【作者】 宋明全；

【导师】 朱金水；

【作者基本信息】 上海交通大学 ， 内科学， 2007， 博士

【摘要】 随着生活水平的提高,饮食习惯的改变,结直肠癌的发病率在我国呈上升态势,越来越多的研究者将注意力转向这种疾病。结直肠癌的临床治疗效果与早期发现密切相关。早期结直肠癌的5年生存率超过90%,甚至部分病人可达到长期无瘤状态。随着纤维结肠镜检查的逐渐普及,内镜微创治疗成为治疗结直肠癌的快捷有效的方法。然而内镜粘膜下剥离术(ESD)或内镜下黏膜切除术(EMR)等内镜微创治疗并非对所有的早癌均适合,对于黏膜下层结直肠癌,由于黏膜下层有丰富的淋巴管和血管,即使内镜完全切除,也仍有29%左右的病例已发生了淋巴结转移,所以这部分病人更适合手术治疗给予淋巴结清扫。病理学分型是指导临床治疗结直肠癌的最可观的标准。目前修订的Vienna病理学分型修订方案是一种能比较准确地提供结直肠癌诊断和相应处理方式的病理学分类方式。Vienna病理学分型对结直肠癌的分类主要依据肠镜钳取的组织活检标本,但是通过肠镜获取的标本通常比较表浅,很难到达黏膜肌层,通常的结肠镜活检标本不能确定结直肠癌细胞是否有黏膜下浸润,更不能确定是否有淋巴结转移,无法明确诊断出结直肠癌是否已经浸润到黏膜下。即内镜活检材料与外科病理手术切除标本不同,前者钳取的组织较少,位置表浅,在判断肿瘤细胞的恶性生物学行为方面(如癌细胞浸润)存在局限,无法准确判断是否能是通过内镜黏膜切除就可以彻底治疗,还是需要手术切除并清扫淋巴结。本实验通过蛋白质组学的方法,鉴定出在Vienna病理学分型中出现黏膜下浸润和无黏膜下浸润的差异蛋白,为结直肠癌病理学分型寻找蛋白标志物;为结直肠癌选择是通过肠镜微创切除还是手术给予淋巴结清扫提供客观参考依据;并可进一步为结直肠癌的生物学治疗寻找新的靶点。本实验首先利用n-甲基-n-亚硝基脲(MNU)诱导出结直肠癌近交系大鼠动物模型;再通过维也纳病理学诊断标准将大鼠结直肠癌的组织标本鉴别出有黏膜下浸润和无黏膜下浸润的两类;应用荧光差异定量双向电泳技术( DIGE )和基质辅助激光解吸/电离-飞行时间质谱(MALDI-TOF-MS)鉴定出这两类大鼠结直肠癌组织的差异蛋白;并应用分子生物学的方法对部分差异蛋白在转录和翻译水平的表达进行进一步观察。DIGE是定量蛋白质组学方面的一个进步,与传统2DE相比,DIGE可以提供更为准确的定性和定量分析。利用DIGE技术分析我们得到了结直肠癌有黏膜下浸润和无黏膜下浸润的1640-1880个蛋白差异点;在T-test Value<0.001和Average Ratio>2.0的条件下,实验最后得到黏膜浸润与无黏膜浸润的差异蛋白5个,这些蛋白均与正常结直肠黏膜组织有显著差异,且黏膜浸润与无黏膜浸润癌组织的显著差异的5个蛋白与正常组织相比较其表达趋势相一致仅仅是表达的程度不同。经过MALDI-TOF-MS鉴定,有黏膜下浸润比较无黏膜下浸润的大鼠结直肠癌组织表达显著上调的蛋白有3个:胶转蛋白(Transgelin)、肽基脯氨酰异构酶A (Peptidylprolyl isomerase A)和Tropomyosin1, alpha isoform d;显著下调的蛋白有2个,为碳酸脱羧酶2(carbonic anhydrase 2,CAⅡ)和一种没有命名的蛋白质。对于应用蛋白质组学方法鉴定出的5种差异蛋白,本实验继续选择了两种分别在黏膜下浸润中表达减少和增多的CAⅡ和Transgelin,应用分子生物学的方法给以进一步研究。结果显示,在结直肠癌有黏膜下浸润时,CAⅡ和Transgelin的转录水平和翻译水平的表达,与蛋白质组学的结果相一致。本实验结果说明近交系大鼠结直肠癌无黏膜下浸润和黏膜下浸润至少存在Transgelin、Peptidylprolyl isomerase A、Tropomyosin1、CAⅡ等5种差异表达的蛋白;可以作为鉴别结直肠癌是否出现黏膜下浸润的标记物。更多还原

【Abstract】 With the improvement of people’s living standard and the change of diet habit, the incidence of colorectal cancer(CRC) trend to ascend straightly in our country. The good therapeutic effect and early detection are closely related to this cancer. Survival rate in 5 years of early CRC was over 90% if was given immediate and appropriate treatment, some patients could have long-time survival. With rapid development and the increasing popularity of colonoscopies,endoscopic minimally invasive treatment has become a quick and effective way to treat early CRC. Endoscopic invasive treatments such as endoscopic submucosal dissection (ESD) or endoscopic mucosal resection (EMR) are ideal methods, but not appropriate in all patients. When CRC with invasive submucosa, because submucosa was rich of lymphatic and blood vessels, even with endoscopic complete resection, there were still at least 29% cases with metastasis to lymphonodus. These cases were especially suitable for operation with lymph-node dissection.Pathology classification is the guide of CRC treatment. Recently Vienna classification has variously categories provide a good basis for differentiated recommendations on how to be further diagnostic and therapeutic measures. This pathologic diagnosis is depend on tissues by endoscopic biopsy. However, those biopsy tissues by biopsy forceps were usually not deep enough to reach the submucusa., so it was not easy to diagnose whether the tumor had invaded submucosa. Judging whether CRC had submucosal invasion obtained by endoscopic biopsy, whether had metastatic lymphonodus, it was not able to known whether CRC had invaded the submucosa. That means these tissues obtained by endoscopic biopsy and by surgical excision were different, the former was less and superficial, these little tissues were not provided evidence with the diagnose of CRC invasion, were unable to provide recommendations of endoscopic mucosal resection or surgical excision with lymph-node dissection.In our study using proteomic technology, we analyzed differentially expressed proteins in CRC with or without submucosal invasion, and so as to find some biomarker of CRC pathological classification. We hope it can give a reference to select a better treatment using endoscopic mucosal resection or surgical excision. And even provide some new theraputic targets in futrue biotherapy.Animal models were made by N-methyl-N-nitrosourea (MNU) enema; then these rars were randomly divided into two groups by Vienna revised classification: submucosal invasion and no submucosal invasion; Using two - dimensional fluorescence difference gel electrophoresis(DIGE) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) we selected some different proteins between submucosal invasion and without submucosal invasion group; and gave further study by molecular biological methods on these different expressing proteins’transcription and translation.DIGE is a progress in quantitative proteomics. Compared with traditional 2DE, DIGE could provide more accurate qualitative and quantitative analyses. We got 1640-1880 differently expressed proteins by DIGE in CRC of submucosal invasion and no submucosal invasion. The condition of T-test Value<0.001 and Average Ratio>2.0, there were 5 proteins between submucosal invasion and no submucosal invasion. These 5 proteins were all significantly different with normal colorectal mucosa, and similar increasing and decreasing tendency with different expression. We found there were 3 upregulated expression proteins in submucosal invasion compared with non-submucosal invasion: Transgelin、Peptidylprolyl isomerase A and Tropomyosin1, alpha isoform d;2 downregulated expression proteins in submucosal invasion: carbonic anhydrase 2 and an unnamed protein identified by MALDI-TOF-MS,We selected 2 proteins from the 5 proteins identified from proteomics, they were CAⅡand Transgelin which were downregulated and upregulated in submucosa invasion, and gave them a further study by molecular biology. The results showed that: in the CRC with submucosa invasion, CAⅡa nd Transgelin in transcription and translation were similar with the results of proteomics.Our studies showed that there were at least 5 proteins in the inbret strain rats model of CRC with submucosa invasion and non-submucosa invasion: Transgelin、Peptidylprolyl isomerase A、Tropomyosin1、CAⅡet al. These proteins could be specific biomarker to identify submucosal invasion in the judgement of early CRC.更多还原