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目的:探讨大鼠腹主动脉血管平滑肌细胞(VSMCs)在缺氧培养时胞内钙库钙释放的变化情况,并探讨胞内钙库钙释放在休克血管对去甲肾上腺素(NE)低反应性中的可能作用,为进一步阐明休克血管低反应性的发生机制提供依据。方法:建立大鼠失血性休克(40mmHg,2h)的在体模型及大鼠VSMCs缺氧细胞模型;采用Fura-3/AM钙离子成像方法测定VSMCs胞浆钙离子浓度([Ca2+])的变化情况及其与IP3受体(IP3R)及雷诺定受体(RyR)介导的钙释放通道的关系;采用离体器官张力测定技术,检测不同内钙释放依赖信号通路在休克血管低反应性形成中的可能作用。结果:在细胞外液无Ca2+的前提下,NE可通过动员内钙释放引起VSMCs胞浆[Ca2+]的明显升高;缺氧培养后VSMCs胞浆[Ca2+]较正常对照组有所升高,由NE诱导的VSMCs胞浆[Ca2+]较对照组有所降低,但均无显著差别;但在缺氧培养的VSMCs,细胞内钙库钙释放功能明显改变,表现为与正常对照组比较,缺氧VSMCs由IP3R敏感钙释放通道开放剂adenophostinA(10-5mol/L)及ATP-Na2(10-4mol/L)诱导的VSMCs胞浆[Ca2+]升高不显著,但RyR敏感钙释放通道开放剂caffeine可诱导缺氧VSMCs胞浆[Ca2+]的明显升高;失血性休克(40mmHg,2h)可引起大鼠腹主动脉血管对由NE诱导的收缩反应性明显降低,IP3R激动剂ATP-Na2(10-4mol/L)并不明显提高休克血管对NE的收缩反应性,但IP3R阻断剂heparin(104U/L)可明显抑制休克血管对NE的收缩反应性;此外,在无钙及含钙的K-H液中,RyR阻断剂ryanodine(10-5mol/L)可部分恢复休克血管对NE的收缩反应性,而RyR激动剂caffeine(10-3mol/L)可进一步降低休克血管对NE诱导的收缩反应性。结论:失血性休克后由RyR介导的内钙释放被激活部分参与了休克血管低反应性的形成。
OBJECTIVE: To investigate the changes of intracellular calcium release during hypoxia in rat abdominal aortic vascular smooth muscle cells (VSMCs), and to explore the effect of intracellular calcium release on low blood pressure of norepinephrine (NE) The possible role of sex, in order to further clarify the mechanism of shock vascular hyporeactivity provide the basis. Methods: The in vivo models of hemorrhagic shock (40 mmHg, 2 h) and the hypoxic cell model of rat VSMCs were established. The changes of cytosolic calcium concentration ([Ca 2+]) in VSMCs were measured by Fura-3 / AM calcium ion imaging And its relationship with IP3R and RyR-mediated calcium release channels. Tissue tension measurement was used to detect the expression of different intracellular calcium-dependent signaling pathways in shock vascular hyporeactivity In the possible role. Results: In the extracellular fluid without Ca2 +, NE could significantly increase [Ca2 +] in the cytoplasm of VSMCs by mobilizing intracellular calcium release. Compared with normal control group, NE increased in hypoxic culture, The cytoplasmic [Ca2 +] induced by NE decreased compared with the control group, but there was no significant difference between the two groups. However, in hypoxic cultured VSMCs, the intracellular Ca2 + release significantly changed, showing as compared with the normal control group Oxygen-loaded VSMCs showed no significant increase of [Ca2 +] in cytoplasm of VSMCs induced by IP3R-sensitive calcium channel openers adenophostin A (10-5mol / L) and ATP-Na2 (10-4mol / L), but RyR-sensitive calcium release channel openers Caffeine induced a significant increase of [Ca2 +] in the cytoplasm of hypoxic VSMCs. Hemorrhagic shock (40mmHg, 2h) induced a significant decrease of contractile responses induced by NE in the abdominal aorta of rats. IP3R agonist ATP-Na2 10-4mol / L) did not significantly increase the contractile response to NE in shocked vessels, but IP3R blocker heparin (104U / L) significantly inhibited the contractile response of NE to contractile shock. In addition, Of KH solution, RyR blocker ryanodine (10-5mol / L) can partially restore the contractile response of neovasculature to NE, while RyR agonist caffeine (10 -3mol / L) can further reduce the shock-induced contractile response of NE to NE. CONCLUSIONS: RyR-mediated activation of intracellular calcium release following hemorrhagic shock is partially involved in the formation of shock vascular hyporesponsiveness.