【作者】 张莺；

【导师】 陈泽奇；

【作者基本信息】 中南大学 ， 中西医结合临床， 2008， 硕士

【摘要】 目的1.建立正常大鼠、高血压肝阳上亢证大鼠及高血压肝阳上亢证平肝潜阳法治疗后大鼠的下丘脑和肾上腺组织双向凝胶电泳图谱。2.观察高血压肝阳上亢证大鼠平肝潜阳法治疗前后下丘脑及肾上腺的蛋白质表达差异,从蛋白质组学水平探讨其降血压和改善肝阳上亢证症状的分子机制,为寻找新的药物标志蛋白提供理论依据。方法1.以SHR大鼠加灌服附子汤法制备高血压肝阳上亢证大鼠模型。2.二维凝胶电泳分离大鼠下丘脑及肾上腺蛋白质,凝胶经考染显色后,PDQuest图像分析软件进行比较分析,获得差异表达蛋白质。3.利用基质辅助激光解吸电离飞行时间质谱和数据库分析鉴定差异表达蛋白质。结果1.动物模型的复制:模型组和PGQYF组大鼠在附子汤灌胃3周后,其易激惹程度发生变化,多表现为Ⅲ级,且大部分出现了结合膜充血;饮水量明显增加;收缩压明显升高。与正常对照组及造模前比较,差异均具有统计学意义(P＜0.01)。经PGQYF治疗后(第6周末),其易激惹程度及结合膜充血均好转,易激惹程度多表现为Ⅰ和Ⅱ级,饮水量及收缩压较治疗前及模型组明显减少,差异均具有统计学意义(P＜0.01)。2.各组总蛋白2-DE图谱的建立及差异蛋白质识别:建立了正常大鼠、高血压肝阳上亢证大鼠及平肝潜阳法治疗后大鼠重复性好的下丘脑和肾上腺组织蛋白质组表达图谱。通过PDQuest图像分析,发现了27个差异表达蛋白质点:和正常组比较,肝阳上亢模型组下丘脑图谱中1、2、4、5、7、8号蛋白质点、肾上腺图谱中2、3、5、6、7号蛋白质点表达上升2倍及以上,且其在PGQYF组中表达下降2倍及以上,大致处于与正常组同一水平或低于正常对照组低;肝阳上亢模型组下丘脑图谱中3、6、9、10、11、12、13、14、15号蛋白质点、肾上腺图谱中1、4、8、9、10、11、12号蛋白质点表达下降2倍及以上,且同一点在PGQYF组中表达上升2倍及以上,大致处于与正常组同一水平或高于正常对照组。3.差异蛋白质点质谱鉴定及蛋白功能分类:选取25个蛋白质点进行质谱鉴定,成功鉴定22个蛋白质点。根据NCBI、MSDB数据库中提供的信息,这些差异蛋白质涉及多种蛋白种类,如参与能量代谢的酶类、抗氧化蛋白、神经内分泌相关蛋白、细胞信号转导蛋白、细胞骨架相关蛋白等。其中硫氧还蛋白过氧化物酶-Ⅱ(PRX-Ⅱ)、热休克蛋白-27(HSP-27)、膜联蛋白-Al(Annexin-Al)等蛋白可能与高血压肝阳上亢证及平肝潜阳治疗法密切相关。结论平肝潜阳法治疗后,高血压肝阳上亢证大鼠症状改善,血压降低,可能与下丘脑和肾上腺组织中某些蛋白质的改变有关。成功鉴定了高血压肝阳上亢证平肝潜阳法治疗前后下丘脑及肾上腺组织差异表达蛋白,为深入研究高血压病肝阳上亢证病理生理机制及平肝潜阳法治疗的作用分子机制奠定基础。更多还原

【Abstract】 SUBJECT1. To establish two-dimensional electrophoresis profiles with high resolution and reproducibility from hypothalamuses and adrenal glands of normal rats、hyperensive rats with hyperactivity of liver-yang and the hypertensive rats with hyperactivity of liver-yang with Pinggan-Qianyang treatment.2. To explore the different expression in hypothalamic and adrener-gdrenic proteins of hypertensive rats with hyperactivity of liver-yang with Pinggan-Qianyang treatment, and probe into the hypertension pathogenesy and mechanism of decreasing the blood pressure and ameliorating the symptoms of Pinggan-Qianyang treatment from the level of proteomics, and provide theories evidences for new medical marked protein.METHODS1. Hypertensive rats with excessive rising of liver-yang model rats were prepared by spontaneous hypertensive rats (SHR) with drenching Aconiti Praeparatae Decoction.2. Hypothalamic and adrenergdrenic proteins was separated by 2D gel electrophoresis (2-DE). After Coomasie briliant blue G250 staining, gel-image analysis was performed by using PDQuest, we get the differential expressed proteins. 3. The differential expressed proteins was identified by matrix_associated laser desorption/ionization time of flight massspectrometry (MALDI-TOF-MS) and database analysis.RESULTS1. The model of hyperensive rats with hyperactivity of liver-yangwere reproduced succeedly, and the curative effect of Pinggan-Qianyang treatment was good.2. The 2-DE Patterns of hypothalamuses and adrenal glands of normal rats、SHR with hyperactivity of liver-yang and the rats with hyperactivity of liver-yang with Pinggan-Qianyang treatment were presented.Gel-image analysis reveals that there are 27 differential protein spots.3. The 22 of total 25 differential protein spots are successfully identified by MALDI-TOF-MS.The functions of these Proteins involve in metabolism, energy generation, transportation, antioxidation, sigal transduction and cytoskeleton etc according to information provided in NCBI and MSDB database. Among them, there are some proteins closely related to hypertension with hyperactivity of liver-yang and the Pinggan-Qianyang treatment, such as Peroxirdoxin-II、Heat shockprotein-27、Annexin-A1.CONCLUSIONThe blood pressure of hyperensive rats with hyperactivity ofliver-yang is reduced with symptoms ameliorated after taking Pinggan-Qianyang treatment, which might be related to the changes of hypothalamic and adrenergdrenic protein. Appreciated hypothalamic and adrenergdrenic differential expressed proteins with Pinggan-Qianyang treatment succeedly. This approachmay lay a foundation for the further investigation of pathogenic mechanisms in hypertension with hyperactivity of liver-yang and curative effect mechanisms of Pinggan-Qianyang treatment.更多还原