【作者】 毕建璐；

【导师】 罗仁；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2011， 硕士

【摘要】 目的：中医“证”是疾病发生过程中不同阶段病因病机的高度概括,既然同一证有共同的临床表现和病理机制,那么其肯定有共同的物质基础,而这种物质基础很有可能反应在基因或蛋白水平上。肾所藏之精禀受于父母,是构成胚胎发育的原始物质,其与现代医学描述的遗传物质(DNA)具有一定的同一性。而基因又要在表达相应蛋白质的情况下才能影响生物的功能。蛋白组学是研究在生命体或细胞的整体水平研究蛋白质的表达和修饰状态,以及蛋白质与蛋白质的相互作用,从而确定人体的功能蛋白并阐明其在细胞中的功能与相互关系,揭示生命活动的本质。肾阴虚证和肾阳虚证是中医学的基本证候,历代医家对肾阴虚证和肾阳虚证的理论与防治研究都颇为重视。肾阴虚证和肾阳虚证作为疾病的某一阶段的主要矛盾,必然受到“病”这一基本矛盾的影响。正是由于不同疾病的特异性,决定了不同疾病相同证侯之间的差异；而同一疾病不同中医证侯之间也存在差异。只有通过对这些差异的研究,进而归纳出证的一般规律,才有可能对肾阴虚证和肾阳虚证有更全面的解析。因此,“病证结合”是研究肾阴虚证和肾阳虚证差异蛋白表达的重要思路。本研究采取“病证结合”的方法,以IgA肾病和亚健康状态的肾阴虚证和肾阳虚证为研究对象,对其各种细胞因子靶蛋白进行抗体芯片表达谱的比较研究,筛选肾阴虚证、肾阳虚证相关蛋白的改变,通过细胞信号转导等方面的研究,结合前期试验结果,寻找主要的细胞因子改变,为确定中医肾虚证证候客观化指标提供依据。方法：1、采用“病证结合”的方法,选取确诊为IgA肾病和亚健康状态的肾阴虚证和肾阳虚证患者,作为实验组；选择健康志愿者作为正常对照组。2、收集样本,制备样品,运用生物素直接标记技术,检测出多种细胞因子的表达水平,研究肾阴虚证和肾阳虚证的细胞因子改变。结果：1、肾阴虚证组与正常对照组比较,获得差异表达蛋白共25条,其中表达水平上调的有2条(BMP5、TIMP4),表达水平下调的有23条(OSM、CXCR4、GCG、CXCR1、CTLA-4、Siglec-9、CCR7、IL-17A、IL-17F、TRADD、IGFBP-6、THBS2、IL-4R、HGFR、IL-RA、Osteoactivine、MMP10、M-CSF R、G-CSF R、CNTF、IL-17C、TGFβ、FGF23)。这些差异表达的蛋白按功能分析进行大体分类,主要涉及到免疫失调、新陈代谢、蛋白质生物合成、氧化应激、损伤修复、细胞凋亡、细胞信号传导有关等方面。肾阴虚证差异表达蛋白的异常变化,与中医肾阴虚证所表现的五心烦热、潮热盗汗、腰膝酸软、头晕目眩、易感外邪、精神萎靡、反应迟钝等症状相符。2、肾阳虚证组与正常对照组比较,获得差异表达蛋白共14条(Csk、BMP5、Frizzled1、Frizzled4、BDNF、CXCR3、LBP、LTBP1、IL-27RA、M-CSF、L-13RαⅡ、PECAM-1、Activin RⅡA、FGF R5),表达水平均上调。主要涉及到免疫应答、新陈代谢、细胞凋亡、细胞黏附、损伤修复、氧化应激、生殖能力等方面。肾阳虚证差异表达蛋白的异常变化,与中医肾阳虚证所表现的腰膝酸软、发脱齿松、耳鸣耳聋、反应迟钝、手足发冷、性欲减退,男子阳痿早泄,女子宫寒不孕等症状相符。3、肾阳虚证与肾阴虚证比较,共获得差异表达蛋白1条(BMP5),表达水平上调。主要涉及细胞凋亡、骨的合成等方面。中医认为,肾藏精,主人体的生长、发育和生殖,在体合骨,生髓。此共同蛋白的改变与中医理论相符。4、中医认为：肾藏精,主人体的生长、发育和生殖,肾主水,主纳气,在体合骨,生髓。肾精气盛衰是机体生、长、壮、老、已的基石,同时又是人体全身阴阳的根本。通过比较分析,我们发现肾阴虚证与肾阳虚证存在明显的靶蛋白改变,主要涉及到免疫失调、新陈代谢、细胞周期、骨骼发育、DNA修复、蛋白质的合成、生殖能力、神经营养、造血功能等方面的功能,与中医基本理论相符。结论：1、肾阴虚证与肾阳虚证差异表达蛋白存在明显不同,提示肾阴虚证与肾阳虚证有着各自不同的蛋白表达谱,这是符合中医理论的。同时,肾阴虚证与肾阳虚证差异表达蛋白可以为肾阴虚证和肾阳虚证的中医辨证分型提供科学依据。2、肾阴虚证与肾阳虚证存在共同的差异表达蛋白,这表明肾阴虚证与肾阳虚证都存在肾虚的现象,这不但为研究中医证侯本质提供科学的思路与方法,也为探讨肾阴虚证和肾阳虚证在蛋白水平的发生机制奠定基础。3、蛋白芯片是研究中医证候相关蛋白的比较理想的一种技术方法。从蛋白组学的角度探讨肾阴虚证与肾阳虚证,有利于揭示肾的本质。更多还原

【Abstract】 Object:The syndrome of Traditional Chinese Medicine is the summarization of pathogenesis in different stages of diseases. Since the same symptom has common clinical manifestation and pathogenesis, then they have common material foundation, which will probably reflect in the gene or protein. The essence in Kidney, coming from the reproduction of the parents, is the basis of embryonic development and identity with DNA in modrn medicine. In the case of the corresponding proteins express can the gene affect its biological functions. Proteomics is the study of protein expression and modification status, and protein-protein interactions under the overall level of living organisms or cells, leading to determine the function of human proteins and to clarify its function in the cell and then revealed the nature of living.Because the Kidney-yin deficiency and Kidney-yang deficiency is the basic syndrome of TCM, ancient physicians paid much more attentions on it. The Kidney-yin deficiency and Kidney-yang deficiency, which are the principal contradiction in a certain stage of a disease, must be influenced by the disease. The differences among varies diseases determined the differences among the same syndrome in varies diseases. Only when studied the differences and concluded the general rules can we analysis the Kidney-yin deficiency and Kidney-yang deficiency. As a result, "disease combined with syndrome" is an important ideas in studying the different proteins expression in the Kidney-yin deficiency and Kidney-yang deficiency.Using "disease combined with syndrome" in this study, we took Kidney-yin deficiency and Kidney-yang deficiency as the research object to screen the changes of cytokine through antibody chips research. We look for a major change of cytokines, to provide the basis for the TCM Kidney deficiency syndrome for objective indicators.Method:1 Using "disease combined with syndrome", selected the observers diagnosed with IgA nephropathy and subhealth condition, which are also in Kidney-yin deficiency and Kidney-yang deficiency, as the experimental group; choose healthy volunteers as the control group.2 Afters the sample collection and preparation, using direct labeling of biotin technique to detect the expression of multiple cytokines, and study the cytokine changes of Kidney-yin deficiency and Kidney-yang deficiency.Result:First,after the comparion between Kidney-yin deficiency group and normal control group, we received a total of 25 differentially expressed factors,2 of which are up(BMP5、TIMP4) and 23 are down(OSM、CXCR4、GCG、CXCR1、CTLA-4、Siglec-9、CCR7、IL-17A、IL-17F、TRADD、IGFBP-6、THBS2、IL-4R、HGFR、IL-RA、Osteoactivine、MMP10、M-CSFR、G-CSF R、CNTF、IL-17C、TGFβ2、FGF23). Classified by function, the differentially expressed factors mainly related to immune disorder, matabolism, protein biosynthesis, oxidative stress, damage repair, apoptosis, signal transduction and so on. The differentially expressed proteins in Kidney-yin deficiency conform to the theory of Kidney-yin deficiency of TCM, such as fever in chestpalms-soles, tidal fever and night sweating, lumbar debility, dizzy, susceptibility pericardium, listlessness, slow in reacting. Secondly, after the comparion between Kidney-yang deficiency group and normal control group, we received a total of 14 differentially expressed factors, all of which are up(Csk、BMP5、Frizzled1、Frizzled4、BDNF、CXCR3、LBP、LTBP1、IL-27RA、M-CSF、L-13RαⅡ、PECAM-1、Activin RⅡA、FGF R5). Classified by function, the differentially expressed factors mainly related to immune response, metabolism, apoptosis, cell adhesion, damage repair, oxidative stress reproductive capacity and etc. The differentially expressed proteins in Kidney-yang deficiency conform to the theory of Kidney-yang deficiency of TCM, such as lumbar debility, hair off and tooth loose, tinnitus and deafness, slow in reacting, brothers chills, hyposexuality, man’s impotence and premature ejaculation, woman’s infertility due to uterus cold.Thirdly, after the comparion between Kidney-yang deficiency group and Kidney-yin deficiency group, we received a total of 1 differentially expressed factors, of which is up(BMP5). Classified by function, the differentially expressed factors mainly related to apoptosis, bone synthesis and etc. TCM thought that Kidney stores essence, which is related to the body’s growth, develepment and reproduction, and can compound the bone. The differentially expressed protein in Kidney-yang deficiency and Kidney-yin deficiency conforms to the theory of Kidney deficiency of TCM.Fourthly, TCM thought that the Kidney can store the essence, and is related to the body’s growth, development and reproduction.The Kidney plays a main role in water metabolism and air lift, as well as the bone formation.The change of kindey essence is the cornerstone of the body’s born,grow and old, which is also the base of yin and yang in the body. The differentially expressed protein in Kidney-yang deficiency and Kidney-yin deficiency are mainly related to immune function, metabolism, cell cycle, bone development, DNA repair, protein synthesis, reproductive capacity, neurotrophic, and other aspects of hematopoietic function, of which are consistent with the basic theory of TCM.Conclusion:Fistly, the differentially expressed protein in Kidney-yang deficiency and Kidney-yin deficiency are great and clearly different, which is conform to the theory of TCM. The differentially expressed protein can also provide scientific evidence to the TCM syndrom differentiation.Secondly, there are the same differentially expressed protein in Kidney-yang deficiency and Kidney-yin deficiency, which can provide scientific ideas and methods in studying the essence of the TCM. It also plays an important role in studying the mechanism of Kidney-yang deficiency and Kidney-yin deficiency.Thirdly, protein chip is an ideal method in the study of TCM-related protein. From the perspective of proteomics to study of Kidney-yang deficiency and Kidney-yin deficiency, we may reveal the essence of the kindey.更多还原