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目的:研究缺氧/复氧(hypoxia/reoxygenation,H/R)刺激是否可通过激活程序性坏死信号通路诱导心肌细胞坏死。方法 :体外培养新生大鼠原代心肌细胞,采用缺氧2 h复氧4 h的方法复制心肌细胞损伤模型,碘化丙腚(propidium iodide,PI)染色检测心肌细胞坏死情况,Western blot和免疫共沉淀方法检测程序性坏死信号通路中受体相互作用蛋白1(receptor interacting protein-1,RIP1)、受体相互作用蛋白3(receptor interacting protein-3,RIP3)的蛋白表达水平和RIP1/RIP3复合物Ⅱ形成情况,以及RIP1、RIP3的泛素化水平。结果:与对照组相比,缺氧/复氧组心肌细胞坏死数量明显增多,RIP1、RIP3蛋白表达水平显著升高,RIP1/RIP3复合物Ⅱ的形成增多,且RIP1与RIP3的泛素化水平也有所增加。结论:缺氧复氧可诱导心肌细胞坏死,其机制可能与激活程序性坏死RIP1/RIP3信号通路有关。
AIM: To investigate whether hypoxia / reoxygenation (H / R) stimulation can induce cardiomyocyte necrosis by activating the programmed necrosis signaling pathway. Methods: Primary cultured neonatal rat cardiomyocytes were cultured in vitro. Cardiomyocytes were harvested by hypoxia for 2 h and then reperfused for 4 h. Cardiomyocyte necrosis was detected by propidium iodide (PI) staining. Western blot and immunofluorescence The coprecipitation method was used to detect the protein expression level of receptor interacting protein-1 (RIP1) and receptor interacting protein-3 (RIP3) and RIP1 / RIP3 complex The formation of Ⅱ, as well as RIP1, RIP3 ubiquitination level. Results: Compared with the control group, the number of cardiomyocyte necrosis in hypoxia / reoxygenation group increased significantly, the expression of RIP1 and RIP3 protein increased significantly, the formation of RIP1 / RIP3 complex Ⅱ increased, and the ubiquitination level of RIP1 and RIP3 It has also increased. Conclusion: Hypoxia and reoxygenation can induce cardiomyocyte necrosis, which may be related to the activation of RIP1 / RIP3 signaling pathway.