【作者】 甘嘉亮；

【导师】 高枫；

【作者基本信息】 广西医科大学 ， 外科学， 2012， 博士

【摘要】 CRC是世界上最常见的三大恶性肿瘤之一,我国CRC的死亡率在常见肿瘤中排在第四位,发病率在逐渐上升。目前临床上早期诊断率不高,就诊的患者大多数已经属于进展期。CEA是临床上应用最广泛的血清肿瘤学标志物,但其敏感性和特异性不高。与CEA~+CRC相比,CEA~-CRC更缺乏血清学诊断和监测指标。因此,很迫切需要寻找一些更有效的CRC分子标志物。稳定同位素标记联合液相串联质谱技术(iTRAQ-LC-MS/MS)的建立和发展,为寻找有效肿瘤分子标志物提供了可靠的技术手段。本研究以CRC为研究对象,分以下几个部分进行,试图筛选出有价值的CRC候选标志物。第一部分血清CEA阴性和阳性结直肠癌的临床和流行病特征比较——附1969例分析本部分的研究目的是通过回顾性调查与CEA相关1969例CRC患者的临床和流行病学特征,了解CEA相关的这两类CRC有何不同以及CEA的血清阳性率情况,为后续基础研究做铺垫。结果显示, CEA的血清阳性率为38.04%,CEA阴性结直肠癌平均发病年龄比CEA阳性结直肠癌年轻2岁多,中位发病年龄较后者提前3岁,发病人群以60岁以下的青壮年为主,其中A型血患者比例较高,而B型血患者比例较低,并发2型糖尿病和结肠肠梗阻比例较低。本研究说明,CEA血清阳性率不高,缺乏敏感性,CEA阴性结直肠癌和CEA阳性结直肠癌在临床和流行病学上确实有一定的差异,为后续深入研究奠定了基础。第二部分CEA阴性和阳性结直肠癌的组织蛋白质组学比较与验证本部分的研究目的是比较血清CEA~-CRC和CEA~+CRC与正常粘膜组织(normal mucosa, NM)之间差异表达蛋白,验证部分有差异表达的蛋白质,试图寻找潜在的候选标志物。结果显示,在筛出的57个差异蛋白质中,大多数细胞功能定位在胞内,生物学过程主要体现为调控和代谢,分子功能主要参与结合。许多差异蛋白之间有联系,尤其是差异蛋白TTN、PRDX1、TAGLN、TP11等,处于蛋白相互作用网络的中心枢纽节点上。采用Western Blot (WB)和免疫组织化学染色验证差异蛋白Annexin A5和Trangelin表达,它们趋势与质谱鉴定信息一致。采用荧光定量PCR验证Lumican蛋白对应基因,mRNA表达水平,结果与质谱信息不一致。本研究说明,iTRAQ定量标记是寻找CRC有效分子标志物的重要方法,但需要验证确认。本实验筛选出一组与CEA相关组织差异蛋白,其中有些可能是CRC潜在的分子标志物。第三部分CEA阴性和阳性结直肠癌的分泌蛋白质组学比较与验证本部分的研究目的是比较血清CEA~-CRC、CEA~+CRC与NM微小组织块培养上清之间差异分泌表达蛋白,验证部分有差异表达的蛋白质,试图寻找可能在血清中出现的的潜在候选标志物。结果显示,在筛出的61个差异蛋白质中,总共有50.82%(31／61)是分泌蛋白,其中有一些是未见报道与肿瘤相关的分泌差异蛋白,Western Blot验证差异蛋白Annexin A5和Trangelin,结果显示它们表达趋势与质谱鉴定信息基本相似,但分泌表达模式与组织表达模式存在分离现象。本研究说明,分泌蛋白组学是组织蛋白组学的有益补充；本实验筛选出一组与CEA相关的分泌差异蛋白,可能是CRC潜在的血清分子标志物。第四部分AnnexinA5和Lumican在结直肠癌的表达和意义本部分的研究目的是扩大样本研究Annexin A5和Lumican在结直肠癌的表达和意义,为下一步进行功能研究进行初步筛选。结果显示,Annexin A5在CRC阳性率为40%(20／50),明显高于正常组织,其表达与CRC分期、转移有关。Lumican在结直肠癌呈高表达,与病理参数无关。本研究说明,Annexin A5和Lumican是结直肠癌的重要候选分子标志物,具体的作用机制还有待深入研究。结论1.CEA血清阳性率不高,缺乏敏感性,CEA阴性结直肠癌和CEA阳性结直肠癌在临床和流行病学上确实有一定的差异。2. iTRAQ定量标记是寻找CRC有效分子标志物的重要方法,但需要验证确认。组织蛋白组学筛选出57个与CEA相关差异蛋白,分泌蛋白组学选出61个与CEA相关差异蛋白,两组差异蛋白有较多重复,但差异蛋白的组织表达模式与分泌表达模式可以不一致。本组筛选出的差异蛋白,有些可能CRC潜在分子标志物。3.改良Alamar blue法是检测组织块活力灵敏有效的新型检测方法之-4. AnnexinA5和Lumican表达与结直肠癌关系密切,是结直肠癌的重要分子标志物之一。创新点：1. iTRAQ定量标记技术应用在结直肠癌分泌蛋白组学研究。2.以血清CEA表达状态为切入点分组,寻找CRC潜在分子标志物,符合临床实际需求。3.设计单位质量吸光度评价指标,把Alamar blue法首次应用在组织块活力评价上。更多还原

【Abstract】 Colorectal carcinoma (CRC) is one of the three common malignant tumors in the world, and ranks fourth in cancer mortality in our country. At present, the incidence rate of CRC is increasing, but early diagnosis level of CRC is poor and majority of CRC belong to the aggressive-phase. carcinoembryonic antigen (CEA) is the most widely used serum marker,but its sensitivity and specificity are far from satisfying. The CEA-negative CRC is more short of serodiagnosis and monitoring marker than the CEA’positive CRC. So, it is urgent to screen for new tumor biomarkers to improve CRC diagnosis. Now, the new tagging technique such as isobaric tag for relative and absolute quantitation (iTRAQ), followed by LC and MS/MS analysis is one of the more powerful methods in the search for disease biomarkers. In this study, CRC is researched in order to screen some worthy tumor biomarkers.Part One Clinicopathologic and epidemiologic analysis of 1696 cases of the colorectal carcinoma related with preoperative carcinoembryonic antigenThis section aimed to understand the differences between CEA-negative CRC group and CEA-positive group, and the positive expression ratio of CEA by exploring the clinicopathologic and epidemiologic characteristics of 1969 cases CRC.The result manifested the positive expression ratio of CEA was 38.04 percent. The mean age in the CEA-negative CRC was younger than in the CEA-positive one, and the median age in the CEA-negative CRC was ahead 3 years of that. The negative CEA group patients were younger than 60 years of age. The blood group A ratio was higher in the negative CEA group than in the positive CEA group, and the blood group B one was lower in the negative CEA group than that. The concomitant ratios of type 2 diabetes and colon obstruction in the negative CEA group, apparently lower than the positive CEA group.This study showed CEA had poor sensitivity for the detection of some CRC, and there really had some differences between the negative CEA group and the positive CEA group, which was the basement of next research.Part Two The analysis and verification of tissue proteomics between the CEA-negative CRC and CEA-positive CRC.This section aimed to screen some potential biomarkers of CRC by comparing the differential proteins in the CEA-negative CRC and CEA-positive CRC to the normal mucosa.Totally 57 proteins were finally identified. Most proteins was intracellular component. The biological process reflected regulation and metabolism. Molecular function mostly participated binding. There had been connected among many differential proteins, especially such as TTN、PRDX1、TAGLN and TP11. These proteins were put in the central key position of network. Annexin A5 and trangelin were chose for identification by western blot and immunohistochemistry stain, lumican was validated by fluorescence quantitative PCR, and majority were proved to be line with the iTRAQ results. Our results proved that iTRAQ results were reliability.This study showed iTRAQ technique can be as effective manner to screen the differential expressed protein, but it was needed to validate. Some of the differential proteins were probably potential markers of CRC. Part Three The analysis and verification of secretome between the CEA-negative CRC and CEA-positive CRC.This section aimed to screen some potential serum biomarkers of CRC by comparing the differential proteins in the CEA-negative CRC supernatant and CEA-positive CRC supernatant to the normal mucosa supernatant.Totally 61 proteins were finally identified. The secretory proteins ratio were 50.82%by bioinformatics. Some secretory proteins had not been mentioned in tumor in the past records. Annexin A5 and trangelin were chose for identification by western blot, and were proved to be basically similar with the iTRAQ results. But the secretory expression model was probably not line with the tissue expression model.This study showed the secretome of CRC was a benefical supplement to the tissue proteomics. These differential secretory proteins were probably potential serum biomarkers of CRC.Part Four The study and significance of annexin A5 and lumican expression in CRCThis section aimed to explore in enlarged sample the annexin A5 and lumican expression in CRC and their significance.The result showed the expression level of annexin A5 and Lumican in CRC were markedly higher than one in normal mucosa. Annexin A5 expression was related with CRC stage and metastasis, but Lumican expression had no this relationship.This study manifested annexin A5 and Lumican were one of important candidate biomarkers in CRC.更多还原