南方红豆杉和银杏叶响应紫外辐射及土牛膝治疗大鼠关节炎的蛋白质组学研究

发布时间：2018-03-23 21:31

  本文选题：蛋白质组学　切入点：代谢组学　出处：《浙江大学》2016年博士论文

【摘要】：蛋白质组学技术是从蛋白质水平对疾病机理、细胞模式、功能联系等方面进行整体探索的科学。我国药用植物资源丰富,但通过现代分子生物学手段阐释其逆境响应或疾病治疗靶点的研究仍比较匮乏。本论文首次采用蛋白质组学技术,结合代谢组学、分子生物学、植物生理学等手段,对南方红豆杉和银杏叶响应紫外辐射的应激和防御机制进行研究,揭示了紫外辐射下两种药用植物各种生理途径的调控网络,寻找到次生代谢相关途径的关键酶,为提高药用植物品质和利用价值提供了一定的理论依据。土牛膝是畲医治疗类风湿性关节炎的常用药材,本论文首次采用蛋白质组学技术,结合天然药物化学、病理学等手段,寻找到滑膜中与类风湿性关节炎病程相关的生物标志物,对了解类风湿性关节炎的发病机制及今后寻找高效低毒的候选药物具有重要意义。本论文的主要研究内容及结果如下：(1)南方红豆杉对UV-A辐射响应和防御机制研究南方红豆杉是中国东南部的特有品种,其主要天然成分紫杉醇具有抗癌活性。为研究南方红豆杉在UV-A下的分子响应和防御机制,采用gel-free/label-free和2-DE蛋白质组学以及GC-MS技术对南方红豆杉叶片中蛋白及代谢物进行分析研究。透射电镜结果显示UV-A照射后红豆杉叶绿体结构受到明显损伤。红豆杉叶片和叶绿体蛋白质组学分析结果表明在UV-A作用下其叶片光合作用、糖酵解、次生代谢、逆境相关、蛋白合成、降解和激活系统相关蛋白受到显著影响。其中47个光系统ⅡI蛋白和6个光系统Ⅰ蛋白发生显著性变化,表明红豆杉叶片中光系统Ⅱ比光系统Ⅰ对UV-A更为敏感。四个糖酵解相关蛋白丰度的显著增加,为次生代谢过程提供必要的前体物质。1-脱氧-D-木酮糖-5-磷酸还原酶(DXR)和4-羟基-3-甲基-2-(E)-丁烯基-4-磷酸还原酶(HDR)在UV-A和暗培养处理后丰度显著增加,最后导致紫杉醇显著增加。通过对紫杉醇生物合成途径中关键基因的mRNA表达水平分析发现在UV-A照射阶段相关基因表达下调,而暗培养过程中基因表达上调。综上所述短时间高剂量的UV-A辐射可以刺激红豆杉防御系统的开启和紫杉醇生物合成的增加。(2)银杏药物成份改进和相关蛋白质组学研究银杏作为传统药用植物已被广泛应用于心血管和脑血管疾病的预防和治疗。黄酮类和萜内酯类化合物是银杏的主要活性成分,而银杏酚酸具有较强的致敏性,在产品质量检测中需要严格控制。在本研究中,比较分析了UV-B辐射下银杏叶中类黄酮和银杏酚酸的含量变化,并分别对处理前后的银杏叶片总蛋白和叶绿体蛋白进行比较蛋白质组学分析。结果表明,UV-B辐射可以显著提高银杏叶片中黄酮的含量,并减少酚酸的含量。UV-B辐射前后银杏叶片总蛋白和叶绿体蛋白分别鉴定到27和43个差异蛋白。UV-B辐射能够激活抗氧化和胁迫响应相关的蛋白,并且减弱银杏叶片的光合作用。本研究证明UV-B辐射可以提高银杏叶片的药学品质。(3)土牛膝治疗关节炎大鼠模型研究及其滑膜候选生物标记筛选类风湿关节炎是一种系统自身免疫缺陷病,病症表现为滑膜组织炎症的不断加重,长期以来我国畲族人民使用土牛膝来治疗此病。为确证土牛膝的抗关节炎活性,寻找其治疗关节炎活性物质及病程相关因子,本文提取并分离土牛膝根中的主要化合物,利用核磁共振对其结构进行鉴定。并建立大鼠胶原诱导关节炎模型,应用土牛膝皂苷对其进行治疗,考察大鼠足关节病变,证实土牛膝皂苷具有显著抗关节炎活性。采用二维差异荧光凝胶电泳技术挖掘大鼠滑膜中炎症病程相关及药物治疗相关的差异蛋白,结果显示纤维蛋白原、原骨胶原、蛋白二硫化物异构酶A3和载脂蛋白A-I在炎症组滑膜组织中均表现出增加趋势而在治疗组滑膜组织中呈降低趋势；此外,α-1-抗蛋白酶和超氧化物歧化酶均在治疗组滑膜组织中显著增加。土牛膝对关节炎过程的抑制作用表明其可以作为今后治疗人类关节炎的一种候选药物,且土牛膝总皂苷为其活性成分。自身抗原(例如纤维蛋白原和胶原)可能对类风湿关节炎的恶化和炎症的加剧起到一定的诱导作用,波形蛋白和蛋白二硫化物异构酶A3可能作为土牛膝皂苷治疗关节炎滑膜的候选生物标记物。
[Abstract]:Proteomics is a cell model of disease mechanisms, from the protein level, functions of overall exploration science. Medicinal plant resources are rich in China, but by means of interpretation of modern molecular biology to study the treatment response to stress or disease target is still relatively scarce. This paper firstly adopts the proteomics technique combined with metabolism proteomics, molecular biology, plant physiology and other means of ultraviolet radiation stress and defense mechanism of Taxus and Ginkgo biloba network reveals the UV response, two kinds of medicinal plants in various physiological pathways under the radiation of regulation, to find the key enzymes related to secondary metabolism pathway, provides a theoretical basis for to improve the quality of medicinal plants and utilization value. Tuniuxi is commonly used herbs she medicine in the treatment of rheumatoid arthritis, this is the first time using proteomics technology, combined with the Natural medicine chemistry, pathology and other means, to find the object associated with the course of rheumatoid arthritis synovial biomarkers, has important significance for understanding the pathogenesis of rheumatoid arthritis in the future and to find efficient and low toxicity drug candidates. The main research contents and results of this paper are as follows: (1) the study of UV-A of Taxus chinensis the radiation response and defense mechanism of Taxus chinensis are endemic species in Southeast Chinese, the main anticancer activity of paclitaxel with natural ingredients. The molecular response and defense mechanism of Taxus chinensis under UV-A, analysis of proteins and metabolites of Taxus chinensis var.mairei leaves by gel-free/label-free and 2-DE in proteomics and GC-MS technology. The results of TEM show the chloroplast structure damage in Taxus UV-A after irradiation. Taxus leaf and chloroplast proteome analysis results Show in UV-A under the action of leaf photosynthesis, glycolysis, secondary metabolism, stress, protein synthesis, protein degradation and related activation system significantly affected. 47 of the photosystem I protein and 6 proteins of photosystem I changed significantly, that of photosystem II in the leaves of Taxus chinensis var.mairei than photosystem I on UV-A was more sensitive. Four significantly increased glycolytic protein abundance, to provide the necessary precursors of.1- deoxy -D- xylulose phosphate reductase -5- secondary metabolism process (DXR) and 4- hydroxy -3- methyl -2- (E) - -4- butenyl phosphate reductase (HDR) in UV-A and dark culture after treatment abundance increased significantly, resulting in significantly increased levels of paclitaxel. Analysis showed that the related gene expression in UV-A irradiation stage down through the key in Taxol Biosynthetic Pathway Gene Expression of mRNA and up-regulated gene expression in the process of dark culture in. The increase of UV-A short time high doses of radiation can stimulate the defense system to open and yew taxol biosynthesis. (2) ginkgo pharmaceutical ingredients and related improvement of proteomics research of ginkgo as traditional medicinal plants have been widely used in cardiovascular and cerebrovascular disease prevention and treatment. Flavonoids and terpene lactones were the main active ingredient of Ginkgo biloba, and ginkgolic acids with strong allergenicity, the need for strict control in product quality testing. In this study, a comparative analysis of flavonoids and Ginkgo biloba UV-B radiation in Ginkgo Leaf phenolic content changes of acid, comparative proteomic analysis and treatment on Ginkgo Biloba Leaves Extract Tablets total protein and the chloroplast protein. The results showed that UV-B radiation can significantly improve the content of flavonoids in Ginkgo Biloba Leaves Extract Tablets, and reduce the content of salvianolic acid.UV-B before and after the radiation of Ginkgo Biloba Leaves Extract Tablets total protein and leaf The green body protein were identified to 27 and 43 differential protein.UV-B radiation can activate the antioxidant and stress response proteins, and reduced Ginkgo Biloba Leaves Extract Tablets photosynthesis. This study proved that UV-B can improve the quality of pharmaceutical radiation Ginkgo Biloba Leaves Extract Tablets. (3) tunniuxi treatment of arthritis rat model and Study on its candidate biomarkers of synovium of rheumatoid arthritis a system of its own immunodeficiency disease manifestations of synovial tissue inflammation has been increasing, our country for a long time she people use on the knee to treat the disease. For anti arthritic activity confirmed tunniuxi, looking for the treatment of arthritis active substance and pathogenesis related factors, the extraction and separation of the main native Achyranthes compound. Its structure was identified by NMR. And the establishment of rat collagen induced arthritis model, the therapeutic application of Achyranthes root saponin, investigation Foot joint lesions in rats, confirmed that soil Achyranthes bidentata saponin has obvious anti arthritis activity. Using two-dimensional difference gel electrophoresis fluorescence mining course of rat synovium inflammation related and drug treatment related proteins showed that fibrinogen, bone collagen, protein two sulfide isomerase A3 and apolipoprotein A-I in synovium inflammation group showed an increasing trend in the synovial tissue in the treatment group decreased; in addition, alpha -1- protease resistant and superoxide dismutase were significantly increased in the synovial tissue in the treatment group. Inhibitory effect of Achyranthes root on arthritis process show that it can be used as a candidate drug for treatment of human arthritis the total saponins of Achyranthes root, and its active ingredients. Self antigens (e.g. fibrinogen and collagen) on rheumatoid arthritis and inflammation aggravate the deterioration to be induced by, Vimentin and protein two sulfide isomerase A3 may be a candidate biomarker for the treatment of arthritis synovium by Achyranthes bidentata saponins.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R29
，

本文编号：1655238