【作者】 吴淼；

【导师】 周忠良；

【作者基本信息】 华东师范大学 ， 动物学， 2006， 硕士

【摘要】 我国是肝癌的高发国家，每年死于肝癌约11万人，占全世界肝癌死亡人数的45％。目前主要的治疗肿瘤的手段有手术治疗、化学治疗及放射治疗等，但它们都存在相当的局限性和不可避免的副作用。近年来兴起的肿瘤导向治疗，即借助高度特异的亲肿瘤物质作为载体携带药物对肿瘤部位进行靶向给药，稳定发挥杀伤作用，成为医学、生物学领域最热门的研究课题之一，正逐步的成为肿瘤治疗的新途径。 目前，临床上主要以单抗作为载体进行抗肿瘤治疗，取得了丰硕成果。但是这些单抗大多为鼠源性的，进入人体后会引起各种排异反应，从而影响携带的肿瘤药物的疗效或产生副作用。寻找新的、与正常组织不结合或很少结合、但与肿瘤组织特异性结合的新的非抗体类载体，是优化导向治疗的策略之一。我们已经利用噬菌体肽库技术进行了三轮筛选，得到了一些对肝癌具有靶向性的单克隆噬菌体肽。以肽类作为药物载体，分子量小，跟目标组织具有很强亲和能力，在血液及其它非靶组织中清除快，能够稳定渗透和穿入组织细胞中。于是我们将噬菌体外壳去除掉，仅对肝癌特异性黏附肽片段进行了化学合成。进一步说，我们以这些肝癌特异性黏附肽为探针，可以找寻到肿瘤相关蛋白或者肿瘤标志蛋白，研究其受体蛋白的生化特性和结合机理，从而优化肽类药物载体，为肝癌发病机理的研究以及肝癌的导向治疗提供新的药靶提供科学依据。 本论文就是利用免疫组织化学和免疫细胞化学方法对化学合成的肝癌特异性肽A54进行体内体外的鉴定；同时利用免疫荧光细胞化学手段对该肽与肿瘤细胞的结合部位进行了精确地定位：通过生物素-亲和素系统进行的亲和层析筛选肝癌特异性黏附肽的受体蛋白，并利用质谱技术和生物信息学软件进行鉴定和分析。 从实验结果来看，肝癌特异性黏附肽A54具有很好的肿瘤靶向性，对肝组织的结合能力较弱，结合荧光素标记肽A54的细胞定位实验，我们可以判定该肽是与肝肿瘤细胞膜结合的，受体蛋白位于肿瘤细胞膜表面。以肝肿瘤细胞膜蛋白为库，亲和筛选出肝癌特异性黏附肽A54的受体蛋白，经鉴定分析为热激蛋白70家族蛋白，用生物信息学软件Swiss-modeler进行同源模建，展示出该蛋白结构中存在多肽结合区域，由4个反相平行的B折叠和一个α螺旋构成。这个结构域使得HSP70具有分子伴侣功能，能够在肿瘤组织中结合多肽，参与机体的肿瘤免疫应答以及协助肿瘤抗原形成的过程。 筛选到与肿瘤免疫相关蛋白HSP70家族结合的肝癌特异性黏附肽，不仅是A54更多还原

【Abstract】 China is a country of high incidency on liver cancer and more than 110,000 persons died from liver cancer every year which takes up 45% of all the liver cancer deaths in the world. At present, the main means on cancer therapy are surgical treatment, chemotherapy and radiotherapy,on which there are still lots of limitations and side effects.Recently,tumor targeting therapy has sprung up,that is making use of a highly special adhere-tumor stuff as a carrier to bring the medicine to the tumor only and stable the medicinal effects.The tumor’s targeting therapy is becoming the most popular reseach topics on biology and medicine,that will be a new way to tumor therapy.Currently, monoclonal antibody as a major tumor medicine carrier has been accepted clinically.But most of the antibodies are generated from rats,so we have to acknowlege their heterology between rats and human-beings.The rejecting reaction would depress the pharmacodynamic action.We had better find a kind of non-antibody-like carrier which can target mainly tumor tissue but normal tissue.We have panned the targeting peptide to tumor for three times with phage-peptide display technique from the random phage peptide library and got several tumor-specific monoclones. Peptide, as a medicine carrier ,has low weight and strong affinity to the cells.In addition ,they can be cleared from blood and non-targeted tissue and penetrate the targeting cells stably.For obtaining the low weights and small peptide,phage shell should be got rid of from the phage-peptide and the left peptides are synthesized chemically. Tumor related protein or tumor signal protein may be researched through the liver-tumor adhere peptide as the detector.Their biochemical characteristics and integration mechanisms with the peptide may be useful for carrier’s optimization.and the therapy of tumor.This paper shows the identification and localization of liver-cancer adhere peptide by immunohistochemistry or immunocytochemistry methods, and the researchment on the peptide’s receptor by biochemistry and mass chromatography techniques.Bioinformatics software Swiss-modler is used to analyse the result.The experiment results show that the liver-cancer adhere peptide A54 can target to the liver tumor tissue or cells specially but normal liver tissue.Exactly it adheres to the membrane surface of the tumor cells from the immunoflucytochemistry更多还原