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目的研究micro RNA-181b(mi R-181b)和胰岛素样生长因子1受体(Insulin-like growth factor 1 receptor,IGF1R)参与血管内皮细胞衰老和血管新生的调节机制。方法体外培养人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs),传代培养计算细胞群体倍增水平(Population doublings,PDL),将PDL≤8 HUVECs定义为年轻细胞,PDL≥44 HUVECs定义为衰老细胞,并检测mi R-181b和IGF1R在年轻(PDL8)和年老(PDL44)HUVECs中的表达。PDL8 HUVECs过表达或抑制mi R-181b后,分别用MTS比色法、划痕(Wound healing)和成管(Tube formation)实验检测内皮细胞的增殖、迁移、成管等血管新生能力。采用双荧光素酶报告系统法检测mi R-181b对IGF1R转录后水平的调控。此外,给予缺氧刺激,观察缺氧应激条件下mi R-181b对IGF1R表达的调控作用。结果过表达mi R-181b可抑制PDL8内皮细胞的增殖能力22%(P<0.001)、迁移能力23%(P<0.001),对成管能力没有显著影响(P>0.05)。与PDL8HUVECs比较,mi R-181b在PDL44衰老内皮细胞中上调64%(P=0.046),IGF1R的m RNA和蛋白水平分别下调39%和45%(P=0.004,P=0.014)。荧光素酶活性实验显示mi R-181b可与IGF1R的3’UTR结合,但过表达mi R-181b对IGF1R的表达水平无显著影响(P>0.05)。在缺氧应激条件下,mi R-181b可上调IGF1R的表达(P=0.005)。结论 Mi R-181b抑制血管内皮细胞增殖、迁移等血管新生能力,这一作用与mi R-181b在缺氧应激条件下上调血管发育相关基因IGF1R的表达相关,其机制仍需进一步研究。
Objective To investigate the regulatory mechanism of micro RNA-181b (mi R-181b) and Insulin-like growth factor 1 receptor (IGF1R) involved in vascular endothelial cell senescence and angiogenesis. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro. Population doublings (PDL) were counted in subcultures. PDL≤8 HUVECs were defined as young cells. PDL≥44 HUVECs were defined as senescent cells , And the expression of mi R-181b and IGF1R in young (PDL8) and old (PDL44) HUVECs was examined. PDL8 HUVECs overexpressing or inhibiting mi R-181b were detected by MTS colorimetry, Wound healing and tube formation assay respectively. The proliferation, migration and angiogenesis of endothelial cells were detected. Dual-luciferase reporter assay was used to detect the transcriptional level of IGF1R by mi R-181b. In addition, given hypoxia stimulation, mi R-181b under hypoxia stress on IGF1R expression regulation. Results Overexpression of mi R-181b could inhibit the proliferation of PDL8 endothelial cells by 22% (P <0.001) and migration ability by 23% (P <0.001), and had no significant effect on tube formation ability (P> 0.05). Compared with PDL8HUVECs, mi R-181b was upregulated by 64% (P = 0.046) in PDL44 senescent endothelial cells and mRna and protein levels were downregulated by 39% and 45%, respectively, in IGF1R (P = 0.004, P = 0.014). Luciferase activity assay showed that mi R-181b could bind to 3’UTR of IGF1R, however, overexpression of mi R-181b had no significant effect on IGF1R expression (P> 0.05). Under hypoxia stress, mi R-181b upregulated IGF1R expression (P = 0.005). Conclusions Mi R-181b inhibits angiogenesis, such as proliferation and migration of vascular endothelial cells. This effect is related to the upregulation of IGF1R by mi R-181b in hypoxia stress. The mechanism remains to be further studied.