Tie-2介导Ang-1、Ang-2调节大鼠失血性休克血管反应性双相变化的作用研究

目的 观察Tie-2在血管生成素-1(angiopoietin-1, Ang-1)、血管生成素-2(angiopoietin-2, Ang-2)调节失血性休克血管反应性双相变化中的作用。方法 采用离体微血管环张力测定技术和western blot技术，观察失血性休克后不同时间点肠系膜上动脉(superior mesenteric artery，SMA)中Tie-2蛋白表达和磷酸化变化、抑制Tie-2对Ang-1和Ang-2调节缺氧早期和晚期血管反应性作用的影响，以及给予Ang-1和Ang-2后缺氧的血管内皮细胞(vascular endothelial cell, VEC)和血管平滑肌细胞(vascular smooth muscle cell, VSMC)混合细胞中Tie-2蛋白表达和磷酸化变化，并观察抑制Tie-2对缺氧早期和晚期的混合细胞NO含量的影响。结果 (1) 肠系膜上动脉Tie-2蛋白表达和酪氨酸磷酸化在正常时很低，失血性休克后逐渐增高，在休克早期(休克10min)，Tie-2蛋白表达变化不大，但酪氨酸磷酸化已明显增高(P<0.01)；随着休克时间延长，Tie-2蛋白表达和酪氨酸磷酸化均进一步显著增高(P<0.01)。(2)Tie-2抑制剂可降低缺氧10min的血管高反应性(NE的Emax由13.479mN降低至10.122mN，P<0.05)，并显著抑制Ang-1对缺氧10min血管反应性的维持作用(Emax由15.283mN降低至10.253mN，P<0.01)；Tie-2抑制剂可改善缺氧4h的血管低反应性(NE的Emax由5.875mN增高至8.003mN，P<0.05)，并显著拮抗Ang-2进一步降低缺氧4h血管反应性的作用(Emax由3.444mN增高至7.643mN，P<0.01)。(3)缺氧10min时，降低血管高反应性的Ang-2可降低Tie-2磷酸化，使其由0.0403降低至0.0123(P<0.01)；缺氧4h时，恢复血管低反应性的Ang-1可降低Tie-2蛋白表达，使其由0.2276降低至0.0851 (P<0.01)，也可以降低Tie-2磷酸化，使其由0.1437降低至0.0359 (P<0.01)。(4) NO含量在缺氧早期显著增高，Ang-2和Tie-2抑制剂显著抑制其增高(P<0.01)；缺氧晚期NO含量较正常对照组增高得更为显著，Ang-1和Tie-2抑制剂可抑制其增高(P<0.01)。结论 Ang-1、Ang-2通过Tie-2受体调节大鼠失血性休克血管反应性双相变化。

Tie-2 Receptor Mediates the Regulation of Ang-1 and Ang-2 on the Biphasic Change of Vascular Reactivity after Hemorrhagic Shock in Rats

OBJECTIVE To observe the role of Tie-2 in the regulation of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) on the biphasic change of vascular reactivity after hemorrhagic shock in rats. METHOD The protein expression and tyrosine phosphorylation of Tie-2 in the superior mesenteric artery (SMA) after hemorrhagic shock were measured via western blot technique, the effect of Tie-2 inhibitor on the vascular reactivity of SMA in the early (hyperreactivity) and late (hyporeactivity) period of hypoxia when treated with Ang-1 and Ang-2 were observed via the isolated organ perfusion system, and the protein expression and tyrosine phosphorylation of Tie-2 in the hypoxia mixture of vascular endothelial cell (VEC) and vascular smooth muscle cell (VSMC) when treated with Ang-1 and Ang-2 were measured via western blot technique. Effect of Tie-2 on the NO concentration of VSMC and VEC mixture in the early and late hypoxia was detected by the NO kit. RESULTS (1) The protein expression and tyrosine phosphorylation of Tie-2 in the superior mesenteric artery (SMA) were low in normal control group, and were increased after hemorrhagic shock, the protein expression of Tie-2 was increased after 30min shock (P<0.01), and the tyrosine phosphorylation of Tie-2 was increased after 10min shock (P<0.01), both protein expression and tyrosine phosphorylation of Tie-2 were further increased as shock went on (P<0.01). (2) Tie-2 inhibitor could decrease the vascular hyperreactivity in 10min hypoxia (the Emax of NE was decreased from 13.479mN to 10.122mN, P<0.05), and repress the maintenance effect of Ang-1 on vascular reactivity in 10min hypoxia (the Emax of NE was decreased from 15.283mN to 10.253mN, P<0.01); Tie-2 inhibitor could improve the vascular hyporeactivity in 4h hypoxia (the Emax of NE was increased from 5.875mN to 8.003mN, P<0.05), and inhibit the decrease effect of Ang-2 on vascular reactivity in 4h hypoxia (the Emax of NE was increased from 3.444mN to 7.643mN, P<0.01). (3) At 10min hypoxia, Ang-2 could decrease the tyrosine phosphorylation of Tie-2 from 0.0403 to 0.0123 (P<0.01), and at 4h hypoxia, Ang-1 could decrease the protein expression of Tie-2 from 0.2276 to 0.0851 (P<0.01), and decrease the tyrosine phosphorylation of Tie-2 from 0.1437 to 0.0359 (P<0.01). (4) The NO concentration in the cells mixture was significantly increased in the early hypoxia, and the increase could be inhibited by Ang-2 and Tie-2 inhibitor(P<0.01); in the late hypoxia, the NO concentration was further increased, and could be inhibited by Ang-1 and Tie-2 inhibitor(P<0.01). CONCLUSION Ang-1 and Ang-2 regulate the biphasic change of vascular reactivity after hemorrhagic shock in rats through Tie-2 receptor.