“醒脑开窍”针刺法对局灶性脑缺血大鼠海马蛋白质组学影响的研究

Effect of Xingnao Kaiqiao acupuncture on proteomics in hippocampus in rats with focal cerebral ischemia

关键词:  “醒脑开窍”针刺法  脑缺血  蛋白质组学  双向电泳  基质辅助激光解析电离飞行时间质谱

Key words:  Xingnao Kaiqiao(restoring consciousness and waking up the patient from unconsciousness) acupuncture  cerebral ischemia  Proteomics  two-dimensional gel electrophoresis(2-DE)  matrix-assisted laser esorption/ionization-time of flight(MALDI-TOF-MS)

摘要:

[目的] 寻找与脑缺血相关及针刺起效的靶蛋白,从蛋白质组学层面探讨脑缺血的病理机制及针刺治疗脑缺血性损伤的作用机制。[方法] 将Wistar大鼠随机分为正常组、假手术6h组,模型6h组和针刺6h组,采用改进的线栓法制备动物模型。在规定时间快速断头取脑,剥离缺血侧海马,提取脑组织总蛋白进行双向电泳,以Image Master 2DV3.01 Elite软件进行图像分析。选取差异蛋白质点进行胶内酶解,以基质辅助激光解析电离飞行时间质谱(MAL-DI-TOF-MS)测肽质量指纹图,检索Swiss-Port数据库,对蛋白质进行鉴定。[结果] 共鉴定出30个差异蛋白质。正常组与假手术组比较无差异蛋白表达;模型组与正常组比较鉴定出差异蛋白质点29个,其中在模型6h组新出现1个,在模型6h表达上调的11个,表达下调的17个;针刺组与模型组比较鉴定出差异蛋白质点21个,其中在针刺组上调有16个,下调的有5个。[结论] 以双向电泳联合质谱技术,初步发现与脑缺血相关以及针刺治疗脑缺血的部分靶蛋白质,有助于深入研究脑缺血性损伤病理机制及针刺治疗脑缺血性损伤的作用机制。

Abstract:

[Objective] To find out the target and effective proteins related with cerebral ischemia and then explore the pathologic mechanism and the acupuncture effect on proteomics in rats with focal cerebral ischemia.[Methods] Wistar rats were randomly divided into normal group, sham operated 6 h group, MCAO 6 h group and acupuncture 6 h group. Nylon monofilament was used to induce middle cerebral artery occlusion (MCAO). After sample preparation, two-dimensional gel electrophoresis was performed. The 2-DE gel was stained with Coomassie brilliant blue R-350. The images were analyzed by software of Image master 2D V3.01 Elite. The peptide mass fingerprints of differentially expressed proteins were detected by MALDT-TOF-MS, then the data base of Swiss-Port to identify the proteins was researched.[Results] There were 30 differentially expressed proteins in normal group, sham operated 6 h group, MCAO 6 h group and acupuncture 6 h group. Compared with normal group, Ubiquinol-cytochrome C reductase iron-sulfur subunit was detected only in MCAO 6 h group; 11 proteins were significantly up-regulated and 17 down-regulated. Compared with acupuncture group, 16 proteins were significantly up-regulated and 5 down-regulated.[Conclusions] From proteomics, we find out a series of proteins related with cerebral ischemia and some parts of target proteins of acupuncture therapy. It can be helpful for deeply researching the ischemic injury mechanism and acupuncture treating mechanism.