本文選題：腺淋巴瘤 + 質(zhì)譜成像技術(shù)�。� 參考：《廣西醫(yī)科大學(xué)》2014年博士論文

【摘要】：腺淋巴瘤（Warthin Tumor）也被稱為乳頭狀淋巴性囊腺瘤或單形性腺瘤，是第二常見的唾液腺良性腫瘤。其結(jié)構(gòu)主要包括兩個(gè)部分：腫瘤上皮組織及淋巴間質(zhì)。淋巴腺瘤組要影響60至70歲人群，吸煙被認(rèn)為于該疾病密切相關(guān)。雖然以往的研究對(duì)于腺淋巴瘤的起病及進(jìn)程進(jìn)行了很多探討，也設(shè)立了很多假說，但其病理機(jī)制至今仍不明確。大多數(shù)學(xué)者對(duì)腺淋巴瘤的研究多著重于基因組學(xué)及蛋白組學(xué)。而對(duì)于脂質(zhì)組學(xué)這一門新興學(xué)科，其在腺淋巴瘤中的研究甚少。尤其是對(duì)于脂質(zhì)在腺淋巴瘤中的分布及組成，尚未發(fā)現(xiàn)任何報(bào)道。 脂質(zhì)是人體細(xì)胞中不可或缺的重要組成部分，其包括脂肪、水溶性維生素、甘油脂類、磷脂類等一系列生物活性物質(zhì)。其不但參與生物膜構(gòu)建，細(xì)胞增值、分化、代謝調(diào)節(jié)及免疫，而且涉及到儲(chǔ)存能量、傳遞信號(hào)等生物活動(dòng)。磷脂酰膽堿，又稱為卵磷脂，為磷脂類的一種，其為細(xì)胞膜磷脂雙分子層的主要成分，并且更多的存在于雙分子層的外側(cè)。磷脂酰膽堿成分在細(xì)胞及細(xì)胞膜內(nèi)的改變被認(rèn)為與細(xì)胞內(nèi)生物信號(hào)的傳送及各類細(xì)胞的新陳代謝有關(guān)。其對(duì)腫瘤的形成、增長及轉(zhuǎn)歸起到重要的作用。磷脂酰膽堿的特性是由其綁定的脂肪酸種類決定，因此，比較正常和病理組織中不同脂質(zhì)的分布，尤其是不同脂肪酸的分布，能夠獲得代謝調(diào)控中關(guān)鍵的脂質(zhì)標(biāo)志物,最終揭示脂質(zhì)在腫瘤活動(dòng)中的作用機(jī)制。 質(zhì)譜成像（Imaging Mass Spetrometry, IMS）是一種最新的原位分析技術(shù)。其利用質(zhì)譜直接掃描生物樣品，分析分子在細(xì)胞或組織中的“結(jié)構(gòu)、空間與時(shí)間分布”信息。此技術(shù)以質(zhì)譜為基礎(chǔ)，不局限分析特異的一種或者幾種生物分子。質(zhì)譜成像不但能夠在短時(shí)間內(nèi)一次性檢測(cè)多種生物分子的空間分布，而且不需要煩雜的樣本準(zhǔn)備過程，更不需要任何免疫技術(shù)進(jìn)行檢測(cè)前的標(biāo)記。作為唯一能夠使綁定在脂質(zhì)上的脂肪酸可視化的技術(shù)，，質(zhì)譜成像能夠?qū)⒔壎ㄓ胁煌舅岱N類的磷脂酰膽堿定位，以直觀的方式觀察各種磷脂酰膽堿在生物樣本上的分布情況。為了更全面更深入地了解腺淋巴瘤的病理特征，為探究此疾病的發(fā)生發(fā)展及病理機(jī)制提供更多的理論依據(jù)。本實(shí)驗(yàn)利用人類腺淋巴瘤病理樣本作為研究對(duì)象，采用質(zhì)譜成像技術(shù)比較樣本的腫瘤及非腫瘤ROI內(nèi)磷脂酰膽堿的分布情況，并獲得腺淋巴瘤特異性信號(hào)分子。進(jìn)一步采用串聯(lián)質(zhì)譜法識(shí)別這些特異性分子，從而獲得腺淋巴瘤內(nèi)磷脂酰膽堿的組成情況并討論其意義。 方法：將術(shù)后獲得的8個(gè)病理樣本采用冰凍切片機(jī)進(jìn)行組織樣本的連續(xù)切片，樣本厚度為10μm。將所獲得的連續(xù)切片分別放置于ITO包埋及MAS包埋的載玻片上，用于IMS檢測(cè)及HE染色。通過HE染色來確認(rèn)腺淋巴瘤及非瘤組織的ROIs。通過對(duì)質(zhì)譜成像儀參數(shù)的設(shè)定，將質(zhì)荷比調(diào)整至m/z460-m/z1000范圍用于脂質(zhì)，特別是磷脂的檢測(cè)。獲得IMS檢測(cè)數(shù)據(jù)后，通過flexImaging軟件比較腫瘤及非腫瘤ROIs的質(zhì)譜，質(zhì)譜中每個(gè)信號(hào)峰即代表一種生物分子。通過SIMtools軟件對(duì)獲得的質(zhì)譜進(jìn)行信號(hào)峰進(jìn)行挑選，同時(shí)觀察相應(yīng)信號(hào)峰對(duì)應(yīng)的生物分子的離子成像圖，判斷各個(gè)生物分子的大概分布趨勢(shì)。隨后通過統(tǒng)計(jì)學(xué)分析比較各個(gè)信號(hào)峰在腫瘤及非腫瘤ROI的信號(hào)強(qiáng)度，以p 0.01為標(biāo)準(zhǔn)，獲得相應(yīng)區(qū)域的特異性信號(hào)即生物分子。最后，運(yùn)用串聯(lián)質(zhì)譜法，通過觀察離子碎片質(zhì)譜以及各個(gè)離子碎片信號(hào)峰之間的質(zhì)量差，即中性丟失，并根據(jù)此離子碎片質(zhì)譜檢索“人類代謝組數(shù)據(jù)庫”(http://www.hmdb.ca/spectra/ms/search)，獲得候選生物分子，從而識(shí)別上述步驟中獲得的特異性生物分子。 實(shí)驗(yàn)結(jié)果: (1) HE染色顯示腺淋巴瘤的典型結(jié)構(gòu)，包括腫瘤上皮及淋巴間質(zhì)。 (2)比較腫瘤及非腫瘤ROI的質(zhì)譜，m/z740-m/z940區(qū)域內(nèi)的信號(hào)峰有較大區(qū)別，此區(qū)域大部分信號(hào)峰在腫瘤ROI有較高強(qiáng)度。經(jīng)過挑選后，此區(qū)域的15個(gè)信號(hào)峰被納入隨后的分析。 (3)離子成像圖顯示的結(jié)果與質(zhì)譜基本一致，15個(gè)信號(hào)中大部分在腫瘤ROI有較高的信號(hào)強(qiáng)度。其中m/z796.5, m/z895.5, m/z897.5and m/z923.5主要分布在非腫瘤ROI，m/z782.5and m/z798.5在腫瘤與非腫瘤ROI的分布未見明顯區(qū)別。而剩下的九個(gè)信號(hào)則在腫瘤ROI有較高的信號(hào)強(qiáng)度。 (4)統(tǒng)計(jì)學(xué)分析比較腫瘤及非腫瘤ROI中15個(gè)信號(hào)的強(qiáng)度，發(fā)現(xiàn)其中10個(gè)信號(hào)峰的信號(hào)強(qiáng)度在腫瘤及非腫瘤ROI有明顯的差異，其差別具有統(tǒng)計(jì)學(xué)意義。其中，m/z796.5的信號(hào)強(qiáng)度在非腫瘤ROI中較高；其余九個(gè)信號(hào): 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，在腫瘤ROI有較高信號(hào)強(qiáng)度。 (5)在所有組織樣本中按上述方法檢測(cè)獲得的九個(gè)信號(hào)的強(qiáng)弱，并確認(rèn)各個(gè)信號(hào)在腫瘤ROI的部位。離子成像圖結(jié)果顯示，m/z741.5及m/z772.5重要分布于腺淋巴瘤的淋巴間質(zhì)區(qū)域，m/z822.5, m/z824.5及m/z826.5則分布在腫瘤上皮和淋巴間質(zhì)中，m/z820.5, m/z844.5, m/z846.5及m/z848.5則主要位于腫瘤上皮區(qū)域。最終，五個(gè)信號(hào)，即m/z772.5，m/z820.5，m/z822.5，m/z844.5及m/z846.5被發(fā)現(xiàn)始終于腫瘤ROI保持高強(qiáng)度，因此被確定為腺淋巴瘤特異性信號(hào)。 (6)通過MS/MS識(shí)別這個(gè)五個(gè)腺淋巴瘤的特異性信號(hào)所代表的生物分子。m/z772.5為PC (diacyl-16:0/16:0)，m/z820.5為PC (diacyl-16:0/20:4)，m/z822.5為PC (diacyl-16:0/20:3)，m/z844.5為PC (diacyl-18:2/20:4)，and m/z846.5為PC (diacyl-18:0/20:5)。 (7)對(duì)照HE染色以及m/z772.5即PC (diacyl-16:0/16:0)的離子成像圖發(fā)現(xiàn)，此磷脂酰膽堿主要分布在腺淋巴瘤的淋巴基質(zhì)區(qū)域，特別集中在淋巴濾泡的部位。 結(jié)論：IMS技術(shù)能成功的檢測(cè)磷脂酰膽堿在腺淋巴瘤的腫瘤及非腫瘤ROI的空間分布及信號(hào)強(qiáng)度。通過此技術(shù)，五種磷脂酰膽堿被發(fā)現(xiàn)在腺淋巴瘤內(nèi)特異性的增高，即m/z772.5為PC (diacyl-16:0/16:0)，m/z820.5為PC(diacyl-16:0/20:4)，m/z822.5為PC (diacyl-16:0/20:3)，m/z844.5為PC(diacyl-18:2/20:4)，and m/z846.5為PC (diacyl-18:0/20:5)。其中PC (diacyl-16:0/20:3)主要位于腫瘤上皮區(qū)域， PC (diacyl-16:0/20:4)， PC (diacyl-18:2/20:4)及(diacyl-18:0/20:5)則位于腫瘤和淋巴間質(zhì)區(qū)域，而PC (diacyl-16:0/16:0)則特異性在腺淋巴瘤淋巴間質(zhì)，特別是淋巴濾泡內(nèi)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.87

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