低压低氧延迟预适应小鼠海马差异表达蛋白的分离及鉴定研究

ISOLATION AND IDENTIFICATION OF DIFFERENTIALLY EXPRESSED PROTEINS IN HIPPOCAMPUS OF MICE DURING HYPOBARIC HYPOXIC DELAYED PRECONDITIONING

目的:探索低压低氧延迟预适应(HHDP)小鼠海马差异表达蛋白。方法:建立小鼠海马HHDP动物模型后,用含尿素的细胞裂解液提取海马总蛋白质。经等电聚焦、SDS-PAGE电泳、考马斯亮蓝R250染色,比较对照组及HHDP组双向电泳图谱上蛋白质点的差异。切取HHDP组表达增高的蛋白质点,经脱色、胰蛋白酶酶切、提取肽片段,进行MALDI-TOF-MS检测。结果:在正常对照组和HHDP组海马总蛋白双向电泳图谱上,分别检测到481±38和477±21个点,匹配点为169±6个。其中HHDP组比对照组增高大于2倍的有33±10个点,降低大于2倍的有21±12个点。电泳图谱平均相关系数为0.7748±0.0267。有12个点在HHDP组显著增高(P<0.05,n=4),对其进行了肽质量指纹图谱分析,其中8个蛋白点得到相应的肽质量指纹图谱。数据库检索显示其中一个为果糖二磷酸醛缩酶A。3个在蛋白质数据库中没有检索到与之相匹配的任何蛋白,可能为新蛋白。另外4个可找到与其有部分匹配的氨基酸序列,但分子量和等电点与对应蛋白相差悬殊,它们可能具有同源性。结论:小鼠海马HHDP时,果糖二磷酸醛缩酶A等多种蛋白表达发生改变,可能是产生HHDP的重要机制之一。

Aim： To explore the differentially expressed proteins between hypobaric hypoxic delayed preconditioning （HHDP） and normal mouse hippocampus. Methods： After the animal model of HHDP was constructed, hippocampal proteins were obtained by a series of abstraction with lysis solution containing high concentration urea. As soon as isoelectric focusing and SDS-PAGE was performed. The resolved proteins in the 2-DE gels were visualized by Coomassie blue R-250. The gels were scanned, and the images were processed with PDQuest software. Differential proteins were exactly excised from the gels, destained and digested with trypsin. The peptides were isolated and sent for MALDI-TOF-MS testing. Database searching was performed using peptide masses obtained from MALDI- TOF-MS. Results： Averages of 481 ± 38 and 477 ± 21 protein spots were detected in control gels and preconditioning gels, respectively. 169 ± 6 protein spots were matched between these two types of gels. Among the matched spots, while the quantities of 21 ±12 spots in control gels increased by above 2 times than that in preconditioning one, the quantities of 33 ±10 spots in preconditioning gels increased by the same times than that in control one. The correlation coefficient between these two patterns were 0. 7748 ±0. 0267. 12 spots in preconditioning gels significantly increased compared with the control（ P〈 0.05, n = 4）. Among 12 spots excised from the gels, perfect peptide mass fingerprinting spectrums of 8 spots were acquired. The results showed that one protein was fructose biphosphate aldolase A. Three proteins matched nothing might be new proteins. The other four proteins just matched the partial sequences of the proteins of database were no coincidence to it＇s isoelectric point and molecular weight. So they might be homological proteins. Conclusion： Many proteins, for example fructose biphosphate aldolase A, has been differentially expressed in hippoeampus of mice during HHDP. This may be one of the molecule mechanisms of HHDP.