發(fā)布時間：2018-04-17 00:22

  本文選題：海洋球石藻 + 病毒感染�。� 參考：《集美大學(xué)》2017年碩士論文

【摘要】：鞘脂類是目前脂類研究的熱點之一,其代謝產(chǎn)物(如神經(jīng)酰胺、鞘氨醇及各種鞘糖脂等)具有較強的生物活性,作為信號分子參與細胞分化、增殖、免疫、凋亡及衰老等重要生理活動,特別是作為一種新型膳食營養(yǎng)成分和藥用成分對人體健康具有重要影響。目前對于鞘脂的研究主要集中在高等動植物,對海洋生物中鞘脂的研究還很少。海洋微藻因其繁殖快、種類多、數(shù)量大,含有多種活性物質(zhì),已成為新型天然產(chǎn)物的主要來源。海洋球石藻(Emiliania huxleyi)是一種海洋真核浮游植物,能夠產(chǎn)生豐富的次級代謝生物活性物質(zhì),特別是該藻被特異性病毒感染后能夠合成新的具有獨特結(jié)構(gòu)的鞘糖脂,該結(jié)構(gòu)的鞘糖脂具有較高的生物學(xué)活性,有望開發(fā)為新型的天然食品醫(yī)藥資源。本研究以海洋球石藻E.huxleyi BOP92和特異性裂解病毒EhV99B1為研究材料,建立了鞘脂標準品的質(zhì)譜檢測方法,初步明確了鞘脂的質(zhì)譜學(xué)規(guī)律;在此基礎(chǔ)上建立了適用于海洋球石藻細胞中粗脂的提取方法和超高效液相色譜-四級桿和軌道阱雜交傅里葉變換超高分辨率質(zhì)譜聯(lián)用(UPLC-Q-Exactive-MS)的分析檢測方法,分析了病毒感染前后,海洋球石藻細胞中鞘脂的組成及含量變化。主要研究結(jié)果如下:首先對海洋球石藻細胞總脂進行了熒光觀察和薄層色譜(TLC)分析。熒光顯微鏡觀察結(jié)果顯示,病毒感染的藻細胞中中性脂質(zhì)積累明顯增加,細胞核遺傳物質(zhì)片段化;TLC結(jié)果顯示藻細胞總脂中中性脂質(zhì)的含量比例較高,且病毒感染在一定程度上影響宿主藻細胞脂質(zhì)的組成和含量,特別是能夠促進中性脂質(zhì)的合成和積累;球石藻細胞總脂粗提物具有一定抗氧化和抗腫瘤活性,病毒感染后藻細胞粗脂具有更強的總還原能力,球石藻粗脂對正常肝細胞LO2和肝癌細胞HepG2的生長具有抑制或刺激作用,且與粗脂的濃度相關(guān)。將鞘脂的標準品直接進行質(zhì)譜分析,在正負離子模式下探索鞘脂的質(zhì)譜學(xué)規(guī)律。結(jié)果顯示,鞘脂更易在正離子模式下離子化,且鞘脂分子發(fā)生類似的結(jié)構(gòu)斷裂,產(chǎn)生的離子碎片或中性丟失可以提供鞘脂的結(jié)構(gòu)信息,因此解析質(zhì)譜圖的離子碎片可以確定鞘脂的種類及結(jié)構(gòu)。鞘脂的特征離子碎片為鞘氨醇骨鏈脫去兩分水時產(chǎn)生的離子碎片,例如鞘脂的長鏈堿基為d18:1、t18:0,在正離子模式下其特征離子碎片的質(zhì)荷比為264.3、282.3。在上述基礎(chǔ)上,進一步建立了UPLC-Q-Exactive-MS分析手段,用于檢測病毒感染前后藻細胞中鞘脂的組成及含量變化情況。結(jié)果鑒定出了13種鞘脂,其中神經(jīng)酰胺的含量最高,可達鞘脂總含量的99%以上,其結(jié)構(gòu)多為帶兩個羥基的C18長鏈堿基和不帶羥基的C20-C23脂肪酸鏈,還有少量的單糖鞘糖脂存在;病毒EhV99B1感染E.huxleyi BOF92后宿主藻細胞鞘脂類的組成沒有發(fā)生改變,但含量均不同程度降低,表明病毒基因組編碼的鞘脂類代謝酶參與并調(diào)節(jié)宿主的鞘脂類代謝途徑。
[Abstract]:Sphingolipid lipid is one of the hot research at present, its metabolites (such as ceramide, sphingosine and various glycosphingolipids etc.) have strong biological activity, as signaling molecules involved in cell differentiation, proliferation, immune, important physiological activities of apoptosis and senescence, especially as a new dietary nutritional and medicinal ingredients is important the impact on human health. The sphingolipid research mainly concentrated in the higher animals and plants, very little has been done to study sphingolipid in marine organisms. Marine microalgae because of its fast breeding, variety, quantity, contains a variety of active material, has become a major source of new natural products. Emiliania huxleyi (Emiliania huxleyi) is a marine eukaryotic phytoplankton, can produce abundant secondary metabolites of biologically active substances, especially the algae are specific to the synthesis of new virus infection with a unique structure of glycosphingolipids, The biological activity of the structure of glycosphingolipids is high, is expected to develop as a natural food and medicine. In this study, the new resources of emiliania huxleyi E.huxleyi BOP92 and specific cleavage of viral EhV99B1 as research materials, established mass spectrometry sphingolipid standard, the sphingolipid mass spectrometry extraction method based on rule; built for crude fat marine coccolithophorid cells and ultra performance liquid chromatography - four bar and hybrid Orbitrap Fourier transform ultra high resolution mass spectrometry (UPLC-Q-Exactive-MS) analysis method, analyzed before and after virus infection, changes in the composition and content of sphingolipids in marine coccolithophorid cells mainly. The results are as follows: firstly, emiliania huxleyi cell total lipids were observed using fluorescence microscopy and thin layer chromatography (TLC) analysis. Fluorescence microscopy showed that neutral algae cells the virus infection Lipid accumulation increased significantly, fragments of genetic material nuclei; TLC results show that the proportion of total lipid content of neutral lipid algal cells in high and virus infection in a certain extent influence the composition and content of lipid host cell, especially to promote the synthesis and accumulation of neutral lipids; coccolithophorid cell lipid extract has some antioxidant and antitumor activity, the total reduction ability of the virus after infection of algal cells have stronger growth of crude fat, crude fat coccolithophorids on normal liver cells and hepatoma cells LO2 HepG2 have inhibition or stimulation, and concentration and crude fat. The standard of sphingolipids directly by mass spectrometry, exploration of sphingolipids MS in the positive and negative ion mode under the law. The results show that sphingolipids more easily ionized in positive ion mode, and the molecular structure of the sphingolipid similar fracture, product ions or neutral can be lost Provide information about the structure of sphingolipids, therefore the mass spectrum of analytical ions can determine the type and structure of sphingolipids. Characteristics of ion fragments of sphingolipids for fragment ions from two water sphingosine bone chain, such as long chain base of sphingolipids is d18:1, t18:0, in the positive ion mode characterized by ion fragment M / Z 264.3282.3. on the basis of the above, we established UPLC-Q-Exactive-MS analysis method for the detection of virus infection before and after the changes of the composition and content of sphingolipids in the algal cells. The results showed that 13 kinds of sphingolipid ceramide, which was the highest, up to 99% of the total content of sphingolipids, its structure is C18 long chain base with two hydroxyl groups and C20-C23 fatty acid chain with hydroxyl groups, there is a small amount of monosaccharide glycosphingolipids; EhV99B1 virus E.huxleyi BOF92 composed of sphingolipids algal cells did not produce host class Change, but the content decreased to different degree, that sphingolipid metabolism sphingolipids metabolism pathway enzymes involved in the viral genome encoding and modulation of the host.

【學(xué)位授予單位】：集美大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O657.63;TS201.2

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