發(fā)布時間：2018-03-23 21:31

  本文選題：蛋白質(zhì)組學(xué)　切入點(diǎn)：代謝組學(xué)　出處：《浙江大學(xué)》2016年博士論文

【摘要】：蛋白質(zhì)組學(xué)技術(shù)是從蛋白質(zhì)水平對疾病機(jī)理、細(xì)胞模式、功能聯(lián)系等方面進(jìn)行整體探索的科學(xué)。我國藥用植物資源豐富,但通過現(xiàn)代分子生物學(xué)手段闡釋其逆境響應(yīng)或疾病治療靶點(diǎn)的研究仍比較匱乏。本論文首次采用蛋白質(zhì)組學(xué)技術(shù),結(jié)合代謝組學(xué)、分子生物學(xué)、植物生理學(xué)等手段,對南方紅豆杉和銀杏葉響應(yīng)紫外輻射的應(yīng)激和防御機(jī)制進(jìn)行研究,揭示了紫外輻射下兩種藥用植物各種生理途徑的調(diào)控網(wǎng)絡(luò),尋找到次生代謝相關(guān)途徑的關(guān)鍵酶,為提高藥用植物品質(zhì)和利用價值提供了一定的理論依據(jù)。土牛膝是畬醫(yī)治療類風(fēng)濕性關(guān)節(jié)炎的常用藥材,本論文首次采用蛋白質(zhì)組學(xué)技術(shù),結(jié)合天然藥物化學(xué)、病理學(xué)等手段,尋找到滑膜中與類風(fēng)濕性關(guān)節(jié)炎病程相關(guān)的生物標(biāo)志物,對了解類風(fēng)濕性關(guān)節(jié)炎的發(fā)病機(jī)制及今后尋找高效低毒的候選藥物具有重要意義。本論文的主要研究內(nèi)容及結(jié)果如下：(1)南方紅豆杉對UV-A輻射響應(yīng)和防御機(jī)制研究南方紅豆杉是中國東南部的特有品種,其主要天然成分紫杉醇具有抗癌活性。為研究南方紅豆杉在UV-A下的分子響應(yīng)和防御機(jī)制,采用gel-free/label-free和2-DE蛋白質(zhì)組學(xué)以及GC-MS技術(shù)對南方紅豆杉葉片中蛋白及代謝物進(jìn)行分析研究。透射電鏡結(jié)果顯示UV-A照射后紅豆杉葉綠體結(jié)構(gòu)受到明顯損傷。紅豆杉葉片和葉綠體蛋白質(zhì)組學(xué)分析結(jié)果表明在UV-A作用下其葉片光合作用、糖酵解、次生代謝、逆境相關(guān)、蛋白合成、降解和激活系統(tǒng)相關(guān)蛋白受到顯著影響。其中47個光系統(tǒng)ⅡI蛋白和6個光系統(tǒng)Ⅰ蛋白發(fā)生顯著性變化,表明紅豆杉葉片中光系統(tǒng)Ⅱ比光系統(tǒng)Ⅰ對UV-A更為敏感。四個糖酵解相關(guān)蛋白豐度的顯著增加,為次生代謝過程提供必要的前體物質(zhì)。1-脫氧-D-木酮糖-5-磷酸還原酶(DXR)和4-羥基-3-甲基-2-(E)-丁烯基-4-磷酸還原酶(HDR)在UV-A和暗培養(yǎng)處理后豐度顯著增加,最后導(dǎo)致紫杉醇顯著增加。通過對紫杉醇生物合成途徑中關(guān)鍵基因的mRNA表達(dá)水平分析發(fā)現(xiàn)在UV-A照射階段相關(guān)基因表達(dá)下調(diào),而暗培養(yǎng)過程中基因表達(dá)上調(diào)。綜上所述短時間高劑量的UV-A輻射可以刺激紅豆杉防御系統(tǒng)的開啟和紫杉醇生物合成的增加。(2)銀杏藥物成份改進(jìn)和相關(guān)蛋白質(zhì)組學(xué)研究銀杏作為傳統(tǒng)藥用植物已被廣泛應(yīng)用于心血管和腦血管疾病的預(yù)防和治療。黃酮類和萜內(nèi)酯類化合物是銀杏的主要活性成分,而銀杏酚酸具有較強(qiáng)的致敏性,在產(chǎn)品質(zhì)量檢測中需要嚴(yán)格控制。在本研究中,比較分析了UV-B輻射下銀杏葉中類黃酮和銀杏酚酸的含量變化,并分別對處理前后的銀杏葉片總蛋白和葉綠體蛋白進(jìn)行比較蛋白質(zhì)組學(xué)分析。結(jié)果表明,UV-B輻射可以顯著提高銀杏葉片中黃酮的含量,并減少酚酸的含量。UV-B輻射前后銀杏葉片總蛋白和葉綠體蛋白分別鑒定到27和43個差異蛋白。UV-B輻射能夠激活抗氧化和脅迫響應(yīng)相關(guān)的蛋白,并且減弱銀杏葉片的光合作用。本研究證明UV-B輻射可以提高銀杏葉片的藥學(xué)品質(zhì)。(3)土牛膝治療關(guān)節(jié)炎大鼠模型研究及其滑膜候選生物標(biāo)記篩選類風(fēng)濕關(guān)節(jié)炎是一種系統(tǒng)自身免疫缺陷病,病癥表現(xiàn)為滑膜組織炎癥的不斷加重,長期以來我國畬族人民使用土牛膝來治療此病。為確證土牛膝的抗關(guān)節(jié)炎活性,尋找其治療關(guān)節(jié)炎活性物質(zhì)及病程相關(guān)因子,本文提取并分離土牛膝根中的主要化合物,利用核磁共振對其結(jié)構(gòu)進(jìn)行鑒定。并建立大鼠膠原誘導(dǎo)關(guān)節(jié)炎模型,應(yīng)用土牛膝皂苷對其進(jìn)行治療,考察大鼠足關(guān)節(jié)病變,證實土牛膝皂苷具有顯著抗關(guān)節(jié)炎活性。采用二維差異熒光凝膠電泳技術(shù)挖掘大鼠滑膜中炎癥病程相關(guān)及藥物治療相關(guān)的差異蛋白,結(jié)果顯示纖維蛋白原、原骨膠原、蛋白二硫化物異構(gòu)酶A3和載脂蛋白A-I在炎癥組滑膜組織中均表現(xiàn)出增加趨勢而在治療組滑膜組織中呈降低趨勢；此外,α-1-抗蛋白酶和超氧化物歧化酶均在治療組滑膜組織中顯著增加。土牛膝對關(guān)節(jié)炎過程的抑制作用表明其可以作為今后治療人類關(guān)節(jié)炎的一種候選藥物,且土牛膝總皂苷為其活性成分。自身抗原(例如纖維蛋白原和膠原)可能對類風(fēng)濕關(guān)節(jié)炎的惡化和炎癥的加劇起到一定的誘導(dǎo)作用,波形蛋白和蛋白二硫化物異構(gòu)酶A3可能作為土牛膝皂苷治療關(guān)節(jié)炎滑膜的候選生物標(biāo)記物。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R29
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本文編號：1655238