【作者】 曾年菊；

【导师】 梁清华；

【作者基本信息】 中南大学 ， 中西医结合临床， 2008， 硕士

【摘要】 研究目的:1.建立脑梗死肝阳化风证、阴虚风动证高分辨率的二维凝胶电泳图谱;2.应用比较蛋白质组学方法分析并鉴定脑梗死肝阳化风证和阴虚风动证的相关蛋白质:3.从腺苷酰环化酶(adenylate cyclase,AC)在脑梗死肝阳化风证及阴虚风动证中的基因表达,探讨中医肝阳化风证与阴虚风动证的关联性与差异性。研究方法:设脑梗死肝阳化风证组、健康人对照组、脑梗死阴虚风动证对照组3组,严格按照疾病诊断、证侯诊断标准,选择脑梗死肝阳化风证患者15例,脑梗死阴虚风动证患者15例,按照纳入排除标准,选择健康人15例,取外周静脉血分离单个核细胞提取蛋白质,经双向凝胶电泳,考马斯亮蓝染色获取凝胶图谱,PDQuestV7.3.1软件比较分析3组图谱并识别脑梗死肝阳化风证与阴虚风动证的差异表达蛋白质点及相同表达蛋白质点。MALDI-TOF-MS质谱仪进行肽质量指纹谱的鉴定,Mascot检索软件检索软件在Swiss-prot数据库搜索进行蛋白鉴定,检索出相关蛋白质的名称、分类及功能。并用RT-PCR技术检测腺苷酰环化酶(adenylate cyclase,AC)在脑梗死肝阳化风证及阴虚风动证中的基因表达情况。研究结果:1.初步建立了脑梗死肝阳化风证、健康人组、脑梗死阴虚风动证患者外周血单个核细胞2-DE图谱,分别获得蛋白质点459个,552个,644个;2.PDQuest V7.3.1软件比较分析,发现了脑梗死肝阳化风证与阴虚风动证的相同蛋白质点8个,差异蛋白质点23个;3.MALDI-TOF-MS质谱分析和MASCOT软件检索Swissprot蛋白质数据库鉴定出27个蛋白质斑点,共20种蛋白质,各种蛋白质点在3组的表达各异,经肝阳化风组与健康对照组,阴虚风动组与健康对照组,以及肝阳化风组与阴虚风动组的相互比较,发现肝阳化风组与阴虚风动组差异蛋白质点23个,鉴定出22个共15种蛋白质,分别为成帽蛋白、血小板凝血酶敏感蛋白-1、腺苷酰环化酶联合蛋白-1、鸟苷二磷酸解离抑制因子2、γ-肌动蛋白、钙结合蛋白、肌球蛋白调节轻链、抗增殖蛋白、谷胱甘肽转移酶ω-1、硫氧还蛋白过氧(化)物酶-1、Visual cortex cDNA、假想蛋白、Ras抑制蛋白、受体相关蛋白/RNA聚合酶相关蛋白、锌结合蛋白;相同表达蛋白质点8个,鉴定出6个蛋白质点共5种蛋白质.分别为核内不均一核糖核蛋白、丝氨酸/苏氨酸特异蛋白激酶、踝蛋白、纤维蛋白素原β-链前体、纤维蛋白素原α链前体,包括细胞骨架蛋白,信号传导蛋白,氧化还原蛋白,凋亡相关蛋白,血液蛋白等多类蛋白质表达异常;4.腺苷酰环化酶(adenylate cyclase,AC)mRNA在脑梗死肝阳化风组中表达缺失,阴虚风动组中表达明显下调,且表达量有显著差异性(P＜0.05)。研究结论:1.初步建立了脑梗死肝阳化风证、脑梗死阴虚风动证及健康人PBMC2-DE图谱,并通过MODI-TOP-MS技术鉴定分析出脑梗死肝阳化风证与阴虚风动证相同表达蛋白与差异表达蛋白,提示本病相类证有共同的物质基础,同病异证有不同的本质内涵。2.RT-PCR验证ACmRNA在脑梗死肝阳化风证与阴虚风动证中的表达与蛋白组学检测结果一致,证明了蛋白组学技术的稳定性和结果的准确性。更多还原

【Abstract】 Objective:1. TO establish cerebral infarction Ganyang-Huafeng Syndrome (GYHF) and Yinxu-Fengdong Syndrome(YXFD) high-resolution map of the two-dimensional gel electrophoresis;2. Using Comparison of proteomics methods to analyze and identify different proteins in cerebral infarction Ganyang-Huafeng Syndrome(GYHF) and Yinxu- Fengdong Syndrome(YXFD);3. From the gene expression of adenylate cyclase (AC) in cerebral infarction Ganyang-Huafeng Syndrome(GYHF) and Yinxu-Fengdong Syndrome(YXFD), to explore the relevance and the difference of Chinese medicine Ganyang-Huafeng Syndrome(GYHF) and Yinxu-Fengdong Syndrome(YXFD).Methods: Design 3 groups, cerebral infarction Ganyang-Huafeng Syndrome group(GYHF), Yinxu-Fengdong Syndrome group (YXFD),and healthy control group,in strict accordance with the disease diagnosis, diagnostic criteria syndrome, cerebral infarction choice of Ganyang-Huafeng Syndrome(GYHF)patients of 15 cases, Yinxu-Fengdong Syndrome (YXFD) patients of 15 cases, in accordance with the exclusion criteria included, choose healthy people in 15 cases, isolated from peripheral blood mononuclear cells extracted proteins. The two-dimensional gel electrophoresis, Coomassie brilliant blue staining patterns of access to gel, PDQuest V7.3.1 software comparative analysis of the three groups and identify differentially expressed proteins and the same expression of proteins of Ganyang-Huafeng Syndrome(GYHF)and Yinxu-Fengdong Syndrome(YXFD). MALDI-TOF-MS mass spectrometer for peptide mass fingerprint identification, Mascot retrieval software in the Swiss-prot database search for protein identification, retrieval related protein’s name, classification and functions. By RT-PCR technology to detect gene expression of adenylate cyclase(AC) in the cerebral infarction Ganyang-Huafeng Syndrome(GYHF)and Yinxu-Fengdong Syndrome(YXFD).Results:1. Initially established PBMC 2-DE maps of Ganyang-Huafeng Syndrome(GYHF) patients and Yinxu-Fengdong Syndrome(YXFD) patients and healthy group, respectively proteins are 459, 552,644;2. PDQuest V7.3.1 software comparative analysis ,and found 8 same proteins and 23 different proteins in Ganyang-Huafeng Syndrome (GYHF) group and Yinxu-Fengdong Syndrome(YXFD)group.3. MALDI-TOF-MS mass Spectrometry analysis and MASCOT software retrieval Swissprot protein database identified 27 protein spots, a total of 20 kinds of proteins, various protein spots express different in different groups,compared with Ganyang-Huafeng Syndrome(GYHF) group and healthy group, Yinxu-Fengdong Syndrome(YXFD)group and healthy group, Ganyang-Huafeng Syndrome(GYHF) group and Yinxu-Fengdong Syndrome(YXFD)group,we found 23 different protein spots, 22 identified with 15 kinds of proteins, they are Capping protein, Thrombospondin-1, Adenylyl cyclase-associated protein 1, Rho GDP-dissociation inhibitor 2,γ-actin, Transgelin-2, Prohibitin, myosin regulatory light chain,Glutathione transferase omega-1, Thioredoxin-dependent peroxide reductase, Visual cortex cDNA, Hypothetical protein NEDD5, Ras suppressor protein,RAP1B protein, ZYX protein [Fragment]. 8 same protein spots, 6 identified with 5 kinds of proteins,they are Heterogeneous nuclear ribonucleo proteinK, serine/threonine-specific protein kinase, Talin-1, fibrinogen beta chain precursor, fibrinogen alpha chain precursor. Including the cytoskeleton protein, signal transduction protein, redox protein, apoptosis-related protein, blood protein, and other types of abnormal proteins.4. Expression of ACmRNA in Ganyang-Huafeng Syndrome(GYHF) group was missing, Yinxu-Fengdong Syndrome(YXFD)group was significantly down-regulated, and the expression have significant difference (P <0.05).Conclusion:1.Initially established cerebral infarction Ganyang-Huafeng Syndrome(GYHF) , Yinxu-Fengdong Syndrome(YXFD) and healthy PBMC 2-DE maps, Through MODI-TOP-MS analysis of Ganyang-Huafeng Syndrome(GYHF) , Yinxu-Fengdong Syndrome (YXFD) , the same protein expression and protein expression of differences, suggesting that the disease similar cards have a common material basis of different ,and have different connotations of the essence.2. RT-PCR verified ACmRNA expression in Ganyang-Huafeng Syndrome(GYHF) and Yinxu-Fengdong Syndrome(YXFD) ,the results is the same with protein test results, Proved the stability of Proteomics technology and the accuracy of the results.更多还原