the Promoting Effect of Blood Activation and Qi Supplement Prescription on the Migration of Human Umbilical Vein Endothelial Cells in Hypoxia and its Related Regulatory Gene mRNA Expression

目的 观察活血益气方及其拆方药物血清对缺氧损伤后人脐静脉内皮细胞(HUVEC)迁移的影响,探讨其促血管新生可能的作用机制及配伍规律。方法 采用三气培养箱建立HUVEC缺氧损伤模型,另设正常对照组常规培养。活血益气方、活血方及益气方组分别给予相应药物血清培养,缺氧组及正常对照组给予正常对照血清培养。干预24h后,采用TranswellInsert检测细胞迁移,实时荧光定量PCR法检测细胞粘着斑激酶(FAK)、应激活化蛋白激酶-2(p38)、MAP激酶活化蛋白激酶(MAPKAPK)、热休克蛋白27(HSP27)mRNA表达水平。结果 与正常对照组比较,缺氧组细胞迁移数显著减少(P<0.01);FAK、p38、MAPKAPK、HSP27mRNA表达水平显著下调(P<0.01)。与缺氧组比较,活血益气方、活血方及益气方可增加缺氧细胞迁移数(P<0.05或P<0.01),其中活血益气方组细胞迁移数显著高于活血方组,差异有统计学意义(P<0.05);活血益气方可上调细胞FAK、p38、MAPAPK、HSP27mRNA表达水平(P<0.05或P<0.01),活血方可上调细胞FAK、p38mRNA表达水平(P<0.05),而益气方则可上调细胞FAK、p38、MAPKAPKmRNA表达水平(P<0.05或P<0.01)。结论 活血益气方及其拆方可不同程度地促进缺氧后HUVEC迁移,其作用机制可能与上调FAK及p38/MAPKAPK/HSP27信号通路活性有关;活血益气方的促迁移效应最显著,活血方和益气方发挥协同作用。

Objective To observe the effect of blood activation and Qi supplement prescription (BAQS) and its disassembled prescriptions medicated serum on the migration of human umbilical vein endothelial cells (HUVEC) induced by hypoxia,and to investigate the possible mechanisms and compatibility law of promoting angiogenesis. Methods The HUVEC hypoxia model was established by three gas incubators,and the normal control group was cultured in the normal condition.The HUVEC of BAQS group,blood activation prescription (BA) group and Qi supplement prescription (QS) group were treated with corresponding medicated serum,and hypoxia group and normal control group were given normal control medicated serum.After 24 hours,cell migration was detected by Transwell Insert,and the level of FAK,p38,MAPKAPK and HSP27 mRNA in HUVEC were detected by real-time fluorescence quantitative PCR. Results Compared with the normal control group,the migration number of HUVEC in hypoxia group was significantly reduced ( P <0.01),and the level of FAK,p38,MAPKAPK and HSP27 mRNA was significantly downregulated ( P <0.01).BAQS,BA and QS can increase the migration number of hypoxia HUVEC ( P <0.05 or P <0.01),and the migration number in BAQS group was significantly higher than that of BA group,and the difference was statistically significant( P <0.05);BAQS can upregulate FAK,p38,MAPAPK,HSP27 mRNA levels,BA can upregulate FAK and p38 mRNA levels,and QS can upregulate FAK,p38, MAPKAPK mRNA levels ( P <0.05 or P < 0.01). Conclusion BAQS and its disassembled prescriptions can significantly promote the migration of HUVEC induced by hypoxia,and the mechanism may be related to its upregulation of FAK and p38/MAPKAPK/HSP27 signaling pathway activity;the promoting effect of BAQS is the most significant,BA and QS play a synergistic effect.