海洋球石藻（Emiliania huxleyi）鞘脂类的液质分析及其活性初探

发布时间：2018-04-17 00:22

本文选题：海洋球石藻 + 病毒感染　；参考：《集美大学》2017年硕士论文

【摘要】：鞘脂类是目前脂类研究的热点之一,其代谢产物(如神经酰胺、鞘氨醇及各种鞘糖脂等)具有较强的生物活性,作为信号分子参与细胞分化、增殖、免疫、凋亡及衰老等重要生理活动,特别是作为一种新型膳食营养成分和药用成分对人体健康具有重要影响。目前对于鞘脂的研究主要集中在高等动植物,对海洋生物中鞘脂的研究还很少。海洋微藻因其繁殖快、种类多、数量大,含有多种活性物质,已成为新型天然产物的主要来源。海洋球石藻(Emiliania huxleyi)是一种海洋真核浮游植物,能够产生丰富的次级代谢生物活性物质,特别是该藻被特异性病毒感染后能够合成新的具有独特结构的鞘糖脂,该结构的鞘糖脂具有较高的生物学活性,有望开发为新型的天然食品医药资源。本研究以海洋球石藻E.huxleyi BOP92和特异性裂解病毒EhV99B1为研究材料,建立了鞘脂标准品的质谱检测方法,初步明确了鞘脂的质谱学规律;在此基础上建立了适用于海洋球石藻细胞中粗脂的提取方法和超高效液相色谱-四级杆和轨道阱杂交傅里叶变换超高分辨率质谱联用(UPLC-Q-Exactive-MS)的分析检测方法,分析了病毒感染前后,海洋球石藻细胞中鞘脂的组成及含量变化。主要研究结果如下:首先对海洋球石藻细胞总脂进行了荧光观察和薄层色谱(TLC)分析。荧光显微镜观察结果显示,病毒感染的藻细胞中中性脂质积累明显增加,细胞核遗传物质片段化;TLC结果显示藻细胞总脂中中性脂质的含量比例较高,且病毒感染在一定程度上影响宿主藻细胞脂质的组成和含量,特别是能够促进中性脂质的合成和积累;球石藻细胞总脂粗提物具有一定抗氧化和抗肿瘤活性,病毒感染后藻细胞粗脂具有更强的总还原能力,球石藻粗脂对正常肝细胞LO2和肝癌细胞HepG2的生长具有抑制或刺激作用,且与粗脂的浓度相关。将鞘脂的标准品直接进行质谱分析,在正负离子模式下探索鞘脂的质谱学规律。结果显示,鞘脂更易在正离子模式下离子化,且鞘脂分子发生类似的结构断裂,产生的离子碎片或中性丢失可以提供鞘脂的结构信息,因此解析质谱图的离子碎片可以确定鞘脂的种类及结构。鞘脂的特征离子碎片为鞘氨醇骨链脱去两分水时产生的离子碎片,例如鞘脂的长链碱基为d18:1、t18:0,在正离子模式下其特征离子碎片的质荷比为264.3、282.3。在上述基础上,进一步建立了UPLC-Q-Exactive-MS分析手段,用于检测病毒感染前后藻细胞中鞘脂的组成及含量变化情况。结果鉴定出了13种鞘脂,其中神经酰胺的含量最高,可达鞘脂总含量的99%以上,其结构多为带两个羟基的C18长链碱基和不带羟基的C20-C23脂肪酸链,还有少量的单糖鞘糖脂存在;病毒EhV99B1感染E.huxleyi BOF92后宿主藻细胞鞘脂类的组成没有发生改变,但含量均不同程度降低,表明病毒基因组编码的鞘脂类代谢酶参与并调节宿主的鞘脂类代谢途径。
[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.

【学位授予单位】：集美大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.63;TS201.2

【参考文献】