腺淋巴瘤内磷脂酰胆碱的分布与其代谢及病理机制的研究

发布时间：2018-06-05 14:39

本文选题：腺淋巴瘤 + 质谱成像技术　；参考：《广西医科大学》2014年博士论文

【摘要】：腺淋巴瘤（Warthin Tumor）也被称为乳头状淋巴性囊腺瘤或单形性腺瘤，是第二常见的唾液腺良性肿瘤。其结构主要包括两个部分：肿瘤上皮组织及淋巴间质。淋巴腺瘤组要影响60至70岁人群，吸烟被认为于该疾病密切相关。虽然以往的研究对于腺淋巴瘤的起病及进程进行了很多探讨，也设立了很多假说，但其病理机制至今仍不明确。大多数学者对腺淋巴瘤的研究多着重于基因组学及蛋白组学。而对于脂质组学这一门新兴学科，其在腺淋巴瘤中的研究甚少。尤其是对于脂质在腺淋巴瘤中的分布及组成，尚未发现任何报道。脂质是人体细胞中不可或缺的重要组成部分，其包括脂肪、水溶性维生素、甘油脂类、磷脂类等一系列生物活性物质。其不但参与生物膜构建，细胞增值、分化、代谢调节及免疫，而且涉及到储存能量、传递信号等生物活动。磷脂酰胆碱，又称为卵磷脂，为磷脂类的一种，其为细胞膜磷脂双分子层的主要成分，并且更多的存在于双分子层的外侧。磷脂酰胆碱成分在细胞及细胞膜内的改变被认为与细胞内生物信号的传送及各类细胞的新陈代谢有关。其对肿瘤的形成、增长及转归起到重要的作用。磷脂酰胆碱的特性是由其绑定的脂肪酸种类决定，因此，比较正常和病理组织中不同脂质的分布，尤其是不同脂肪酸的分布，能够获得代谢调控中关键的脂质标志物,最终揭示脂质在肿瘤活动中的作用机制。质谱成像（Imaging Mass Spetrometry, IMS）是一种最新的原位分析技术。其利用质谱直接扫描生物样品，分析分子在细胞或组织中的“结构、空间与时间分布”信息。此技术以质谱为基础，不局限分析特异的一种或者几种生物分子。质谱成像不但能够在短时间内一次性检测多种生物分子的空间分布，而且不需要烦杂的样本准备过程，更不需要任何免疫技术进行检测前的标记。作为唯一能够使绑定在脂质上的脂肪酸可视化的技术，，质谱成像能够将绑定有不同脂肪酸种类的磷脂酰胆碱定位，以直观的方式观察各种磷脂酰胆碱在生物样本上的分布情况。为了更全面更深入地了解腺淋巴瘤的病理特征，为探究此疾病的发生发展及病理机制提供更多的理论依据。本实验利用人类腺淋巴瘤病理样本作为研究对象，采用质谱成像技术比较样本的肿瘤及非肿瘤ROI内磷脂酰胆碱的分布情况，并获得腺淋巴瘤特异性信号分子。进一步采用串联质谱法识别这些特异性分子，从而获得腺淋巴瘤内磷脂酰胆碱的组成情况并讨论其意义。方法：将术后获得的8个病理样本采用冰冻切片机进行组织样本的连续切片，样本厚度为10μm。将所获得的连续切片分别放置于ITO包埋及MAS包埋的载玻片上，用于IMS检测及HE染色。通过HE染色来确认腺淋巴瘤及非瘤组织的ROIs。通过对质谱成像仪参数的设定，将质荷比调整至m/z460-m/z1000范围用于脂质，特别是磷脂的检测。获得IMS检测数据后，通过flexImaging软件比较肿瘤及非肿瘤ROIs的质谱，质谱中每个信号峰即代表一种生物分子。通过SIMtools软件对获得的质谱进行信号峰进行挑选，同时观察相应信号峰对应的生物分子的离子成像图，判断各个生物分子的大概分布趋势。随后通过统计学分析比较各个信号峰在肿瘤及非肿瘤ROI的信号强度，以p 0.01为标准，获得相应区域的特异性信号即生物分子。最后，运用串联质谱法，通过观察离子碎片质谱以及各个离子碎片信号峰之间的质量差，即中性丢失，并根据此离子碎片质谱检索“人类代谢组数据库”(http://www.hmdb.ca/spectra/ms/search)，获得候选生物分子，从而识别上述步骤中获得的特异性生物分子。实验结果: (1) HE染色显示腺淋巴瘤的典型结构，包括肿瘤上皮及淋巴间质。 (2)比较肿瘤及非肿瘤ROI的质谱，m/z740-m/z940区域内的信号峰有较大区别，此区域大部分信号峰在肿瘤ROI有较高强度。经过挑选后，此区域的15个信号峰被纳入随后的分析。 (3)离子成像图显示的结果与质谱基本一致，15个信号中大部分在肿瘤ROI有较高的信号强度。其中m/z796.5, m/z895.5, m/z897.5and m/z923.5主要分布在非肿瘤ROI，m/z782.5and m/z798.5在肿瘤与非肿瘤ROI的分布未见明显区别。而剩下的九个信号则在肿瘤ROI有较高的信号强度。 (4)统计学分析比较肿瘤及非肿瘤ROI中15个信号的强度，发现其中10个信号峰的信号强度在肿瘤及非肿瘤ROI有明显的差异，其差别具有统计学意义。其中，m/z796.5的信号强度在非肿瘤ROI中较高；其余九个信号: m/z741.5, m/z772.5, m/z820.5, m/z822.5, m/z824.5, m/z826.5,m/z844.5, m/z846.5and m/z848.5，在肿瘤ROI有较高信号强度。 (5)在所有组织样本中按上述方法检测获得的九个信号的强弱，并确认各个信号在肿瘤ROI的部位。离子成像图结果显示，m/z741.5及m/z772.5重要分布于腺淋巴瘤的淋巴间质区域，m/z822.5, m/z824.5及m/z826.5则分布在肿瘤上皮和淋巴间质中，m/z820.5, m/z844.5, m/z846.5及m/z848.5则主要位于肿瘤上皮区域。最终，五个信号，即m/z772.5，m/z820.5，m/z822.5，m/z844.5及m/z846.5被发现始终于肿瘤ROI保持高强度，因此被确定为腺淋巴瘤特异性信号。 (6)通过MS/MS识别这个五个腺淋巴瘤的特异性信号所代表的生物分子。m/z772.5为PC (diacyl-16:0/16:0)，m/z820.5为PC (diacyl-16:0/20:4)，m/z822.5为PC (diacyl-16:0/20:3)，m/z844.5为PC (diacyl-18:2/20:4)，and m/z846.5为PC (diacyl-18:0/20:5)。 (7)对照HE染色以及m/z772.5即PC (diacyl-16:0/16:0)的离子成像图发现，此磷脂酰胆碱主要分布在腺淋巴瘤的淋巴基质区域，特别集中在淋巴滤泡的部位。结论：IMS技术能成功的检测磷脂酰胆碱在腺淋巴瘤的肿瘤及非肿瘤ROI的空间分布及信号强度。通过此技术，五种磷脂酰胆碱被发现在腺淋巴瘤内特异性的增高，即m/z772.5为PC (diacyl-16:0/16:0)，m/z820.5为PC(diacyl-16:0/20:4)，m/z822.5为PC (diacyl-16:0/20:3)，m/z844.5为PC(diacyl-18:2/20:4)，and m/z846.5为PC (diacyl-18:0/20:5)。其中PC (diacyl-16:0/20:3)主要位于肿瘤上皮区域， PC (diacyl-16:0/20:4)， PC (diacyl-18:2/20:4)及(diacyl-18:0/20:5)则位于肿瘤和淋巴间质区域，而PC (diacyl-16:0/16:0)则特异性在腺淋巴瘤淋巴间质，特别是淋巴滤泡内增加。
[Abstract]:Warthin Tumor, also known as papillary lymphadenoma or monomal adenoma, is the second common benign tumor of the salivary glands. Its structure consists mainly of two parts: tumor epithelial tissue and lymphoplasm. Lymphadenoma group should affect 60 to 70 years of age, smoking is considered to be closely related to the disease. Although previous studies Many hypotheses have been established for the onset and process of adenoma lymphoma, but its pathological mechanism is still unclear. Most scholars have focused on genomics and proteomics in the study of adenosine, and for the new subject of liposome, it is rarely studied in adenomas. The distribution and composition of substance in adenoma have not been reported.
Lipid is an indispensable part of human cells, which includes a series of bioactive substances, such as fat, water-soluble vitamins, glycerol lipids, phospholipids and so on. It not only participates in biological membrane construction, cell proliferation, differentiation, metabolic regulation and immunity, but also involves the storage of energy, transmission of signals and other biological activities. Phosphatidylcholine, also known as phosphatidylcholine, is also called Phosphatidylcholine, one of the lecithin, is the main component of the phospholipid class, the main component of the phospholipid biolecular layer, and is more present on the lateral of the biolecular layer. The changes in the phosphatidylcholine component in the cell and cell membrane are considered to be related to the transmission of intracellular biological signals and the metabolism of various cells. The characteristics of phosphatidylcholine are determined by the type of fatty acids bound by them. Therefore, the distribution of different lipids in normal and pathological tissues, especially the distribution of different fatty acids, can be used to obtain the key lipid markers in the metabolic regulation and ultimately reveal the mechanism of the action of lipid in the tumor activity.
Imaging Mass Spetrometry (IMS) is a latest in situ analysis technique. It uses mass spectrometry to scan biological samples directly to analyze the "structure, space and time distribution" of molecules in cells or tissues. This technique is based on mass spectrometry, and is not limited to analysis of a specific or several biological molecules. Mass spectrometry imaging not only It can be used to detect the spatial distribution of a variety of biological molecules in a short time, without the need for a complex sample preparation process, and no immune technology is needed before detection. As the only technology that can visualize fatty acids binding on lipids, mass spectrometry can be used to bind phospholipids with different kinds of fatty acids. The distribution of all kinds of phosphatidylcholine in biological samples was observed in an intuitive way. In order to understand the pathological features of adenomas more thoroughly and more thoroughly, more theoretical basis was provided to explore the development and pathological mechanism of the disease. The distribution of phosphatidylcholine in the tumor and non tumor ROI samples was compared by mass spectrometry, and the specific signal molecules of adenomas were obtained. The specific molecules were identified by tandem mass spectrometry, and the composition of phosphatidylcholine in the adenocarcinoma was obtained and the significance of it was discussed.
Methods: the 8 pathological samples obtained after the operation were sectioned by a frozen section machine. The thickness of the sample was 10 m., and the continuous slices were placed on the ITO coated and MAS coated slides, respectively, for IMS detection and HE staining. By HE staining, the ROIs. of the adenomas and non tumor tissues was confirmed by the mass spectrum of mass spectrometry. The parameters of the imager are set, the mass ratio is adjusted to the m/z460-m/z1000 range for lipid, especially the detection of phospholipids. After obtaining the IMS detection data, the mass spectra of the tumor and non tumor ROIs are compared by flexImaging software. Each signal peak in the mass spectrum is represented by a kind of biomolecule. Through the SIMtools software, the signal of the obtained mass spectrometry is detected. At the same time, the ion imaging of the corresponding biomolecules corresponding to the corresponding signal peaks was observed, and the approximate distribution trend of each biomolecule was judged. Then the signal intensity of each signal peak in the tumor and non tumor ROI was compared by statistical analysis, and the specific signal of the corresponding region was obtained by P 0.01 as the standard. The mass spectrometry, by observing the mass difference between the ion fragmentation mass spectra and the peaks of each ion fragment signal, is a neutral loss, and the candidate biomolecules are obtained by retrieving the "human metabolic group database" (http://www.hmdb.ca/spectra/ms/search) based on the ion fragmentation mass spectrometry to identify the specific biomolecules obtained in the above steps.
Experimental results:
(1) HE staining revealed typical structures of adenocarcinoma, including epithelial and lymphoid stroma.
(2) comparing the mass spectrum of the tumor and non tumor ROI, the signal peaks in the m/z740-m/z940 region have a large difference. Most of the signal peaks in this region have high intensity in the tumor ROI. After selection, the 15 signal peaks in this region are included in the subsequent analysis.
(3) the results of the ion imaging show the same as mass spectrometry. Most of the 15 signals have high signal intensity in the tumor ROI. Among them, m/z796.5, m/z895.5, m/z897.5and m/z923.5 are mainly distributed in non tumor ROI, and m/z782.5and m/z798.5 is not significantly different in the distribution of tumor and non tumor ROI. The remaining nine signals are in the tumor RO. I has high signal intensity.
(4) statistical analysis was made to compare the intensity of 15 signals in tumor and non tumor ROI. It was found that the signal intensity of 10 signal peaks was significantly different in tumor and non tumor ROI, and the difference was statistically significant. Among them, the signal intensity of m/z796.5 was higher in non tumor ROI; the remaining nine signals were m/z741.5, m/z772.5, m/z820.5, m/z822.5. M/z824.5, m/z826.5, m/z844.5, m/z846.5and m/z848.5 showed higher signal intensity in tumor ROI.
(5) the strength of the nine signals obtained by these methods was detected in all the tissue samples, and the various signals were identified at the site of the tumor ROI. The results of the ion imaging showed that m/z741.5 and m/z772.5 were distributed in the lymphoid regions of adenomas, and m/z822.5, m/z824.5 and m/ z826.5 were distributed in the epithelial and lymphoplasm of the tumor, m/z820.5, M/z844.5, m/z846.5 and m/z848.5 are mainly located in the epithelial area of the tumor. Finally, five signals, namely, m/z772.5, m/z820.5, m/z822.5, m/z844.5 and m/z846.5, have been found to be at high strength and are identified as a specific signal of Adeno lymphoma.
(6) the identification of the specific signals of the five adenomas by MS/MS is PC (diacyl-16:0/16:0), m/z820.5 is PC (diacyl-16:0/20:4), m/z822.5 is PC (diacyl-16:0/20:3), and m/z844.5 is PC.
(7) it was found that the phosphatidylcholine was mainly distributed in the lymphatic matrix area of adenomas, especially in the location of the lymphoid follicles, in contrast to the HE staining and the ion imaging of m/z772.5 PC (diacyl-16:0/16:0).
Conclusion: IMS technique can successfully detect the spatial distribution and signal intensity of phosphatidylcholine in adenomas of lymphoma and non tumor ROI. Through this technique, five kinds of phosphatidylcholine have been found to be more specific in adenomas, m/z772.5 is PC (diacyl-16:0/16:0), m/ z820.5 is PC (diacyl-16:0/20:4), m/z822.5 is PC (diacyl-16:0/). 20:3), m/z844.5 is PC (diacyl-18:2/20:4) and and m/z846.5 is PC (diacyl-18:0/20:5). PC (diacyl-16:0/20:3) is mainly located in the epithelial area of the tumor, PC (diacyl-16:0/20:4), and is located in the tumor and the mesosotic region of the lymphoma. The increase in lymphatic follicles, especially in the lymphatic follicles.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.87

【相似文献】