【作者】 邢俊杰；

【导师】 孟荣贵；

【作者基本信息】 第二军医大学 ， 外科学， 2009， 博士

【摘要】 近年来,我国大肠癌的发病率呈上升趋势,且早期症状少,待出现症状后才明确者多属中晚期,故其是一种恶性程度较高的消化道肿瘤,死亡率位居西方发达国家恶性肿瘤死亡率的第2、3位,在发展中国家发病率也呈逐年升高趋势。据调查显示,目前大肠癌在中国大部分地区已经成为发病率上升最快的恶性肿瘤之一。2002年以后上海市大肠癌发病率已经超越胃癌跃居第2位,仅次于肺癌。当前,我们治疗大肠癌的方法相对成熟,主要是以手术切除为主,辅助以化疗、放疗或免疫治疗,以及中药治疗等。但大肠癌术后5年内的复发率、死亡率仍然保存在一个较高的水平。早期发现并采取积极有效的治疗措施成为广大医务工作者的共同目标,因此寻找作为大肠癌早期诊断的新型肿瘤标志物及新的大肠癌治疗靶标差异蛋白,成为目前大肠癌基础及临床研究的热点。本研究我们关注的是膜联蛋白A家族的一个重要成员--膜联蛋白A5。膜联蛋白是一种以钙离子依赖性方式与酸性磷脂结合的蛋白质,在大多数生物的细胞中,膜联蛋白是一种高丰度蛋白,占整个细胞蛋白质含量的0.5%～2%,其中膜联蛋白A家族成员广泛分布于高等脊椎动物中。大量的研究显示,膜联蛋白在胞吞、胞吐作用、细胞间以及细胞与细胞外基质间的相互作用、离子通道形成、磷脂酶A2活性抑制等多种生理过程中起作用,但是二十多年来的研究并没有能够就其生理功能得到一个明确结论。长期以来人们发现膜联蛋白A家族成员与不同来源的良性、恶性肿瘤的发病机制相关,研究显示在多种恶性肿瘤组织中A家族某些成员的表达水平都发生了明显的上调,然而另外一些资料显示在某些恶性肿瘤生成、分化以及转移过程中膜联蛋白的下调可能起着重要的作用。遗憾的是迄今为止膜联蛋白A家族成员与肿瘤的发生、发展的确切关系及具体作用机制仍然不明确。相关研究显示膜联蛋白A家族某些成员的表达在不同特征的大肠癌组织中发生了变化,提示它们可能在大肠癌的发生、发展过程中扮演者重要角色,然而并没有强有力的证据证实它们在大肠癌发病过程中所起的作用,甚至于不同的研究者得到了互相矛盾的结果。因此我们认为有必要借助于一些较先进的技术手段,对膜联蛋白A家族成员在大肠癌中的表达情况及其作用机制进行系统、深入的探讨。首先,我们由正常人胎盘组织中克隆得到了膜联蛋白A5的基因编码区全长序列,并通过亚克隆技术将其装载入真核表达载体pcDNA3.1(+)。我们收集了51例病例完整、背景清楚的散发性大肠癌患者组织样本作为研究对象,同时获得了51例非恶性肠道疾病患者的组织样本作为对照。通过免疫组织化学实验,我们首先对肿瘤样本中p53、Ki-67这两个肿瘤临床诊断上较为常用的标志物进行了分析,并分别根据p53的突变情况和Ki-67增殖指数对51例肿瘤组织样本进行了归类。随后我们选择了目前功能较为清楚的膜联蛋白A5作为切入点,借助荧光实时定量PCR和免疫组织化学等实验手段,分别从mRNA水平和蛋白质水平上对膜联蛋白A5在组织样本中的表达情况进行了检测。研究结果显示:膜联蛋白A5的表达水平在散发性大肠癌组织中发生了上调;同时A5在肿瘤组织中的表达变化与p53的突变和Ki-67增殖指数分别呈正相关。这一结果提示我们膜联蛋白A5与大肠癌的恶化、增殖等特征具有相关性,可能在其中起着重要的作用,并且膜联蛋白A5能够作为一种肿瘤标志物应用于大肠癌的临床诊断,因此具有临床应用价值。在此基础上,我们扩大样本量,选择了176例病例完整、背景清楚的散发性大肠癌患者组织样本作为研究对象。手术切除标本后,分别配对取其大肠癌变部位组织以及正常组织。然后通过实时定量PCR和免疫组化染色的方法鉴定每个标本的膜联蛋白A5的mRNA和蛋白表达情况。实时定量PCR结果显示肿瘤中的膜联蛋白A5的mRNA水平上调。免疫组化结果显示膜联蛋白A5在40.5%的肿瘤之中高表达。同时进行严格详细的术后随访。通过统计数据判断膜联蛋白A5的表达与病理分期和预后之间的关系。我们的研究结果显示,膜联蛋白A5的表达情况可以作为一个有价值的分子标记物,用来预测患者外科手术切除肿瘤后的预后。无论是进行单变量分析还是多变量分析,我们的研究数据都显示,膜联蛋白A5的表达情况和患者5年生存率的降低以及肿瘤复发率的提高都有很强的相关性。多变量分析显示,膜联蛋白A5的表达情况可以作为一个独立的预测因子用来预测预后。因此,不考虑肿瘤的病理分期而仅用肿瘤组织中的膜联蛋白A5表达水平也可以预测患者的术后预后。另外,膜联蛋白A5表达水平也可作为术前预测治疗效果的一个预测因子。我们的研究提示,膜联蛋白A5在大肠癌的生长、进展以及转移等过程中可能扮演了一个相当重要的角色。在组织水平研究的基础之上,我们选择了两种遗传背景相同转移潜能不同的大肠癌细胞系(高转移潜能:SW620、低转移潜能:SW480)。首先,通过Transwell小室实验对两种细胞系的侵袭、迁移能力进行检测,以验证之前对它们转移潜能的分析。随后通过荧光实时定量PCR和Western blot从mRNA水平和蛋白水平对膜联蛋白A家族重要成员A1、A2和A5的表达情况进行了检测。结果显示不同的A家族成员在具备不同转移潜能的大肠癌细胞系中的表达情况互有差异(A1在高转移潜能的细胞中较高、A2在低转移潜能的细胞系中较高、A5的表达与细胞转移潜能不具明显相关性),这一结果提示我们A家族膜联蛋白与肿瘤细胞的转移、凋亡等多个方面具有相关性。我们设想膜联蛋白A家族不同成员在各生理过程中的重要性并不相同,可能存在功能上的互补性。综上所述,本研究对膜联蛋白A5在不同特征的散发性大肠癌中的表达情况进行了系统的检测,对其在大肠癌发生、发展中的作用机制进行了初步分析,并且通过分析膜联蛋白A5的表达与肿瘤分期及临床病理特征之间的相关性,探讨了膜联蛋白A5作为标志物预测患者预后的潜在可能性。并为更加深入地探讨膜联蛋白A家族与大肠癌的发生、发展关系以及膜联蛋白的生理功能研究提供较好的理论依据。更多还原

【Abstract】 Carcinoma of the colon and rectum is one of the most common alimentary malignancies in developed nations, and cancer mortality among the first places 2,3. Even in developing countries, morbidity is with a increasing trend. Now, CRC has become one of malignancies with the fastest rising incidence in most parts of China. In Shanghai, 2002, colorectal cancer incidence has gone beyond the first two ranks, second only to lung cancer. Today , operation is the most important treatment of CRC, supported by chemotherapy, radiotherapy or immunotherapy, as well as traditional Chinese medicine treatment. However, colorectal cancer after 5 years the relapse rate, mortality still remains at a higher level. To take positive measures for early detection and effective treatment has become the common goal for medical workers. So looking for new tumor markers for early diagnosis of colorectal cancer and new protein for therapeutic targets is a hot spots for basic and clinical research of CRC.This study we are concerned is one important member of annexin A family ---Annexin A5. The annexins are characterized as a family of proteins capable of binding to acidic phospholipids in a Ca2+-dependent manner and they appear to be widely distributed throughout nature. Up to this day, there are total of twelve annexins have been found in higher vertebrates and form the A family of the annexins. In most biological contexts studied, the annexins are highly abundant proteins, reaching the level as high as 0.5% to 2% of the total cellular proteins and all these proteins present a homologous core structure and a highly variable N-terminus. Based on their Ca2+-dependent interaction with phospholipids and membranes, many physiological functions have been described for the annexins including endocytosis, exocytosis, cell-cell or cell-matrix interaction, formation of ion channels, inhibition of phospholipase A2 and so on. Unfortunately, there has not been a definitive description of any physiological role played by these annexins in more than 20 years of study. Although any exact in physiological function has not been described for the annexins, there are great deals of evidence to suggest that they are involved in the pathogenesis of many different cancers. In many cancers, there is sharp up-regulation of annexins in both mRNA and protein levels. On the other hand, there is data indicating that down-regulation of annexins may play a significant role in tumorigenesis and metastasis of other types of cancer. However, the precise role played by the annexins in the pathogenesis of tumor is still unknown. Related studies have shown that expression of some members of annexin A family has changed in the colorectal cancer tissues with different characteristics. That means these members have been playing important roles in the occurrence and the development process of colorectal cancer. Nevertheless, no strong evidences had shown they had affected incidence of colorectal cancer, even different researchers had conflicting results.Therefore, in our opinion, it is necessary to analyze the expression of A family members in colorectal cancer and their mechanism in tumorigenesis.Above all, we succeeded in cloning full length sequence of human annexin A5 coding regions of from human placenta by RT-PCR. Then we constructed and identified recombinant eukaryotic expression vectors of annexin A5.51 tumor samples with complete clinical data were selected. At the same time 51 normal tissues (obtained from patients with benign colorectal diseases) were chosen as normal control. We firstly detected p53 and Ki-67 in tumor tissues by immunoassaying and split all tumor samples into two groups separately according to the detection of p53 or Ki-67. Then the mRNA and protein levels of annexin A5 in each sample were analyzed by real-time quantitative PCR and immunohistochemistry. We found that there is an up-regulation of annexin A5 in colorectal cancer. We also found the up-regulation has a positive correlation with the mutation of p53 or Labeling Index (Ki-67) in tumor tissues. Besides, the mutation of p53 in tumor tissues has a negative correlation with Labeling Index (Ki-67). Our study indicates that annexin A5 might be used as an assistant clinicopathological biomarker in cancer diagnosis and the up-regulation of annexin A5 in colorectal cancer might be related to the malignant proliferation of tumor cells.We investigated the expression of ANXA5 in colorectal adenocarcinoma. This study included 176 consecutive patients with sporadic colorectal cancer. Paired colorectal tissue samples and corresponding nonmalignant tissues were obtained by surgical resection. ANXA5 mRNA and protein expression in each tissue were assessed by real-time RT-PCR and immunohistochemical staining. Real-time RT-PCR showed that there is an up-regulation in mRNA level of ANXA5 in tumors . Immunohistochemical study revealed that high ANXA5 expression was present in 40.58% of tumors. Data were statistically correlated with pathological parameters and clinical outcome. In this study, we have shown that expression of ANXA5 can be used as a potential molecular marker to predict patient outcome in patients treated with surgical resection alone. Our data showed that high ANXA5 expression has a strong correlation with decreased five-year survival and increased tumor recurrence both in univariate and multivariate analyses. Multivariate analysis indicated that ANXA5 expression is an independent prognostic factor for poor survival. Thus, tumor levels of ANXA5 regardless of tumor stage can be used to determine patient outcome. In addition, ANXA5 expression level can potentially be used as a prognostic factor before surgery can be done. Our findings also suggest that ANXA5 plays an important role in tumor growth, progression, and metastasis in colorectal adenocarcinoma.We selected two human colon adenocarcinoma cell lines and analyzed their metastatic potentials by invasion/migration assay (higher metastatic potential: SW620; lower metastatic potential:SW480). Then we analyzed expression of annexin A1, A2 and A5 mRNA and protein levels in cell lines by real-time PCR and western blot. We found that the order of A1, A2, A5 expression level from high to low was separately SW620 >SW480, SW480 > SW620, and SW480≥SW620. Our results indicated that annexin A1 and A2 may play important roles in tumor metastasis and annexin A5 may not work in metastasis but apoptosis. We also imagine that different annexins may be complementary in functionWe analyzed expression of annexin A5 in spocitic colorectal cancer and its mechnium in tumorigenesis. We determined its prognostic significance by correlating ANXA5 expression with tumor stage and clinicopathologic features.Our work would make the groundwork for further researches on physiological functions of the annexins and their role in tumorigenesis.更多还原