喉癌及喉正常粘膜组织的差异蛋白质组学分析

【作者】 刘玲；

【导师】 赵长青； 肖健云；

【作者基本信息】 山西医科大学 ， 耳鼻咽喉科学， 2005， 硕士

【摘要】 背景与目的:喉癌是头颈部常见的恶性肿瘤,发病率高且呈不断增长趋势,如果早期诊治可获得较好预后。肿瘤在很早期即可伴随分子标志物的改变,检测其含量变化有助于肿瘤的早期发现。但目前尚未找到理想的喉癌标志物。主要原因在于多数研究未从整体上把握喉癌相关物质的改变,或停留在基因和转录水平,而蛋白质才是生命活动真正的体现者。蛋白质组学即从整体研究不同时间和空间发挥功能的特定蛋白质组成、表达水平、翻译后修饰、功能及蛋白质间相互作用的一门新学科,其技术已广泛用于肿瘤相关蛋白的联合检测,但在喉癌领域尚处于起步阶段,目前包括血清及组织蛋白质组的分析。已证实喉癌病人与正常人血清、喉癌与癌旁组织的蛋白表达谱之间确实存在差异,但尚未发现具高度特异性和灵敏度的喉癌标志物;对已识别的喉癌相关蛋白未进行全面的鉴定及验证。鉴于喉癌生物学特征主要表现在组织蛋白质表达异常,血清蛋白成分易受各种因素干扰,以及双向凝胶电脉(2-DE)是目前唯一能在一块胶上同时分离上万种组分的方法,本实验用喉癌和正常喉粘膜组织为样本分别抽提总蛋白质,应用2-DE、质谱分析(MS)和生物信息学等技术寻找鉴定差异表达的蛋白质(群),旨在建立喉癌特异性蛋白质组数据库,初步筛选喉癌肿瘤标志物。研究方法:①标本取自病理确诊的9 例喉癌组织及同一患者正常喉粘膜组织。患者43～65 岁男性,声门区癌,T1～2N0M0,术后病理证实均为高分化鳞癌。正常黏膜取自对侧正常喉室或室带黏膜, 病理证实为无肿瘤细胞侵犯的鳞状上皮组织。取新鲜离体组织提取组织总蛋白质。用2D Quant Kit试剂盒测蛋白质总浓度。确定上样量,保存于-70oC 备用。②P H 梯度双向凝胶电泳:第一向固相等电聚焦参照IPGphor 等电聚焦系统使用指南进行,上样量400 μg 总蛋白。等电聚焦结束,迅速取出IPG胶条,平衡后进行第二向垂直SDS-PAGE 电泳。用银染试剂盒进行硝酸银染色使凝胶中的蛋白质点可视化。每一标本重复实验三次。③扫描凝胶,PDquest 图象分析软件进行点检测、背景消减、校正、匹配、量化获取斑点相关信息, 寻找差异蛋白点。更多还原

【Abstract】 Background and Objective: Laryngeal cancer is common in head and neck malignant tumor. Its high incidence keeps on rising. Once early diagnosing is made, the patients will get good results. Tumors always arise and develop accompanied by changes of tumor markers (TM) in very early time. By checking them we can detect tumor in initial period. However, no ideal TM is found for laryngeal cancer by now because most researches are unilateral or on the level of gene and mRNA. But it is proteins that really embody actual life. Proteomics is a new subject that study composing, expression, modification, and interaction of entire function proteins of special kind of cell or tissue at different time and different position. Although proteomic technique has been widely used to detect joint molecules related with tumor, it’s just a beginning in laryngeal cancer field, which involves tissue and serum study. It is confirmed that there are proteomic differences between laryngeal cancer and normal tissue beside tumor, and also between diseased serum and healthy serum, but no symbol molecule of high specificity and sensitivity have been found out, even those differential proteins found out have not been completely checked and identified. As tissue proteins can best embody characters of tumor, and serum proteins are too easy to be influenced by disturbing factors, we choose laryngeal cancer and normal laryngeal epithelial tissue as objects to extract total protein, used 2-DE which is the only method to separate thousands of proteins on one glue, associate MS and bioinformatics to search and identify different proteins of these two kinds of tissue, aims at setting up a proteomic database of laryngeal cancer characteristic and roughly filter TM of laryngeal cancer. Method: ①Laryngeal cancer tissue and pair normal laryngeal tissue specimens were obtained from nine 43-65 years old, male glottis cancer patients in T1～2N0M0 period. All diseased tissue were diagnosed by histopathology in First Xiangya Hospital, Central South University as highly differentiated Squamous cell carcinoma. Normal epithelial tissue was from opposite side of larynx ventricle and false vocal cord which had been diagnosed by histopathology as healthy. We extracted total protein from fresh specimens and measured their concentration respectively with 2D Quant Kit reagent in order to make certain the amount need to administrate in electrophoresis. After that, the extractions were stored at -70oC. ②IPG-2D PAGE: Conducts were according to the manual described by manufacturer and Gorg. IEF was performed using IPG strip on IPGphor? Isoelectric Focusing System with totally 400μg proteins were administrated. After that, took out IPG glue quickly and made it equilibrium before entering Second-dimension SDS–PAGE electrophoresis. The protein spots were visualized by silver-based staining technique with the protein silver stain kit. The stained 2-DE gels were scanned on a scanner. In order to measure the reproducibility, 2-DE for the tumor and normal-tissues from the same patient was repeated three times, respectively. ③Analysis the images with PD Quest software. The differential protein spots were detected between laryngeal cancerous tissues and normal mucosa tissues. ④Selected 37 well-resolved differential spots, cut them from glue. Respectively bring them into decoloration, reversion, enzyme digestion, extraction, desalinization in turn, and then drop them on stainless steel plate. After dried up, they were analysed and identified by MALDI–TOF mass spectrometry. Then, we obtained their peptide mass finger printing (PMF). ⑤Recognized the apexes with Mascot distiller software, and search for database under certain condition. Results: ①We got clear, reproducible and well-resolved silver stained 2-DE patterns of human laryngeal cancer tissues and normal mucosa epithelial tissue protein expression. ②The image analysis in PDquest software showed that these 2-DE maps were reproducible. For laryngeal carcinoma tissues, a total of 1504±122 spots were detected, 1305±143 spots更多还原