【作者】 孙晓敏；

【导师】 罗仁；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2008， 博士

【摘要】 【研究背景与目的】肾阴虚证是中医学的基本证候之一,历代医家对肾阴虚证的理论与防治研究都颇为重视。特别是近几十年来,众多学者运用酶学、细胞生物学、免疫学以及分子生物学等现代技术和方法研究发现,肾阴虚证与血液流变学、内分泌、免疫、物质与能量代谢、微量元素、生化、酶以及基因表达等多方面异常有关,初步证实其具有现代的病理生理学基础,并部分阐释了其中医理论的某些细节及其科学性,取得了一定的成绩。本课题组在先前的肾阴虚证相关基因研究过程中,通过西医辨病与中医辨证相结合,从糖尿病、慢性肾炎、狼疮性肾病以及亚健康状态等肾阴虚证入手,应用RNA微量扩增、抑制性消减杂交、基因克隆等技术和方法,成功地构建了肾阴虚证的cDNA文库;继而根据“以药测证”理论,应用基因芯片研究发现有46条基因可能与肾阴虚证密切相关。而基因又要表达为相应的蛋白质才能影响生物功能,其表达类型与表达程度均受外界环境及内在状态变化的影响。蛋白质组研究正是从蛋白质水平的特异性和表达模式上反映了疾病发生时机体的内在变化。运用蛋白质组学技术,对同一证候不同疾病的蛋白质组表达差异和功能的研究,只有通过对这些差异的研究,从而归纳出“证”的一般规律,才能对肾阴虚证有全面的解析。血浆是取材容易、应用广泛、富含蛋白质的研究样品,其蛋白水平的变化与多种疾病有关。血浆蛋白质组学可以在整体水平上研究血浆蛋白质的表达水平、翻译后修饰、相互作用等,并由此在血浆蛋白水平获得疾病过程和药物干预过程的外在、综合的认识,在医学基础和应用研究中都有着重要意义。因此,本研究拟在前期肾阴虚证流行病学调查和基因组学研究的基础上,采用“病证结合”,开展肾阴虚证的血浆蛋白质组学研究,寻找肾阴虚证的差异蛋白,探讨肾阴虚证在蛋白质水平可能的发生机制。【方法】1、以正常人血浆样品为研究对象,对白蛋白/IgG球蛋白去除后血浆样品、全血浆和热SDS法全血浆样品进行PH梯度双向凝胶电泳分离,凝胶经硝酸银染色后,利用PDQuest软件对2-DE图谱进行重复性分析,并对不同处理方法的全血浆2-DE图像的结果进行比较,以确定后续肾阴虚证双向凝胶电泳方法。2、采用“病证结合”手段,依据本课题组对肾阴虚证的临床流行病学调查及有关文献调研结果,选取亚健康、慢性肾小球肾炎以及IgA肾病。肾阴虚证患者作为研究对象,确诊为肾阴虚证患者,作为实验组;确诊为肾阳虚证患者,为阳性对照组;选择健康志愿者作为正常对照组。运用双向凝胶电泳技术对其进行血浆蛋白质差异表达双向凝胶电泳分析。3、结合软件分析和手工筛选,从有或无和表达差异蛋白质点中选取点清晰且表达水平改变明显的蛋白质点作为最终质谱鉴定的对象;采用肽质量指纹图谱(PMF)的方法,并使用国际互联网上的蛋白质数据库(http://www.expasy.org)提供的检索程序对差异蛋白质进行鉴定。【结果】1、与全血浆直接上样和白蛋白/IgG球蛋白去除后血浆样品上样双向电泳图谱比较,热SDS法全血浆上样双向电泳蛋白丢失较少,凝胶硝酸银染色显示蛋白质点染色深、分散清楚,并且避免了在白蛋白/IgG球蛋白去除过程中低丰度蛋白非特异性吸附所造成的人为差异。从而建立了热SDS法全血浆上样进行血浆双向电泳的方法,获得分辨率较高且重复性好的人全血浆蛋白质双向凝胶电泳图谱,为分离高质量的蛋白质点进行肾阴虚证血浆蛋白组学研究奠定了良好的工作基础。2、应用双向电泳技术对亚健康态、IgA肾病和慢性肾小球肾炎肾阴虚证、肾阳虚证与正常人进行了差异表达血浆双向凝胶电泳和数字化图像分析发现,与正常人相比:(1)在亚健康肾阴虚证血浆中高表达的蛋白质斑点73个,低表达的蛋白质斑点90个,特异出现的蛋白质斑点10个,缺失的蛋白质斑点14个;在亚健康状态肾阳虚证血浆中高表达的蛋白质斑点67个,低表达的蛋白质斑点191个,特异出现的蛋白质斑点17个,缺失的蛋白质斑点19个。(2)在慢性肾小球肾炎肾阴虚证血浆中高表达的蛋白质斑点109个,低表达的蛋白质斑点82个,特异出现的蛋白质斑点36个,缺失的蛋白质斑点9个;在慢性肾小球肾炎肾阳虚证血浆中高表达的蛋白质斑点112个,低表达的蛋白质斑点79个,特异出现的蛋白质斑点39个,缺失的蛋白质斑点14个。(3)在IgA肾病肾阴虚证血浆中高表达的蛋白质斑点122个,低表达的蛋白质斑点120个,特异出现的蛋白质斑点21个,缺失的蛋白质斑点19个;在IgA肾病肾阳虚证血浆中高表达的蛋白质斑点131个,低表达的蛋白质斑点111个,特异出现的蛋白质斑点18个,缺失的蛋白质斑点21个。3、综合对亚健康、慢性肾小球肾炎、IgA肾病肾阴虚证及肾阳虚证患者与正常人全血浆2-DE图谱定量差异表达分析结果,有29个蛋白质斑点在各肾阴虚证组均较正常组和肾阳虚证组有显著差异。其中在肾阴虚证血浆蛋白质双向凝胶电泳图谱中绝对高表达的蛋白质斑点9个,绝对低表达的蛋白质斑点11个;定性差异表达分析发现肾阴虚证血浆蛋白质双向凝胶电泳图谱中特异出现的蛋白质斑点6个,缺失的蛋白质斑点7个。4、采用2-DE分离技术结合MALDI-TOP质谱鉴定了7个与肾阴虚证可能相关的蛋白,其中在肾阴虚证患者血浆高表达的有:α1微球蛋白、血浆视黄醇结合蛋白、转甲状腺素蛋白以及热休克蛋白27;在肾阴虚证患者血浆低表达的则是纤维蛋白原重链、α1-抗胰蛋白酶、补体C4-B。差异蛋白的功能主要涉及机体激素调节、免疫应答、氧化应激、信号传导、细胞骨架等。【结论】1、热SDS法全血浆上样双向凝胶电泳分析法具有蛋白丢失少、操作简便、重复性好的特性,是证侯血浆蛋白质组学研究的有效手段。2、采用“病证结合”,开展肾阴虚证的血浆蛋白质组学研究,筛选并鉴定出了可能与肾阴虚证相关的7个血浆蛋白,为探讨肾阴虚证在蛋白质水平的发生机制奠定基础。更多还原

【Abstract】 【Background】As one of the basic syndrome,Kidney-yin deficiency is,family history of kidney-yin deficiency medical certification and control of the theory are quite seriously.Especially in recent decades,many scholars use of enzymes,cell biology, immunology and molecular biology,and other modern technologies and methods study found that kidney-yin deficiency syndrome concerned with the hemorheology, endocrine,immune,material and energy metabolism,Trace elements,biochemistry, enzymes and abnormal gene expression,and so on,confirmed its preliminary modern basis of the pathophysiology,and some of its interpretation of certain details of the theory of Chinese medicine and science,have made certain achievements.cDNA Lib of Kidney-yin deficiency(including diabetes,chronic nephritis, lupus nephropathia and sub-health)was successfully set up in our prior study works by some techniques and methods,such as RNA microamount-amplification, differential hybridization and gene cloning.We also found that fourty six genes were closely related with Kidney-yin deficiency in our prior study about gene array based on the theory of Chinese medicine measuring Chinese syndrome.It is well known that a gene should express a certain protein to take a part in some biological action.Type and degree of gene expression are affected by both outside environment and internal condition.The study of proteome is exactly aimed to found internal changes of organism in morbid condition.The changes is focused on the specificnesses and differences of protein expression.On the base of the specificnesses and differences resulted from making use of techniques of proteome, a general regularity of syndrome can be summed up to analyze Kidney-yin deficiency in all aspects.The blood plasma is a very good sample which is charactered with obtaining easily,being used widely and protein containing highly.The expression level, post-translational modification and post-translational interaction of protein in blood plasma can be studied in the whole by the techniques of proteome.Then the disease process and medicine therapy will be well known from results of the studies which is very important in foundation study and exploratory development.【Object】The study is aimed to find different proteins between patients of Kidney-yin deficiency and healthy people and approach the machenism of Kidney-yin deficiency in protein level.【Methods】1.The samples were normal human plasmas.The samples were treated with three different ways:removing albumin and IgG globulin,boiling and no treating. Then all samples were analyzed by two-dimensional gel electrophoresis in different pH grad.After Staining by argenti nitras,the gels were scaned and analyzed by PDQuest software.According to the results,the best method of two-dimensional gel electrophoresis was established. 2.The patients with Kidney-yin deficiency were divided into experimental group.While the patients with Kidney-yang deficiency were divided into negative control group and those healthy people were divided into normal control group.All the plasma samples in different treating ways were analyzed by two-dimensional gel electrophoresis.3.Eighteen clear points were chosen from the gels to be identified and analyzed by the method of PMF in mass spectrogram.The points being picked up should be clear and specific.【Results】1.The electrophoregram of normal human plasmas was obtained by two-dimensional gel electrophoresis.By silver nitrate staining,the proteins SDS hot plasma sample of the entire two-dimensional gel electrophoresis staining Deep, scattered clear,and to avoid in the albumin/IgG globulin in the process of removing the low abundance of non-specific absorption caused by man-made differences.The electrophoregram with high-resolution and good-reproducibility was the foundation for choosing good protein points.2.With two-dimensional electrophoresis technology to sub-health state,IgA nephropathy and chronic kidney-yin deficiency card glomerulonephritis,a deficiency syndrome and normal expression of the difference between two-dimensional gel electrophoresis and plasma digital image analysis,and the normal Compared to people:①There are 73 spots which expression levels were increased in the plasma protein of the sub-health kidney-yin deficiency,and 90 spots were decreased.10 protein spots specific appeared,14 protein spots missed.About kidney-yang deficiency of sub-health,the expression levels of 67 protein spots were increased, and 191 protein spots were decreased.The number of specific protein spots was 17, the missing protein spots was 19.②There are 109 spots which expression levels were increased in the plasma protein of the Chronic Glomerulonephritis kidney-yin deficiency,and 82 spots were decreased.36 protein spots specific appeared,9 protein spots missed.About kidney-yang deficiency of Chronic Glomerulonephritis, the expression levels of 112 protein spots were increased,and 79 protein spots were decreased.The number of specific protein spots was 39,the missing protein spots was 14.③There are 122 spots which expression levels were increased in the plasma protein of the IgA nephropathy kidney-yin deficiency,and 120 spots were decreased.21 protein spots specific appeared,19 protein spots missed.About kidney-yang deficiency of IgA nephropathy,the expression levels of 131 protein spots were increased,and 111 protein spots were decreased.The number of specific protein spots was 18,the missing protein spots was 221.3.The above results,twenty-nine protein points in Kidney-yin deficiency group were different from control groups.Among all these different points,nine protein points showed high-expression and eleven showed low-expression.Seven protein points were only found in healthy people and six protein points were only found in patients with Kidney-yin deficiency.4.Seven proteins were found to be closely related with Kidney-yin deficiency through the techniqueof two dimensional gel electrophoresis and MALDI-TOP mass spectra.Among these seven proteins,four proteins,includingα1-microglobulin, retinol binding protein,tetraiodothyronine transferring protein and heat shock protein,were highly expressed in Kidney-yin deficiency group.The other three proteins,including H-chain of fibrinogen,α1-antitrypsin and complement C4-B, were highly expressed in normal control group.The function of the different proteins was related with hormonal modulation,immune response,oxidative stress,signal conduction and cystoskeleton construction. 【Conclusions】1.Because of little-losing,simple and good repeatability,the method of two-dimensional gel electrophoresis using heating-SDS whole plasma sample is an effective means of the syndrome plasma proteomics research.2.In the plasma proteomics research,we screenned and identificated seven plasma specific differences proteins of the kidney-yin deficiency card of a kidney-yin.It was the foundation on to explore the kidney-yin deficiency mechanism in the protein level.更多还原