Akt调节切应力诱导骨髓间充质干细胞Bmi-1基因的表达

目的 探讨流体切应力对骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）中Bmi-1基因表达的影响及可能的信号机制。方法 原代体外分离培养大鼠BMSCs，应用平行平板流动腔系统，给BMSCs施加不同强度（0.5、1.5、3.0 Pa）和不同加载时间（1、2、6、24 h）的层流切应力，用实时定量 RT-PCR法检测Bmi-1基因的表达水平，用免疫印迹法检测磷酸化Akt和ERK1/2的表达水平，利用Wortmannin（PI3K特异性抑制剂）、PD98059（ERK1/2 MAPK特异性抑制剂）信号阻断剂探讨信号转导途径。结果BMSCs在1.5 Pa切应力作用1 h后Bmi-1基因表达即明显增强，24 h达高峰。不同强度切应力都会刺激Bmi-1基因表达，其中3.0 Pa最强。切应力能显著激活磷酸化Akt和ERK1/2表达。Wortmannin而不是PD98059可以明显抑制Bmi-1基因的表达。结论 切应力可诱导BMSCs中Bmi-1基因表达，其表达量与刺激时间和切应力的强度密切相关，这种作用可能通过Akt信号调节。
Objective To investigate the effect of fluid shear stress (FSS) on the expression of B lymphoma Mo-MLV insertion region 1 (Bmi-1) in bone mesenchymal stem cells (BMSCs) and possible signal transduction mechanism. Methods BMSCs were isolated from SD rats and FSS at different magnitude (0.5, 1.5, 3.0 Pa) and under different time phase (1, 2, 6, 24 h) were loaded by parallel-plate flow chamber system. The expression of Bmi-1 was measured by real-time RT-PCR at mRNA level and the levels of phosphorylated Akt (p-Akt) and extracellular signalregulated kinase 1/2 (p-ERK1/2) were detected by Western blotting. The signaling inhibitors, wortmannin (PI3K specific inhabitor) and PD98059 (ERK1/2 specific inhabitor), were used to investigate possible mechanical signal transduction pathway. Results Bmi-1mRNA expression increased when BMSCs were exposed to 1.5 Pa FSS for 1 h and reached the peak at 24 h. All FSS with different magnitude could increase Bmi-1 expression, especial at high FSS (3.0 Pa). Meanwhile, FSS resulted in a significant activation of p-Akt and p-ERK1/2 in BMSCs. After treated with wortmannin, the expression of Bmi-1 was inhibited prominently, however, PD98059, the expression of Bmi-1 did not change. Conclusions FSS can activate the expression of Bmi-1, the amount of Bmi-1 expression was closely related to the stimulating time and the magnitude of FSS, and Akt signal molecule plays an important role during the process. These findings provide significant references for studying the mechanical biological mechanisms of stem cell differentiation.