PI3K/Akt信号转导通路对脑微血管内皮细胞生物学行为的影响

Effect of PI3K/Akt signaling pathway on the biological behaviors of brain microvessel endothelial cells

目的观察PI3K特异性抑制剂LY294002对脑微血管内皮细胞迁移、增殖和血管发生改变及PI3K/Akt信号途径的影响。方法不同浓度(5、10及20μmol/L)LY294002处理后,用体外"伤口愈合"实验、细胞增殖试剂盒和缺乏生长因子的Matrigel胶观察内皮细胞迁移、增殖和血管发生情况,并用Western印迹法检测磷酸化PI3K和磷酸化Ak(t苏氨酸308)的表达变化。结果与正常对照组相比,实验组脑微血管内皮细胞迁移明显减少(均P<0.01)、细胞存活率降低(均P<0.01)、血管发生改变(包括血管床面积、平均血管长度、毛细血管数和血管分支点数)及磷酸化PI3K、磷酸化Akt(苏氨酸308)的表达均降低(均P<0.05或P<0.01),且随LY294002浓度增高抑制作用增强(均P<0.05或P<0.01)。结论 PI3K/Akt信号途径下调可抑制内皮细胞的迁移、增殖和血管发生。

【Objective】 To investigate the effects of LY294002 inhibitor on migration,proliferation and angiogenesis,and role of pathogenesis in brain microvessel endothelial cells （BMEC）.【Methods】Cultivated rat brain microvessel endothelial cells were cultured in LY294002 at 5 μmol/L,10 μmol/L and 20 μmol/L of concentrations,the migration rate of the cells was measured by wound healing test and the cell proliferation was examined with MTT method.Matrigel was spread on 96-well plate,and culture of BMEC with LY294002 and mannitol of different concentrations were added.Microscopic photography was used to calculate the total area of vascular bed,average vessel length,vessel number,branch points,so as to observe the angiogenesis.Western blotting was used to detect the expressions of p85/PI3K,p-PI3K and p-Akt （Threonine308）.【Results】5μmol/L,10 μmol/L,and 20 μmol/L of LY294002 dose-dependently decreased the BMEC migration rates and cell viability,respectively （all P 0.01）.LY294002 decreased the numbers of total area of vascular bed,average tubule length,number of capillaries,and number of vessel branch point formed on the matrigel.LY294002 dose-dependently inhibited the angiogenesis （P 0.05 or P 0.01）.LY294002 dose-dependently inhibited the phosphorylation of p85/ P13K and Akt （P 0.05 or P 0.01）.【Conclusion】LY294002 would inhibit PI3K/Akt signaling pathway leading to migration,proliferation and angiogenesis dysfunction of endothelial cells.