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標題: | 開發細胞脂質體學之濃度校正方法與使用鞘脂體學方法研究缺血引發之神經細胞損傷 Development of the Concentration Normalization Method for Cellular Lipidomic Studies and Sphingolipidomic Investigation of Ischemia Induced Neuron Injury |
作者: | Hsi-Chun Chao 趙錫均 |
指導教授: | 郭錦樺 |
關鍵字: | 鞘質體學,缺血性中風,神經損傷,濃度校正方法, Sphingolipidomics,Stroke,Ishchemia,Neuron Injury,Concentration Normalization Strategy, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 鞘脂是生物中脂類的一種,研究指出其和許多病理生理功能相關,例如細胞凋亡。中風是一個與腦血管相關的嚴重疾病,而在文獻報導中,鞘脂有可能與缺血所引發的神經受損相關,然而缺乏全面性的鞘脂分析於缺血性神經受損的關聯,使研究者難以分辨不同鞘脂,對於缺血性中風的影響,因此,一個能夠完整且無偏差的鞘脂分析平台,對於鞘脂之代謝途徑以及缺血引起神經元受損研究,是非常重要的,藉此才能有效分析討論鞘脂對於缺血性神經受損所牽涉之生理功能。
在本論文第一章,我們建立了一個鞘脂體的分析平台,並且開發了一個針對細胞脂質體學的校正方法。我們利用液相層析搭配電噴灑離子源串聯質譜儀(LC-ESI-MS)能夠偵測87種不同的鞘脂,並且改良了萃取方法,達到快速且和具良好回收率的萃取結果。我們也開發了一個新的細胞脂質體學的濃度校正方法,使用流動注射搭配電噴灑離子源串聯質譜儀(FIA-ESI-MS)搭配先驅離子掃描(Precursor ion scan, PIS)來偵測荷質比(m/z)為184的離子(PIS of 184),藉此來估計樣品相對濃度,並以此為基準進行樣品稀釋,使不同細胞樣品能夠在相似濃度下進行,而減少研究結果的誤差。此利用PIS of 184的方法經準確度與同日以及異日間精密度測試,其準確度在不同稀釋細胞倍數為93.0% 至105.6%,同日間精密度誤差皆小於5%,而異日間精密度誤差也小於10%,並且我們利用HCT-116的細胞株與其他常用細胞濃度校正方法進行比較,本方法和細胞計量以及DNA濃度的方法之校正結果皆具有高度關聯性。 在本論文第二章,我們利用建立的鞘脂體學分析平台以及新的細胞脂質體學的校正方法,針對經過大腦中動脈阻塞之小鼠模型以及氧糖剝奪之神經細胞模型來進行缺血及復氧的鞘脂體學分析。在使用建立的鞘脂體分析平台,共偵測得47個不同鞘脂於小鼠腦樣品以及28個不同鞘脂於神經細胞樣品。針對不同鞘脂分析,結果顯示小鼠模型和神經細胞模型具有類似趨勢,因此我們再針對神經細胞進行鞘脂分析比對而得,在Sphinganine、dihydroceramides、短碳鍊的ceramides (碳側鍊小於20個碳) 和sphingosine在氧糖剝奪後有顯著的增加,而 sphingomyelins和長碳鍊ceramides則是顯著的減少,同時我們發現不同的鞘脂會隨著氧糖剝奪與復氧,在代謝上會隨著不同的時間而有變化,我們基於此結果建構出了鞘脂類在缺血下可能的代謝途徑,並且同時針對此現象討論可能造成神經損傷的原因。 本論文主要貢獻在於建立一個鞘脂體學分析平台以及開發一個利用PIS of 184針對細胞脂質體學的校正方法,並且成功應用於探討鞘脂在缺血所引起之神經損傷。 Sphingolipids (SPLs) are a type of lipid subgroup, and some SPLs have been reported to be associated to many different pathological pathways such as cell apoptosis. Stroke is a serious cerebrovascular-associated disease. Previous studies showed that SPLs play important roles in ischemic stroke induced neuron injury. However, the lack of comprehensive analysis between sphingolipids metabolism and ischemia induced neuron injury has hindered the identification of specific pathophysiological roles of different sphingolipids. Therefore, it is important to have an efficient and comprehensive analytical platform to elucidate the relationship between ischemia induced neuron injury and SPLs concentration changes. We established a sphingolipidomic analytical platform and a concentration normalization method for cellular lipidomic studies in the first chapter of this thesis. Using the optimized LC-ESI-MS conditions, the established sphingolipidomic analytical platform is able to identify 87 sphingolipids. The sphingolipid extraction method provided comparable recoveries when compared with previous methods, but required shorter extraction time. We further developed a new normalization method to improve data integrity for cellular lipidomic studies. The flow-injection-analysis coupled with electro-spray ionization read by a triple quadruple mass spectrometer (FIA-ESI-QQQ) method using precursor ion scan (PIS) of ion 184 was used to estimate relative concentration. Samples were further diluted to analyze all samples at a similar concentration level. The “PIS of 184” method was validated with accuracy, intraday and intermediate precision. We used HCT-116 cell models for normalization method comparison and the results showed good correlations with cell counts (R=0.97) and DNA concentration (R=0.95) methods. In the second chapter, we further investigate ischemia induced neuron injury through the application of the established sphingolipid analytical platform to a C57BL/6J mice model receiving middle cerebral artery occlusion (MCAO) and primary cultured rat neurons with oxygen and glucose deprivation (OGD) for ischemia-reperfusion (I/R) study. A total of 47 sphingolipids were detected in the brain and 28 sphingolipids were detected in neuron cells. The mice brain results showed similar trends to the neurons in terms of SPL concentration changes, and we therefore used primary neurons with OGD-treatment to study SPL concentration changes in association with ischemia induced neuron injury. Sphinganine, dihydroceramides, short-chain ceramides (acyl chain < 20C), and sphingosine were significantly increased, and sphingomyelins and long-chain ceramides were decreased after OGD treatment. Temporal changes of SPLs after OGD treatment were used to investigate the roles of SPLs in ischemia induced neuron injury. The observed changes in the metabolic pathway of SPLs after ischemia induced neuron injury were also reported in this section of the thesis. In conclusion, we established a sphingolipidomics analytical platform with a new normalization strategy using PIS of 184 and successfully applied the method to the investigation of ischemia induced neuron injury. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51794 |
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