摘要
应用蛋白质双向凝胶电泳（two-dimensional polyacrylamide gel electrophoresis，2-DE）技术，分析在急性重度失血性休克（refractory hemorrhagic shock，RHS）条件下，大鼠肝脏蛋白质组表达的差异．16只雄性Wistar大鼠随机分成正常对照组（sham hemorrhage shock，SHS）和RHS模型组，每组8只．采用股动脉放血的方法制备模型，在规定时间内处死大鼠并分离肝脏，提取肝脏总蛋白质后进行2-DE．运用Image Master 2D Platinum v 5．0凝胶图像分析软件对2-DE凝胶图像进行差异表达分析．有意义的差异蛋白质点用基质辅助激光解析电离飞行时间质谱进行肽质量指纹图谱分析，借助Swiss-prot数据库进行蛋白质搜索和鉴定．SHS组和RHS组肝脏的2-DE图谱，分别平均识别到698±11和700±13个蛋白质点，SHS组和RHS组肝脏间平均匹配率达88％～92％．共发现10个差异有意义的蛋白质点，鉴定出了肿瘤抑制性抗原gp96、葡萄糖调节蛋白58、过氧还蛋白Ⅰ、细胞色素b5、谷胱甘肽转移酶、ATP合酶8亚单位、二磷酸果糖酶B、三磷酸甘油醛脱氢酶等8种蛋白质．结果表明，以2-DE技术得到重复性和分辨率都较好的2-DE图谱，并初步鉴定急性重度失血性休克后大鼠肝脏的差异表达蛋白质，为深入研究失血性休克的生理病理机制及寻找失血性休克预防和治疗的生物标志物提供了依据．
To screen the differential expression proteins in the rat liver under refractory hemorrhagic shock （RHS） by two-dimensional polyacrylamide gel electrophoresis （2-DE） , sixteen male wistar rats were randomly divided into sham hemorrhage shock （SHS） group and RHS group equally. RHS was induced by withdrawing blood through the femoral arterial catheter within 15 min until the mean arterial blood pressure （MABP） decreased to 35-40 mm Hg. The MABP was maintained at this level. The animals were sacrificed at 80 min after hemorrhage. The total liver proteins of SHS group and RHS group were extracted and separated by 2-DE. Then the gels were stained by Coomassie brilliant blue R-250, and analyzed using Image Master 2D Platinum v 5.0 software. The differential expression proteins were cut from the gels, analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）, and identified through searching Swiss-prot database with Mascot software. About 698 ± 11 protein spots were detected by Image Master analysis software in SHS group and 700 ± 13 protein spots in RHS group. It was 88%-92 % that the ratio of protein spots matched between SHS group and RHS group. Ten protein spots with significant difference were found,and upregulated or downregulated or appeared after the stimulation of refractory hemorrhage. Eight differential proteins were successfully identified. These proteins included tumor rejection antigen gp96, glucose regulated protein 58, ATP synthase beta subunit, Aldob protein, Gapd protein, glutathione transferase, peroxiredoxin I , cytochrome b5 etc, which involved in transcription regulation, antioxidation, signaling pathway and so on. The better resolution and repetitive 2-DE patterns of SHS group and RHS group were established and some differential proteins between the two groups were found. These results may provide scientific datum for screening the molecular biomarker used to diagnose and treat hemorrhagic shock, as well as to elevate the patient＇s prognosis and provide new clue for the research on pathogenic mechanism of hemorrhagic shock.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2007年第4期316-322,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
军事医学科学院学科培育项目~~
