乳腺癌和肺癌细胞膜脂分子的质谱研究
发布时间：2019-03-21 10:06
【摘要】：在大多数细胞的膜脂质中,磷脂占总量的70%以上而且种类繁多。细胞膜中的脂质成分与细胞特性密切相关。本研究采用基质辅助激光解吸／电离-傅里叶变换离子回旋共振质谱法(MALDI-FTICR MS),对不同肿瘤细胞进行原位脂类分子研究,旨在快速寻找区分不同细胞之间的差异脂类分子。本研究主要包括3部分：1、MALDI-FTICR MS原位表征人乳腺癌细胞的膜脂采用MALDI-FTICR MS,分别对人类乳腺上皮细胞(MCF-10A)和六种不同乳腺癌细胞系(即BT-20、MCF-7、SK-BR-3、MDA-MB-231、MDA-MB-157和MDA-MB-361)的膜脂质组分在没有经过脂质提取和分离的前提下进行原位检测。偏最小二乘判别分析(PLS-DA),相关性分析及聚类分析的结果表明膜脂水平的改变与乳腺细胞的类型密切相关。研究发现在乳腺上皮细胞MCF-10A中的多不饱和脂类水平比六种乳腺癌细胞的高；来源于原发肿瘤的BT-20癌细胞中的多不饱和脂类水平比其他五种来源于转移性癌细胞的高。免疫印迹分析表明五种脂肪生成相关的酶(脂肪酸合成酶1(FASN1)、硬脂酰辅酶A去饱和酶1(SCD1)、硬脂酰辅酶A去饱和酶5(SCD5)、胆碱激酶(CKα)和鞘磷脂合酶1(SMS1))的表达与乳腺细胞类型相关。在MCF-7细胞中SCD1表达水平相对于其他乳腺细胞系显著增加。我们的研究结果表明,在乳腺癌细胞中FASN1、SCD1、SCD5和CKα表达水平的升高可能与其饱和和单不饱和脂类水平的增加密切相关。2、MALDI-FTICR MS原位表征人肺癌细胞的膜脂为了进一步将膜脂表型与非小型细胞肺癌(NSCLC)的分类联系起来,采用MALDI-FTICR MS对来源于三种亚型的NSCLC细胞系(A549、H1650、H1975、H157、 H1703和H460)的膜脂质组分进行原位检测。PLS-DA分析结果表明,其中15个脂质分子(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))对应的投影重要性指标(variable importance in the projection, VIP)值1.0并认为是区分不同NSCLC细胞的重要变量。在肺腺癌细胞特别是H1975细胞中观察到多不饱和脂肪酸(C20:4,C22:4,C22:5和C22:6)与多不饱和磷脂(PE(36:4)口PI(38:4))的正相关性明显。同时,聚类分析结果表明,基于Cl8:1,C20:1,C20:2,C20:5和C22:6的表达不同可将三株肺腺癌细胞(A549、H1650和H1975)从其他NSCLC细胞中区分开来。3、采用MALDI-FTICR MS动态原位监测DHA处理的乳腺癌细胞的膜脂质变化二十二碳六烯酸(C22:6,DHA)是一种n-3多不饱和脂肪酸并具有抗癌作用。本研究选取低剂量的DHA(0,10和40gM)用于研究DHA对乳腺上皮细胞(MCF-10A)和乳腺癌细胞(MDA-MB-231)的膜脂质分布的影响。随着DHA处理时间的延长,DHA使癌细胞中的脂肪酸(C20:5,C22:4,C20:3和C20:1)水平、脂肪酸延长酶指标(C22:4/C20:4, C22:5/C20:5和C20:1/C1s:1)以及磷脂酰胆碱(PC(30:0), PC(32:0), PC(38:6)和PC(40:7))水平变化明显,而DHA处理对正常上皮细胞MCF-10A的影响较小。即在乳腺癌细胞中因DHA处理而引起的脂类重构现象更加明显。
[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 伪 (CK 伪) 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 伪 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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9;R734.2
，
本文编号：2444839
[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 伪 (CK 伪) 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 伪 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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9;R734.2
，
本文编号：2444839
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