【作者】 刘晓丽；

【导师】 陈正堂；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2011， 硕士

【摘要】 肺癌是一种在全世界范围内严重危害人类健康与生命的疾病,其起病隐袭,生存率低,发病率和死亡率高。肺癌按病理类型分为小细胞肺癌和非小细胞肺癌,其中非小细胞肺癌比例高达80%-85%,而非小细胞肺癌中腺癌占60%-80%。肺癌总的5年生存率至今仍在10%或略高。尽管医务工作者们多年来一直在孜孜不倦地致力于寻求能够改善肺癌预后的方法,但我国肺癌的发病率和死亡率仍逐年攀升。寻找有效的途径,对其进行早期诊断从而改善预后,是目前医务工作者们关注和研究的热点之一。从阐明肿瘤的起源着手寻找包括肺癌在内的恶性肿瘤的早诊方法,可能是一个值得努力的方向。肿瘤的发病机制有多种学说,目前研究得较多且较公认的是“肿瘤干细胞学说”。干细胞理论认为,肿瘤可能是一种干细胞疾病。肿瘤干细胞是存在于肿瘤中的一小部分具有干细胞性质的细胞群体,具有自我更新和无限增殖能力,可以产生与上一代完全相同的子代细胞,并且具有多种分化潜能,产生不同表型的肿瘤细胞,从而造成肿瘤细胞的群体漂移,形成肿瘤的异质性,并使肿瘤在体内不断扩大,或形成新的肿瘤病灶。因此,肿瘤的发生,及其侵袭转移、复发、耐药、抗辐射等恶性表型特征都与肿瘤干细胞有关。如能在肿瘤发生的始动阶段找到若干肿瘤干细胞的表面标记物,则将大大推进肿瘤的早期诊断,进而改善其预后。目前常用的肿瘤表面标记物如CEA、CA-199、NSE等虽然对肺癌的诊断有一定帮助,但缺乏特异性。研究表明,上皮细胞粘附分子(EpCAM),又名ESA或CD326,是一个常见的高表达性上皮肿瘤相关抗原,在人类大多数上皮组织肿瘤中的表达强度远远超过正常组织,可作为上皮来源肿瘤的诊断和预后监测的特异性标志,且已被用于多种肿瘤研发基于EpCAM相关的抗体或疫苗的临床试验中。近年来的研究表明:EpCAM是多种上皮肿瘤干细胞的分子标志物,被认定可作为肿瘤起源干细胞的一种标志,使其成为诊断和治疗上皮来源肿瘤的更有意义的靶标。如,Eramo等利用人类肺癌干细胞中的EpCAM高表达,联合CD133作为分子表型(EpCAM+CD133+)对肺癌干细胞进行分选,通过各种方法证实分选出的细胞具有自我更新、无限增殖等干细胞样细胞的特性。因此如能制备出EpCAM特异性抗体,对于靶向鉴定肺癌干细胞,继而最终实现肺癌早诊将具有重要意义。尽管杂交瘤技术生产了许多有价值的单克隆抗体,但必须经过动物免疫、细胞融合及克隆筛选等繁琐、费时的过程。噬菌体展示技术在体外成功模拟抗体生成过程,不经免疫就可以制备抗体,尤其是单链抗体(ScFv),具有分子量小,无抗体分子的Fc段,免疫原性低,组织穿透力强以及体内循环半衰期短等优点。因此,本课题在尚无有效的肺癌干细胞特异性抗体的制备方法的情况下,拟采用噬菌体展示技术探讨制备人源化肺癌干细胞抗体的新途径,使之成为靶向肺癌干细胞的一种有效方法。目的通过噬菌体表面展示技术构建鼠抗人EpCAM单链抗体(ScFv),初步研究其免疫活性,为后续课题构建肺腺癌噬菌体抗体库和筛选肺腺癌干细胞特异性抗体做好前期实验工作。方法1.在Genbank查询鼠抗人EpCAM VH、VL序列,设计EpCAM ScFv;2.采用噬菌体展示技术构建鼠抗人EpCAM ScFv ;3.随机挑取重组pCANTAB-5E/EpCAM ScFv进行测序分析验证;4.采用ELISA方法、免疫细胞化学方法检测EpCAM ScFv的免疫活性。结果1.成功设计并合成了EpCAM ScFv序列;2.成功进行了EpCAM ScFv的噬菌体表面展示;3.对挑取克隆进行筛选并制备单克隆噬菌体抗体,用ELISA及免疫细胞化学方法测定其抗原结合活性及特异性,筛选后的单链抗体能与肺腺癌A549细胞及A549干细胞样细胞特异性结合。结论1.用噬菌体展示技术成功制备了针对上皮细胞抗原的单克隆噬菌体抗体EpCAM ScFv,较传统的免疫动物方法更具简便、省时等优点。2.EpCAM ScFv单链抗体具有良好的免疫活性,可望成为靶向检测上皮来源的肿瘤细胞乃至癌干细胞的有用工具,并为下一步构建肺腺癌噬菌体抗体库以筛选肺腺癌干细胞特异性抗体打下了良好的前期实验基础。更多还原

【Abstract】 Lung cancer is a kind of serious disease worldwide which endangers human life and health. It has the features of insidious onset, low survival rate and high morbidity and mortality. According to pathology, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer, in which the proportion of non-small cell lung cancer is up to 80% - 85%, and the morbidity of the adenocarcinoma of lung makes up 60% - 80% of non-small cell lung cancer. Up to now, the 5-year survival rate of lung cancer is still to remain 10% or a little more. Although medical workers have been committing to finding ways to improve the prognosis of lung cancer, the morbidity and mortality in China are still rising gradually. Finding effective ways for early diagnosis has been the concern for medical workers.It is worthwhile to find out the early diagnosis method of malignant cancer including lung cancer from elucidating the origin of tumor. There are many hypotheses on tumor pathogenesis, and the cancer stem cell theory is the widely accepted one. According to the theory, tumor may be a stem cell disease. Cancer stem cells are cell populations existing in tumor with stem cell properties, such as self-renewal and unlimited proliferation, generating daughter cells with identical properties to previous generation, having a variety of differentiation potential, producing different phenotype of tumor cells, resulting in tumor cell population drift, thus formed heterogeneity, which causing expanding constantly or forming new tumors nidus. Malignant phenotype features of tumor such as recurrence, invasion, metastasis, drug resistance are relevant to cancer stem cells. Scholars speculated: eradication of cancer stem cells maybe mean elimination of the tumor since cancer stem cells are the source of tumors. Thus, the membrane markers of lung cancer stem cells can be used for early diagnosis, so as to promote early diagnosis of cancer, thereby improving the prognosis.It’s now known that surface markers of lung cancer such as CEA, CA-199, NSE, etc. have some contribute to the diagnosis of lung cancer, but lacking of specificity. Epithelial cell adhesion molecule (EpCAM), also known as ESA, or CD326, is a common high-level expression of the epithelial tumor-associated antigen whose expression in most human epithelial tumor is far stronger than in normal tissue, which can be used as the specific marker of diagnosis and prognosis of epithelial tumor, and it has already been used in a variety of tumors based on antibodies or vaccine-related clinical trials. Recently study indicate that EpCAM is the molecular marker of variety epithelial origin tumor stem cell. It has been identified as an additional marker of stem cells– the origin of the tumor, which make it become a more meaningful target of epithelial origin tumor for diagnosis and treatment. For example, it has been confirmed that the cells separated from lung carcinoma stem cell by means of molecular phenotype(EpCAM+CD133+)which is conformed of EpCAM highly expressed in lung carcinoma stem cell of human associated with CD133, have the features of stem cell, such as self-renewal and unlimited proliferation, by Eramo with a varity of methods. Thus, if specific antibody against EpCAM can be prepared, it will play an important role in targeted identification of lung cancer stem cells and then early diagnosis of lung cancer.Though hybridoma technology has generated a lot of valuable monoclonal antibodies, it must go through a cumbersome and time-consuming process which include animal immunity, cell fusion and clone screening. Phage-displaly technology successfully simulates the generation process of antibody in vitro and can be prepared to produce antibodies without immune, especially single-chain antibodies which have small molecular weight, Fc-free antibody molecules fragment, weak immunogenicity, better tissue penetration ability, and short half-life in vivo, etc. Therefore, in the condition of no effective ways to prepare specific antibodies targeted lung cancer stem cells, this study is to use phage-display technology to explore the new avenue to preparing humanized lung cancer antibodies and make it an effective method for the diagnosis of tumors.ObjectiveThe aim of this study is to construct mouse anti-human EpCAM single-chain antibody (ScFv) by phage display technology, and study preliminarily its immunity activity. It is expected to provide preliminary experimental work for the following construction of phage antibody library of lung adenocarcinoma and screening specific antibodies of lung adenocarcinoma stem cells.Methods1.To construct EpCAM ScFv by phage-display technology. 2 . To analyze the EpCAM ScFv sequence by randomly selected recombinant pCANTAB-5E/ EpCAM ScFv.3.The immunity activity identifying of EpCAM ScFv.Results1.The EpCAM ScFv sequence was desgined and synthesized successfully.2.The EpCAM ScFv was displayed successfully on phage surface.3.Positive reactions to EpCAM antigen were detected in 1 of 5 randomly-selected clones, with a positive rate of 20% using ELISA. The selected ScFv specifically bound to human lung adenocarcinoma A549 cells.Conclusion1.The monoclonal phage antibody EpCAM (ScFv) to aim directly at epithelial antigen was prepared successfully by the means of phage-display technology. Compared with traditional immunized animals method, this method has advantages such as being simple and time-saving.2.The EpCAM ScFv has good immunity activity, and could be an important useful tool to targeted diagnosis tumor cells of epithelial origin and even cancer stem cells. It provide good pre-experimental basis for the next construction of phage antibody library associated with lung adenocarcinoma and screening specific antibodies of lung adenocarcinoma stem cells.更多还原