腮腺多形性腺瘤及恶性肿瘤的唾液蛋白组学初探

发布时间：2018-09-19 16:04

【摘要】：目前临床上已经广泛应用化验尿液等体液中的成分来进行疾病诊断。唾液是人体的一种外分泌液,主要由三对大唾液腺(腮腺、颌下腺、舌下腺)所分泌。唾液中的有机、无机成分非常丰富,中含有一千多种蛋白质和大量的无机盐。唾液能够通过许多的无创性手段简便快速获取,然而,应用唾液进行常规的疾病诊断目前还没有实现。有研究证实,当机体存在系统性疾病或者口腔局部病变时,许多唾液蛋白的表达都将产生变化,例如:糖尿病、舍格伦综合征、牙周炎以及口腔鳞状细胞癌。那么,腮腺作为分泌唾液的三大唾液腺之一,当其发生肿瘤时,唾液蛋白成分理论上会产生变化,这些改变对于腮腺肿瘤的诊断是否有参考价值呢?为了探究腮腺多形性腺瘤(pleomorphic adenoma,PMA)和腮腺恶性肿瘤(parotid malignant tumors,PMT)存在时腮腺所分泌的唾液蛋白的表达异常,本实验使用液相色谱电喷雾电离串联质谱(Liquid chromatography electrospray ionization tandem mass spectrometry,LC-ESI-MS/MS)技术来对腮腺肿瘤患者的唾液蛋白进行蛋白组学对比分析,并使用ELISA技术进行差异蛋白验证。研究目的:1.通过测定不同腮腺肿瘤患者及正常人群唾液蛋白组,找到能够反应腮腺肿瘤存在的蛋白标志物。2.证实质谱检测技术所鉴定的重要蛋白在唾液中真实存在。3.通过比较不同腮腺肿瘤患者唾液蛋白组分,找到具有代表性的标志物,辅助术前明确肿瘤良恶性或肿瘤性质。实验方法:分别采集腮腺多形性腺瘤、腮腺恶性肿瘤及正常人群的腮腺唾液标本各5例,使用质谱分析方法筛选差异表达蛋白。肿瘤组采集患侧唾液样本,正常对照组随机采集一侧,提取、纯化唾液蛋白,超滤浓缩、裂解、脱盐后,进行质谱分析检测,分析数据找出差异蛋白后,加大样本量,使用ELISA试剂盒选择性鉴定多形性腺瘤组显著性增高的galectin-7。实验结果:总共有21个蛋白在三组样本间的表达量存在1.5倍或更大改变。其中galectin-7在PMA组中的表达量显著性高于PMT组及对照组;SPARCL1(Hevin)在PMT组中的表达量显著性低于PMA组及对照组;Azurocidin及ARPC4-TTLL3在PMA组和PMT组中的表达量显著性高于对照组;Cystatin C和Lipocalin 1在PMA组和PMT组中的表达量则显著性低于对照组。进一步对18个PMA样本、6个正常样品及5个恶性肿瘤样本中的galectin-7进行ELISA检测,多个独立样本非参数检验及秩和检验分析结果显示:唾液中galectin-7含量在多形性腺瘤组中较对照组(P=0.002)及恶性肿瘤组(P=0.008)均显著性增高,而对照组与恶性肿瘤组之间无显著性差异(P=0.193)。实验结论:1.质谱检测能够特异性地筛选出腮腺肿瘤患者唾液中有显著性改变的蛋白。2.通过质谱检测技术及ELISA检测均证明,唾液galectin-7在多形性腺瘤组中表达明显高于对照组及恶性肿瘤组,而对照组与恶性肿瘤组之间相比较无显著性差异。3.galectin-7有望在多形性腺瘤的术前无创性诊断中提供重要参考。
[Abstract]:Saliva is an exocrine fluid of the human body, secreted mainly by three large salivary glands (parotid glands, submandibular glands, sublingual glands). Saliva is rich in organic and inorganic components, containing more than a thousand proteins and a large number of inorganic salts. Conventional diagnosis of diseases using saliva has not been achieved yet. Studies have shown that when systemic diseases or local oral lesions occur, the expression of many salivary proteins changes, such as diabetes, Schoegren's syndrome, periodontitis and oral squama. So, parotid gland as one of the three salivary glands secreting saliva, when the tumor occurs, the salivary protein composition will theoretically change, these changes for the diagnosis of parotid gland tumors have reference value? In order to explore parotid pleomorphic adenoma (PMA) and parotid malignant tumor (parotid malignancy) Ors (PMT) exists abnormal expression of salivary proteins secreted by parotid glands. In this study, the Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) technique was used to analyze the salivary proteins in parotid tumor patients by proteomic analysis and the use of ELISA technology. Objectives: 1. To find protein markers that can reflect the presence of parotid gland tumors by determining the salivary protein groups of different parotid gland tumor patients and normal people. 2. To confirm the existence of important proteins in saliva identified by mass spectrometry. 3. To find out the salivary protein components of different parotid gland tumor patients by comparing the salivary protein components. Methods: Samples of parotid gland saliva from 5 patients with pleomorphic adenoma, 5 patients with malignant tumor of parotid gland and 5 patients with normal salivary gland were collected, and the differentially expressed proteins were screened by mass spectrometry. On one side, salivary protein was extracted, purified, concentrated, lysed and desalted, then analyzed by mass spectrometry. After differentially expressed proteins were identified, the sample size was increased and the Galectin-7 significantly increased in pleomorphic adenoma group was identified by ELISA kit. The expression of Galectin-7 in PMA group was significantly higher than that in PMT group and control group; the expression of SPARCL1 (Hevin) in PMT group was significantly lower than that in PMA group and control group; the expression of Azurocidin and ARPC4-TTLL3 in PMA group and PMT group was significantly higher than that in control group; the expression of Cystatin C and Lipocalin-1 in PMA group and PMT group was significantly higher than that in PMT group. Galectin-7 was detected by ELISA in 18 PMA samples, 6 normal samples and 5 malignant tumor samples. The results of non-parametric and rank sum tests showed that Galectin-7 content in saliva was higher in pleomorphic adenoma group than in control group (P = 0.002) and malignant tumor group (P = 0.008). There was no significant difference between the control group and the malignant tumor group (P = 0.193). Conclusion: 1. Mass spectrometry can specifically screen out salivary proteins with significant changes in parotid tumor patients. 2. Mass spectrometry and ELISA detection showed that the expression of Galectin-7 in saliva was significantly higher in pleomorphic adenoma group. There was no significant difference between the control group and the malignant tumor group. 3. Galectin-7 is expected to provide an important reference for the preoperative non-invasive diagnosis of pleomorphic adenoma.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8

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