【摘要】：在大多數(shù)細胞的膜脂質中,磷脂占總量的70%以上而且種類繁多。細胞膜中的脂質成分與細胞特性密切相關。本研究采用基質輔助激光解吸／電離-傅里葉變換離子回旋共振質譜法(MALDI-FTICR MS),對不同腫瘤細胞進行原位脂類分子研究,旨在快速尋找區(qū)分不同細胞之間的差異脂類分子。本研究主要包括3部分：1、MALDI-FTICR MS原位表征人乳腺癌細胞的膜脂采用MALDI-FTICR MS,分別對人類乳腺上皮細胞(MCF-10A)和六種不同乳腺癌細胞系(即BT-20、MCF-7、SK-BR-3、MDA-MB-231、MDA-MB-157和MDA-MB-361)的膜脂質組分在沒有經過脂質提取和分離的前提下進行原位檢測。偏最小二乘判別分析(PLS-DA),相關性分析及聚類分析的結果表明膜脂水平的改變與乳腺細胞的類型密切相關。研究發(fā)現(xiàn)在乳腺上皮細胞MCF-10A中的多不飽和脂類水平比六種乳腺癌細胞的高；來源于原發(fā)腫瘤的BT-20癌細胞中的多不飽和脂類水平比其他五種來源于轉移性癌細胞的高。免疫印跡分析表明五種脂肪生成相關的酶(脂肪酸合成酶1(FASN1)、硬脂酰輔酶A去飽和酶1(SCD1)、硬脂酰輔酶A去飽和酶5(SCD5)、膽堿激酶(CKα)和鞘磷脂合酶1(SMS1))的表達與乳腺細胞類型相關。在MCF-7細胞中SCD1表達水平相對于其他乳腺細胞系顯著增加。我們的研究結果表明,在乳腺癌細胞中FASN1、SCD1、SCD5和CKα表達水平的升高可能與其飽和和單不飽和脂類水平的增加密切相關。2、MALDI-FTICR MS原位表征人肺癌細胞的膜脂為了進一步將膜脂表型與非小型細胞肺癌(NSCLC)的分類聯(lián)系起來,采用MALDI-FTICR MS對來源于三種亞型的NSCLC細胞系(A549、H1650、H1975、H157、 H1703和H460)的膜脂質組分進行原位檢測。PLS-DA分析結果表明,其中15個脂質分子(PE(36:1), PI(38:4), SM(42:2), PE(36:4), PE(36:2), PC(36:2), SM(34:1), PA(38:3),C18:0, C22:4, PA(34:2), C20:5, C20:2, C18:2和CerP(36:2))對應的投影重要性指標(variable importance in the projection, VIP)值1.0并認為是區(qū)分不同NSCLC細胞的重要變量。在肺腺癌細胞特別是H1975細胞中觀察到多不飽和脂肪酸(C20:4,C22:4,C22:5和C22:6)與多不飽和磷脂(PE(36:4)口PI(38:4))的正相關性明顯。同時,聚類分析結果表明,基于Cl8:1,C20:1,C20:2,C20:5和C22:6的表達不同可將三株肺腺癌細胞(A549、H1650和H1975)從其他NSCLC細胞中區(qū)分開來。3、采用MALDI-FTICR MS動態(tài)原位監(jiān)測DHA處理的乳腺癌細胞的膜脂質變化二十二碳六烯酸(C22:6,DHA)是一種n-3多不飽和脂肪酸并具有抗癌作用。本研究選取低劑量的DHA(0,10和40gM)用于研究DHA對乳腺上皮細胞(MCF-10A)和乳腺癌細胞(MDA-MB-231)的膜脂質分布的影響。隨著DHA處理時間的延長,DHA使癌細胞中的脂肪酸(C20:5,C22:4,C20:3和C20:1)水平、脂肪酸延長酶指標(C22:4/C20:4, C22:5/C20:5和C20:1/C1s:1)以及磷脂酰膽堿(PC(30:0), PC(32:0), PC(38:6)和PC(40:7))水平變化明顯,而DHA處理對正常上皮細胞MCF-10A的影響較小。即在乳腺癌細胞中因DHA處理而引起的脂類重構現(xiàn)象更加明顯。
[Abstract]:Phospholipids account for more than 70% of the total membrane lipids in most cells and are diverse. Lipid composition in cell membrane is closely related to cell characteristics. In this study, matrix-assisted laser desorption / ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS),) was used to study in situ lipid molecules in different tumor cells. The aim of this study is to quickly search for differentially differentiated lipid molecules between different cells. The main contents of this study are as follows: 1. The membrane lipids of human breast cancer cells characterized in situ by MALDI / FTICR MS were treated with MALDI-FTICR MS, on human breast epithelial cells (MCF-10A) and six different breast cancer cell lines (BT-20,MCF-7,), respectively. The membrane lipid components of SK-BR-3,MDA-MB-231,MDA-MB-157 and MDA-MB-361 were detected in situ without lipid extraction and separation. Partial least squares discriminant analysis (PLS-DA), correlation analysis and cluster analysis showed that the change of membrane lipid level was closely related to the type of breast cells. It was found that the levels of polyunsaturated lipids in breast epithelial cells MCF-10A were higher than those in six breast cancer cells, and the polyunsaturated lipid levels in BT-20 cells derived from primary tumors were higher than those from other five metastatic cancer cells. Western blot analysis showed that five fatty acid production related enzymes (fatty acid synthetase 1 (FASN1), stearoyl coenzyme A desaturase 1 (SCD1), stearoyl coenzyme A desaturase 5 (SCD5) were identified. The expression of cholinkinase 偽 (CK 偽) and sphingomyelin 1 (SMS1) were correlated with breast cell type. The expression level of SCD1 in MCF-7 cells was significantly higher than that in other breast cell lines. Our results suggest that the increased expression of FASN1,SCD1,SCD5 and CK 偽 in breast cancer cells may be closely related to the increase in saturated and monounsaturated lipids. MALDI-FTICR MS in situ characterization of human lung cancer cell membrane lipid in order to further link the membrane lipid phenotype with the classification of non-small cell lung cancer (NSCLC), MALDI-FTICR MS was used to identify three subtypes of NSCLC cell lines (A549, H1650, H1975, H157, H157, A549, H1650, H1975, H157, respectively). PLS-DA analysis showed that 15 lipid molecules, (PE (36:1), PI (38:4), SM (42:2), PE (36:4), PE (36:2, were detected in situ. Projection importance indicators (variable importance in the projection, for PC (36:2), SM (34:1), PA (38:3), C18 / 0, C22 / 4, PA (34:2), C20 / 5, C20 / 2, C18 / 2 and CerP (36:2) VIP) is considered to be an important variable in distinguishing different NSCLC cells. A positive correlation was observed between polyunsaturated fatty acids (C20 / 4, C22 / 4, C / 22 / 5 and C / 22 / 6) and polyunsaturated phospholipid (PE (36:4) / mouth PI (38:4) in lung adenocarcinoma cells, especially in H1975 cells. At the same time, cluster analysis showed that three lung adenocarcinoma cell lines (A549, H1650 and H1975) could be distinguished from other NSCLC cells based on the different expression of Cl8:1,C20:1,C20:2,C20:5 and C22 / 6. Dynamic in situ monitoring of membrane lipid changes in breast cancer cells treated with DHA was performed by MALDI-FTICR MS. 22 Carbohexaenoic acid is a kind of polyunsaturated fatty acid and has anticancer effect on breast cancer cells. In this study, low doses of DHA (0, 10 and 40gM) were used to study the effects of DHA on membrane lipid distribution in breast epithelial cells (MCF-10A) and breast cancer cells (MDA-MB-231). With the prolongation of DHA treatment time, DHA increased the levels of fatty acids (C20, C22, C20, and C20) and the index of fatty acid prolongation enzyme (C22) in cancer cells (C22, C20, and C20). C22:5/C20:5 and C20:1/C1s:1), as well as phosphatidylcholine (PC (30:0), PC (32:0), PC (38:6) and PC (40:7). However, DHA treatment had little effect on MCF-10A of normal epithelial cells. That is, the lipid remodeling caused by DHA treatment in breast cancer cells is more obvious.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.9;R734.2
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本文編號：2444839