督脉电针对不同时段急性颅脑损伤大鼠差异蛋白组学的研究

【作者】 李真姬；

【导师】 李志刚；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2010， 博士

【摘要】 研究背景颅脑损伤(craniocerebral injury, CCI)是一种致死率和致残率很高的疾病,在中国的发生率以及由其致伤致残、致死的伤员也逐年增加。目前,现代医学对颅脑损伤进行了大量研究,但没有重大突破,而针灸和中药具有一定的优势。随着人类基因组草图序列测定的完成,基因组研究的重点也由结构基因组转向功能基因组,生命科学进入了蛋白质组学时代。差异蛋白组学的研究有利于人类更细致地研究疾病相关信息变化所导致的蛋白质种类和数量变化,有利于更深入地研究蛋白质的性质和功能,对探索疾病的真正发生机制具有极其重要的意义。在本课题的研究中,我们不是以颅脑损伤大鼠的血清为研究对象,而是对其脑组织进行差异蛋白组学研究,这更有利于揭示针刺治疗颅脑损伤的作用机制。实验方法135只SD雄性大鼠,随机分为三个时间段,空白组(24h、48h、6d)、模型组(24h、48h、6d)、电针组(24h、48h、6d),每组15只大鼠,共45只。参照Feeney等研制的鼠颅脑创伤模具改良式打击装置造模。电针“百会”、“印堂”督脉穴。术后24h、48h、6d取损伤区颅脑组织称重,保存。对样本蛋白进行提取、定量、蛋白双向电泳图像分析,找出差异点进行质谱分析和数据库检索。实验结果24h时间段有八个差异蛋白点共鉴定出6种蛋白质：烯醇化酶、白蛋白、腺苷酸激酶、结蛋白、半胱氨酸天冬氨酸特异性蛋白酶、锌指蛋白。48h成功鉴定出7种蛋白质：假定蛋白、热休克蛋白8、微管蛋白β-5、锌指蛋白233、分泌颗粒素Ⅱ、醛缩酶A、热休克蛋白70。6d时间段六个差异蛋白点共鉴定出4种蛋白质即电压依赖性阴离子通道1、细胞角蛋白、锌指蛋白365、人转铁蛋白。结论通过本动物实验可见,电针可逆转颅脑损伤大鼠脑内多数差异蛋白表达的变化,其作用可能是通过抑制神经细胞凋亡、改善细胞能量代谢、调节异常蛋白等途径促进颅脑损伤的修复。随着时间和针刺次数的增加,电针可能逐渐侧重于发挥改善细胞能量代谢和抑制其凋亡的作用,而对受损蛋白的调节作用在减弱。更多还原

【Abstract】 BackgroundCraniocerebral injury (craniocerebral injury, CCI) is a high mortality rate and morbidity of diseases, the incidence in our country and its injured and maimed, wounded also increased year by year of death. Currently, modern medicine has done a lot of traumatic brain injury, but no major breakthrough, but acupuncture and Chinese medicine has a definite advantage.With the draft human genome sequence of the complete genome research shifted the focus from structural genomics functional genomics, life sciences into the proteomics era. Proteomics research is conducive to a more detailed study of human disease-related information resulting from changes in the type and quantity of protein changes that contribute to a more in-depth study of the nature and function of proteins, to explore the mechanism of disease, the real place of great importance.In the study of this topic, we do not craniocerebral rats serum to study the object, but modifications to differentiate brain tissue proteomics science research, it more conducive to revealing the effect of acupuncture treatment of brain injury mechanisms.Objectiveto observe the effect of electroacupuncture(EA) on the differentially expressed proteins in the brain at different time courses after craniocerebral injury (CCI) in the rat, so as to study its underlying mechanism in improving craniocerebral injury.MethodsA total of 135 male SD rats were randomized into normal control, model24h,EA24h,mode48h, EA48h, mode6d, EA6dgroups, with15 cases in each. CCI model was established by using modified Feeney’smethod. EA(2Hz,2-5mA)wasappliedtoBaiHui(GV20)and yintang(EX-4)for 30min. The injured brain tissue was collected 24h.48h and 6d after CCI and EA treatment, weighted and stored under-80℃till detection. Two dimensional gel electrophoresis(22DE) was used to separate total proteins of the brain tissue, followed by protein extraction and quantitation,2_D gel image analysis, matrix assisted laserdesorptionionisationtime-of-flightmassspec-trometry(MALDI-TOF-MS).Results24h successful identification of the six kinds of proteins:enolase、albumin、adenylate kinase、Desmin、Caspase、zinc finger protein;48h: successful identification of the seven kinds of proteins:hypothetical protein, heat shock protein 8, tubulinβ-5,233 zinc finger protein, secretory granulesⅡ, and Aldolase A, heat shock protein 70.6d period of six protein spots were identified four kinds of protein that is voltage dependent anion channel 1, cytokeratin, zinc finger protein 365, human transferrin.ConclusionProteome analysis indicates that in CCI rats, some differentially expressed proteins involving energy metabolism, neuronal apoptosis reduction, protein degradation inhibition may contributeto the effect of EA in repairing the craniocerebral injury更多还原