孤儿核受体Nur77对缺/复氧损伤中心肌细胞自噬的调节

目的:研究孤儿核受体Nur77对缺/复氧损伤中心肌细胞自噬的调节作用。方法:差速贴壁法分离乳鼠心肌细胞,经免疫荧光染色鉴定纯度。缺氧(1%O_2、5%CO_2和94%N_2)培养12 h后,常氧培养2 h构建心肌细胞缺/复氧损伤。实时定量PCR和western blot的方法检测Nur77的表达变化。通过siRNA转染抑制心肌细胞nur77表达,通过自噬标志蛋白表达改变作为细胞自噬水平的变化。结果:原代分离的心肌细胞纯度95%以上。缺氧12 h和缺/复氧(12 h/2 h)刺激后,心肌细胞中Nur77表达都明显升高(P0.01)。与缺氧组相比,缺/复氧组细胞质中的水平明显增加(P0.01),细胞核中Nur77水平无明显变化。抑制Nur77后,缺/复氧组自噬水平明显降低,缺氧组心肌细胞自噬水平无明显变化。结论:Nur77参与缺/复氧损伤中心肌细胞自噬水平的调节。

Regulatory Role of Orphan Nuclear Receptor Nur77 in Cardiomyocytes Autophagy during Hypoxia/Reoxygenation Injury

ABSTRACT Objective: To study regulation of orphan nuclear receptor Nur77 on cardiomyocytes autophagy during hypoxia/reoxygenation injury. Methods: Primary neonatal rat cardiomyocytes were obtained by differential adherence method, and were identified purity by immunofluorescence staining. Cardiomyocytes hypoxia/reoxygenation injury was established by culturing in hypoxic condition (1% O₂, 5% CO₂ and 94% N₂) for 12 h, followed by reoxygenation for 2 h. Expression of nur77 was determined by real-time PCR. Cardiomyocytes Nur77 expression was inhibited by siRNA transfection. Autophagy level was determined according to the expression of autophagy marker. Results: The purity of separated cardiomyocytes reached by more than 95 %. The expression of Nur77 was significantly increased in cardiomyocytes after hypoxia (12 h) and hypoxia/reoxygenation (12 h/2 h) treatment (P<0.01). Compared with hypoxia group, Nur77 was detected significantly increase in cytoplasm from hypoxia/reoxygenation treated cardiomyocytes, but no changes in nucleus. By Nur77 inhibition, the autophagy level was significantly decreased in hypoxia/reoxygenation treated cardiomyocytes, but no significantly change was found in hypoxia treated cardiomyocytes. Conclusion: Nur77 was involved in regulation of cardiomyocytes autophagy during hypoxia/reoxygenation injury.