【作者】 杜冰；

【导师】 钱旻；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 原发性肝癌是一种分布范围较广、危害严重的致死性疾病。我国一直以来都是肝癌高发地区，每年至少有12万人死于原发性肝癌，因此建立起一套有效的肝癌临床检测和治疗体系也就成为了一项关系到人民生命健康的重要工程。 目前肝癌靶向治疗领域普遍采用的依然是单抗及其片段作为治疗药物的特异性载体，由于单克隆抗体特异性的识别能力和所特有的免疫学功能，在整个肿瘤靶向治疗领域已经取得了极大的成功，甚至某些单克隆抗体已经通过美国FDA的认证正式走向临床应用。尽管如此，单克隆抗体技术的应用依然存在一些难以逾越的障碍：分子量大以及异源性所诱导机体产生不良的免疫反应，由此对患者的身体造成伤害，同时还会大大降低靶向药物的生物半衰期，进而影响到整个的肿瘤治疗效果。 本论文拟利用体内噬菌体展示肽库技术，筛选到在体内具有特异性结合能力的肝癌肿瘤组织特异性粘附肽，进而利用这些特异性高、结合能力强的肝癌特异性粘附肽来取代传统靶向治疗治疗领域中单克隆抗体的作用，充分利用多肽靶向载体的分子量低、免疫原性小的特点，弥补单克隆抗体的不足，建立一套切实有效的基于多肽的肝癌鉴定和靶向治疗系统。 本论文主要分成以下几个部分： 一、 体内噬菌体肽库技术筛选肝癌组织特异性粘附肽 1．利用广泛采用的NEB公司的噬菌体线性12肽文库，在本实验室构建的荷肝癌细胞Bel-7402细胞株的荷瘤裸鼠体内，通过三轮的体内筛选，得到大量在体内具有特异性靶向能力的噬菌体克隆。 2．在每轮筛选的过程中，利用噬菌体效价的测定及时监测和比较裸鼠体内各个组织中噬菌体的数目；同时采用免疫组织化学的方法，对组织中噬菌体的分布情况进行观察。结合噬菌体数目的定量分析和免疫组织化学的定性分析，可以看出：通过三轮的体内筛选，特异性结合的噬菌体在肿瘤组织中得到了有效的富集。 3．在对筛选得到的噬菌体进行单克隆化后，随机选取了46个噬菌体克隆进行测序，并对所得到的DNA序列进行比对和分析。通过分析，我们将得到的噬菌体根据所含相同序列的特点初步分为PSS／PTT、PPS、RW以及PRA组，并依次确定了每组中最具代表性的多个候选阳性克隆。更多还原

【Abstract】 Human hepatocellular carcinoma (HCC) is one of the most common and deadly cancers in the world. China is one of the most widespread countries of HCC. At least 120,000 people in China were dead from it every year. So it is very important to establish a convenient and reliable system in clinical diagnosis and treatments of hepatocarcinoma.Now the monoclonal antibody and its fragment is still the common used carrier in hepatocarcinoma targeting therapy. Because of the specifically discernment and immunity functional the MAb has become most succeed one in the tumor targeting therapy field. Even some MAb have got the authentication of FDA in USA, the MAb still has its inborn shortcomings: higher molecular weight and immunogenic could induce some bad responses to human body, and these responses will hurt to patients obviously. Then the shortcomings could also decrease the half life of immunotoxin and the therapy effect will also been reduced eventually.In this paper, we will try to screen some specifically binding peptides to hepatocarcinoma tissues by using in vivo phage display technology. Then we can use this peptides which have high specifically and strong binding ability to take the place of monoclonal antibody in traditional tumor targeting therapy. Making the most use of specifically binding peptides’ lower molecular weight and immunogenic to make up the shortcomings of MAb and to establish a feasible peptides system for diagnosis and targeting therapy to hepatocarcinoma.This paper can be divided into three parts:A. The panning of specifically binding peptides to hepatocarcinoma by in vivo phage display technology.a) The common used Ph.D.-12 Phage Display Peptide Library Kit which produced by New England Biolabs Inc was chosen to injected into the nude mouse with hepatocarcinoma tumors on their backs, after three rounds panning a lot of candidate specifically binding phages were obtained.b) Accompanying with each round panning, the phages’ titering was carried out as the kit manual to inspect the number of phages in each tissues, and the immunohistochemistry assay was also performed to appraise the distribution of phages in each tissue. From the data of titering and immunohistochemistry we can say that after the three rounds panning the specifically binding phages were enriched effectively to the tumor tissue.c) Then the specifically binding clones were picked out for random amplification. There are 46 clones were chosen for sequencing eventually and the sequence of those phage clones were compared and analyzed. From the sequence we can say that also the data are all complex we still更多还原