【作者】 郑雯；

【导师】 田景奎； 张琳；

【作者基本信息】 浙江大学 ， 生物医学工程， 2016， 博士

【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。更多还原

【Abstract】 Proteomics technology is a scientific method for gloable research of disease pathogenesis, cell model, and functional connection at protein level. China has abundant resources of medicinal plants, however, the research on stress response and treated target of medicinal plants using morden molecular biology is rarely reported. In this paper, proteomics, metabolomics, molecular biology, and plant physiology were combined to reveal the defense mechanisms of response to UV radiation and regular network on physiological pathway in Taxus chinensis and Ginkgo biloba leaves at first time. Several key enzymes were identified and confirmed related to secondary metabolism. That provided a theoretical basis for quality improment and value in use of medicinal plants. Achyranthes aspera is commonly used for treatment of rheumatoid arthritis as ethnodrug. We first reported biomarkers relevant to rheumatoid arthritis pathogenesis and targets of Achyranthes aspera treatment using Proteomics and other modern medical methods. It is benefit for searching efficient and low toxicity candidate medicines. The main contents and results of this paper are as following:(1) Response and Defense Mechanisms of Taxus chinensis leaves under UV-A Radiation are Revealed Using Comparative Proteomics and Metabolomics AnalysesTaxus chinensis var. mairei is an endemic species to southeastern China and one of the natural sources for the anticancer medicine paclitaxel. To investigate the molecular response and defense mechanisms of T. chinensis leaves to enhanced ultraviolet-A (UV-A) radiation, gel-free/label-free and gel-based proteomics and GC-MS analyses were performed. The transmission electron microscopy results indicated damage on chloroplast under UV-A radiation. Proteomics analyses in leaves and chloroplast showed that photosynthesis-, glycolysis-, secondary metabolism-, stress-, and protein synthesis, degradation, and activation-related systems were mainly changed under UV-A radiation. Forty-seven PS II proteins and six PS I proteins were identified changed in leaves and chloroplast under UV-A treatment. It indicated that PS Ⅱ was more sensitive to UV-A than PS Ⅰ as UV-A light target. Enhanced glycolysis, with four glycolysis-related key enzymes increased, provided precursors for secondary metabolism. The 1-deoxy-D-xylulose-5-phosphate reductoisomerase and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase were identified significantly increased during UV-A radiation, which resulted in paclitaxel enhancement. Additionally, mRNA expression levels of genes involved in the paclitaxel biosynthetic pathway indicated a down-regulation under UV-A irradiation and up-regulation in dark incubation. These results reveal that a short-term high dose of UV-A radiation could stimulate the plant stress defense system and paclitaxel production.(2) Improved Metabolites of Pharmaceutical Ingredient Grade Ginkgo biloba and the Correlated Proteomics AnalysisGinkgo biloba is an attractive and traditional medicinal plant, and has been widely used as a phytomedicine in the prevention and treatment of cardiovascular and cerebrovascular diseases. Flavonoids and terpene lactones are the major bioactive components of Ginkgo, whereas the ginkgolic acids (GAs), with strong allergenic properties, are strictly controlled. In this study, we tested the content of flavonoids and GAs under ultraviolet-B (UV-B) treatment and performed comparative proteomic analyses to determine the differential proteins that occur upon UV-B radiation. That might play a crucial role in producing flavonoids and GAs. Our phytochemical analyses demonstrated that UV-B irradiation significantly increased the content of active flavonoids, and decreased the content of toxic GAs. We conducted comparative proteomic analysis of both whole leaf and chloroplasts proteins. In total,27 differential proteins in the whole leaf and 43 differential proteins in the chloroplast were positively identified and functionally annotated. The proteomic data suggested that enhanced UV-B radiation exposure activated antioxidants and stress-responsive proteins as well as reduced the rate of photosynthesis. We demonstrate that UV-B irradiation pharmaceutically improved the metabolic ingredients of Ginkgo, particularly in terms of reducing GAs. With high UV-absorption properties, and antioxidant activities, the flavonoids were likely highly induced as protective molecules following UV-B irradiation.(3) Identification of Candidate Synovial Membrane Biomarkers after Achyranthes aspera Treatment for Rheumatoid ArthritisRheumatoid arthritis (RA) is a systemic autoimmune disease whose main symptom is a heightened inflammatory response in synovial tissues. Despite its common occurrence, the pathogenic mechanism of RA is still relatively unknown. Historically, Achyranthes aspera L. has been used in the clinical treatment of rheumatoid arthritis in the She ethnic minority group of China. To verify the anti-arthritic activities of A. aspera and its possible therapy-related factors on the pathogenesis of RA, the main compounds in A. aspera root were isolated and identified by nuclear magnetic resonance (NMR). Using the collagen-induced arthritis (CIA) model, Wistar rats were then immunized with type II collagen and the effects of Achyranthes saponins were evaluated by rats’paw swelling, radiological, and histopathological examinations. Two-dimensional DIGE (2D-DIGE) was then performed to determine the differentially expressed proteins in either inflamed or drug-treated synovium of CIA rats. Treatment with Achyranthes saponins resulted in dramatically decreased paw swelling, proliferation of inflammatory cells, and bone degradation. Fibrinogen, procollagen, protein disulfide-isomerase A3, and apolipoprotein A-I were all increased in inflamed synovial tissues and were found to decrease when administered drug therapy. Furthermore, Alpha-1-antiproteinase and manganese superoxide dismutase were both increased in drug-treated synovial tissues. The inhibition of RA progression shows that A. aspera is a promising candidate for future treatment of human arthritis. Importantly, that the total saponins found within A. aspera are the active component. Finally, that autoantigens such as fibrinogen and collagen could act as inducers of RA due to their aggravation of inflammation. Given this, it is possible that the vimentin and PDIA3 could be the candidate biomarkers specific to Achyranthes saponins therapy for rheumatoid arthritis in synovial membrane.更多还原