摘要
目的 利用神经元损伤模型和蛋白双向凝胶电泳技术研究亚低温脑保护机制.方法 细胞裂解液浸融法建立神经元损伤模型,分别置于33℃和37℃温控培养箱中培养,测定培养上清液中乳酸脱氢酶含量,TUNEL细胞凋亡法比较两种培养温度下细胞凋亡情况.提取细胞总蛋白,蛋白双向凝胶电泳技术筛选差异蛋白质点,质谱分析法完成蛋白鉴定,Western blot方法进行蛋白质确认,统计学方法解析检测结果.结果 处于亚低温培养条件下的受损神经细胞乳酸脱氢酶水平和发生原位凋亡的细胞数量均明显低于37℃培养组（p＜0.05）.利用双向凝胶电泳共筛选出14个差异蛋白质点,其中12个得到有效认证,它们参与细胞能量代谢、再生、氧化应激等多种病理生理过程.结论 亚低温通过干预调节细胞能量代谢、再生、氧化应激等多个分子事件发挥肯定的神经保护作用.
Objective To study the neuroprotection mechanism of mild hypothermia using a neuronal injury model and protein two-dimensional gel electrophoresis.Methods A neuron damage model was induced by the cell lysates soak assay.Neurons were cultured in temperature-controlled incubator at 33 ℃ and 37 ℃ respectively.The lactic acid dehydrogenase level in the culture supernatant was tested.TUNEL apoptosis assay was use to compare the cell apoptosis under the 2 culture temperatures.The total cellular protein was extracted.The protein two-dimensional gel electrophoresis technique was used to screen the differential protein spots.Mass spectrometry was used to complete the protein identification.Western blot was used to conduct protein confirmation.Statistical methods were used to analyze the test results.Results The impaired neuronal lactate dehydrogenase level and the cell numbers of occurring in situ apoptosis were significantly lower than those of the 37 ℃ culture group under the low temperature condition （P 〈 0.05）.A total of 14 differential protein spots were screened using the two-dimensional gel electrophoresis,12 of them were identified effectively.They were involved in a variety of pathophysiological processes,including cellular energy metabolism,regeneration,and oxidative stress.Conclusion Mild hypothermia plays a certain neuroprotective effect by intervening and regulating multiple molecular events,including cellular energy metabolism,regeneration,and oxidative stress.
出处
《中华神经外科杂志》
CSCD
北大核心
2015年第8期846-850,共5页
Chinese Journal of Neurosurgery
基金
基金资助：天津市卫生局科技基金项目（10K6210）
塘沽区科委科研基金项目（2011MS06-05）
滨海新区卫生局科研基金资助项目f2011BHKL002）
关键词
低温
人工
颅脑损伤
免疫电泳
双向
蛋白质组学
Hypothermia,induced
Craniocerebral trauma
Immunoelectrophoresis,two-dimensional
Proteomics
