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目的探讨细胞红蛋白(cytoglobin,CYGB)在体外培养神经元缺氧时的表达,为研究缺氧缺血性脑病(hypoxic ischemic encephalopathy,HIE)的生理病理机制及寻求该病新的治疗方法提供实验基础。方法运用无血清培养技术进行胎鼠皮质神经元原代培养6d;先用神经元特异性烯醇化酶免疫组织化学方法纯度鉴定,然后随机分配为4组,将其中3组(第1组作为对照)置于0.5%~0.9%的O2中,用CCK8试剂盒测试细胞生存率,同时用Real-timePCR和Westernblot观察CYGB在神经元正常及缺氧不同时间点(8h,16h,24h)核酸及蛋白的表达情况。结果神经元纯度鉴定纯度大于99%;缺氧后神经元生存率随着时间推移降低,但绝大部分神经元仍存活;CYGB核酸及蛋白的表达随缺氧时间增长而递增,且每组之间差异有统计学意义(P<0.05)。结论在体外培养的神经元细胞中,缺氧可以促进CYGB的表达,提示CYGB在缺氧性神经损伤中可能发挥某种特定的生理功能。
Objective To investigate the expression of cytoglobin (CYGB) during neuronal hypoxia in vitro and provide experimental evidence for studying the pathophysiological and pathological mechanism of hypoxic ischemic encephalopathy (HIE) basis. Methods Primary cultured fetal rat cortical neurons were cultured with serum-free medium for 6 days. The cells were identified by neuron-specific enolase immunohistochemical method and then randomly divided into 4 groups. Group 1 (control group 1) ) Were placed in 0.5% ~ 0.9% O2. Cell viability was tested by CCK8 kit. Real-time PCR and Western blotting were used to observe the effect of CYGB on neuron normal and hypoxia (8h, 16h, 24h) The expression of the situation. Results The purity of neurons was more than 99%. After hypoxia, the survival rate of neurons decreased with time, but the majority of neurons still survived. The expression of CYGB nucleic acid and protein increased with the increase of hypoxia time, The difference was statistically significant (P <0.05). Conclusion In vitro cultured neurons, hypoxia can promote the expression of CYGB, suggesting that CYGB may play a specific physiological function in hypoxic nerve injury.