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6 d;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(8 h,16 h,24 h)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。
Abstract
Objective To examine the expression of cytoglobin(CYGB) of neurons in vitro when suffered hypoxia,which provides an experimental basis for exploring the physiological and pathological mechanism and seeking new treatments of hypoxic-ischemic encephalopathy. Methods The primary cortical neurons from embryonic rat were cultured in a serum-free culture system of B27-supplemented neurobasal medium.The neurons were identified by polyclonal antibody against neuron specific enolase(NSE) on the 6th day.They were divided into 4 groups randomly for assessing cell viability by CCK8 and observing the expression of CYGB by Real-time PCR and Westernblot at 0、8、16、24 h after hypoxic model(oxygen concentration between 0.5% and 0.9%) was induced. Results The purity of neurons was greater than 99%;and cell viability were slightly reduced after hypoxia.Expression of RNA and protein of CYGB was increased in a time-dependent manner.And there was statistical differenc in each group (P<0.05). Conclusions CYGB can up-regulate in neurons in vitro after hypoxia,indicating CYGB may play certain important roles in neurons under hypoxia.
关键词
细胞红蛋白 /
原代神经元 /
缺氧缺血性脑损伤
Key words
cytoglobin /
neurons /
hypoxic-ischemic encephalopathy
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基金
广东省科技计划项目(2008B080701036);广东省医学科研基金立项(B2009180)
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