失血性休克大鼠血浆蛋白质的差异分析

Analysis of Differential Protein Expression in Rat Plasma under Acute Severe Hemorrhagic Shock

目的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（acute severe hemorrhagic shock，ASHS）条件下大鼠血浆蛋白质组表达的差异。方法16只雄性Wistar大鼠随机分成对照组（sham hemorrhage shock，SHS）和ASHS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收集血浆，去除血浆中的高丰度蛋白后进行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。结果SHS组和ASHS组肝脏的2-DE图谱分别平均识别到（570．0±0．9）和（578．0±1．4）个蛋白质点，SHS组和ASHS组肝脏间平均匹配率达83．6％-86．2％，共发现8个差异有意义的蛋白质点，鉴定出了白蛋白、甲状腺激素结合蛋白-D链蛋白和信号素-3D前体等3种蛋白质。结论以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血浆去除高丰度蛋白后的差异表达蛋白质，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。

Objective To screen the differential expression of proteins in rat plasma under acute severe hemorrhagic shock （ASHS） by means of two-dimensional polyacrylamide gel eleetrophoresis （2-DE） . Methods Sixteen male wistar rats were randomly divided into sham hemorrhage shock （SHS） group and ASHS group, with 8 rats for each group. ASHS was induced by withdrawing blood through femoral arterial catheter within 15 min until the mean arterial blood pressure （MABP） was decreased to and maintained at 35-40 mm Hg. The animals were sacrificed 80 min after hemorrhage. The harvested plasma for both SHS group and ASHS group was treated with Aurum TM Serum Protein Mini Kit to remove high-quality of proteins, and the remaining plasma proteins were then separated respectively by 2-DE. The 2-DE gel was 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 gel, 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. Results As shown by Image Master Analysis software, 570. 0±0. 9） protein spots were detected in SHS group and 578.0 ± 1.4 protein spots in ASHS group. The machting ratio of protein spots between SHS group and ASHS group reached to 83.6-86.2 %. Of them, ten protein spots displayed significant difference, either to be upregulated, downregulated or newly visualized after stimulation of acute severe hemorrhage shock. Three differential proteins were identified, including Albumin, Chain D of rat Transthyretin and Semaphorin-3D precursor. Conclusion In this study, the repetitive 2-DE patterns with good resolution were well established. Several differentially expressed proteins between SHS group and ASHS group were preliminarily identified. These results may help to screen molecular biomarkers for diagnosis and treatment of hemorrhagic shock and provide new clue for pathogenic mechanism study of hemorrhagic shock.