【作者】 张彦琼；

【导师】 何剪太；

【作者基本信息】 中南大学 ， 生物医学工程， 2007， 硕士

【摘要】 肝细胞癌（Hepatocellular carcinoma，HCC）是严重威胁人类健康的恶性肿瘤之一，发病率呈不断上升趋势。目前，它的治疗主要以手术、化疗和放疗为主，患者5年生存率不到50％，且不能切除肿瘤的患者5年生存率为0。因而迫切需要有创新性、突破性的早期治疗手段，而肿瘤基因疗法为此提供了一条新思路。其中，以纳米载体作为基因治疗的运载体，已逐渐成为此领域中的一个十分重要的研究方向。然而，无论是离体基因治疗（ex-vivo），还是体内基因治疗（in-vivo），都必须解决如何使纳米载体携带的目的基因能够靶向转移并高效表达的问题。迄今，肝癌基因治疗的效果仍然不能令人满意，缺乏可利用的针对肝癌细胞的高靶向性分子是其重要的障碍。为此，筛选肝癌细胞特异性靶向分子，并将其与纳米基因载体结合，已成为提高纳米基因载体肝癌治疗效果的关键问题之一。为实现纳米基因载体的肿瘤主动靶向治疗，目前主要有两种载体表面靶向修饰的策略可被利用。第一种策略是将能与肿瘤细胞表面高表达受体结合的小分子化合物（如叶酸）作为纳米载体的靶向性分子。第二种策略是通过特异性肿瘤细胞单克隆抗体完成纳米载体与细胞的定向结合。近年来，用能与肿瘤组织高亲和力结合的小分子肽（如RGD）作为靶分子进行药物靶向转运的成功经验，已让我们看到了在此基础上发展第三种纳米载体靶向肿瘤策略的希望。为此，本文首先通过噬菌体展示肽库，筛选获得一批能够与肝癌细胞特异性结合的短肽，并鉴定它们与肝癌细胞的结合特异性。我们的研究为进一步研制用于治疗肝癌的靶向纳米基因载体奠定了初步的实验基础。第一章人肝癌细胞特异性结合短肽的筛选目的：通过噬菌体展示肽库，筛选人肝癌细胞结合短肽，验证它与肝癌细胞特异性结合的能力。方法：（1）以人正常肝细胞（L-02）为阴性消减细胞，人肝癌细胞（HepG2）为筛选靶细胞，对噬菌体随机七肽库(PHD-7^TM)进行全细胞消减筛选。（2）体外筛选4轮后，随机挑取50个阳性噬菌体克隆进行序列测定，并进行短肽序列的同源性分析。（3）以ELISA法鉴定噬菌体与HepG2细胞的结合活性，排除假阳性或无差异结合的噬菌体。根据序列分析及ELISA鉴定的结果，选择其中一个具有较高亲合力的噬菌体克隆进行结合特异性的分析。结果：（1）经过4轮体外筛选，可见噬菌体在靶细胞HepG2上出现明显富集。结果表明，回收噬菌体的滴度由第一轮的0.6×10⁴pfu增加到第四轮的5.856×10⁶pfu，增加了976倍；同时，回收率也显著提高，由第一轮的0.3×10^-7增至第四轮的292.8×10^-7。相对而言，对照细胞L-02的回收率则由第一轮的3.48×10^-7降至第四轮的0.61×10^-7。（2）以蓝白斑实验随机挑取50个携带噬菌体的大肠杆菌克隆，经测序得到各噬菌体单克隆的多肽插入序列（S1～S50），其中，编号为S1的克隆重复14次，S4重复13次，S13重复8次，S8重复3次，S9、S14各重复2次，S20出现1次，因而最终产生7条核苷酸序列，分别为S1、S4、S13、S8、S9、S14、S20。根据氨基酸密码子，将上述插入序列翻译成氨基酸，即为噬菌体展示短肽-HCBP（Hepatoma Cells Binding Peptide）。分别被命名为HCBP1、HCBP4、HCBP13、HCBP8、HCBP9、HCBP14，HCBP20。经BLAST分析发现，在已知蛋白质数据库中未发现与这些短肽序列完全一致或具有较好同源性的蛋白质分子。（3）酶联免疫分析（ELISA）鉴定显示，上述几个阳性克隆在HepG2细胞和L-02细胞上有差异性结合，其中S1噬菌体克隆对两种细胞的结合差异性最大。结合测序分析，本文选择S1噬菌体克隆做进一步鉴定。第二章HCBP1结合短肽特异性的鉴定目的：分析S1噬菌体克隆及人工合成短肽HCBP1与肝癌细胞结合的特异性。方法：（1）通过体外结合实验、免疫细胞化学染色，鉴定S1噬菌体克隆与肝癌细胞结合的特异性；通过免疫组织化学染色，鉴定S1噬菌体克隆与人肝癌组织结合的特异性。（2）人工合成该噬菌体所呈现的短肽，采用竞争ELISA实验证明合成短肽对噬菌体与靶细胞结合的竞争性抑制作用。（3）利用免疫荧光技术观察人工合成肽HCBP1与肝癌细胞的结合特异性。结果：（1）体外结合实验、免疫细胞化学染色及免疫组织化学染色均显示，S1噬菌体克隆对肝癌细胞的结合靶向性明显高于对照细胞，并能与肝癌组织特异性结合。（2）竞争ELISA实验显示，S1噬菌体克隆与HepG2细胞的结合能被其插入序列的人工合成肽HCBP1竞争抑制。（3）免疫荧光显色证实，FITC-HCBP1能特异性地结合于肝癌细胞的胞膜及核周胞浆。结论：（1）本实验获得了7个在HepG2细胞、L-02细胞上有差异性结合的噬菌体克隆（S1、S4、S13、S8、S9、S14、S20），并证实S1噬菌体克隆所呈现的七肽HCBP1能与靶细胞高亲合性结合。（2）本实验证实人工合成的七肽HCBP1能够与肝癌细胞高靶向性结合，它有可能成为抑制肝癌的靶向分子，为进一步研制用于治疗肝癌的高靶向性纳米药物奠定了实验基础。更多还原

【Abstract】 Hepatocellular carcinoma （HCC） is the third leading cause of cancer death in the world because of the lack of targeting drugs which only kill carcinoma cells but not normal cells in chemotherapy. Therefore, tumor-associated moleculars based research have become one of the most promising and actively explored fields for the development of therapeutic agents against HCC and a variety of other tumor types.Chapter one Screening for hepatocellular carcinoma cell specific binding peptidesObjective :To screening the peptides binding specifically to the hepatoma cells using phage display peptides library and study the affinity of these peptides to hepatoma carcinoma cells.Methods :（1）Whole-cell subtractive screening with phage display 7^TM peptide library was performed on HepG2 cells and normal L-02 cells, and the latter were used as controls.（2）After 4 rounds of panning in vitro, 50 phage clones picked randomly were sequenced to identify the consensus sequence.（3）The affinity of 7 phages with hepatoma cells was examined by enzyme-linked immunosorbent assay （ELISA） to exclude false positive clones and those binding with equal affinity to target cells and controls. According to the sequence analysis and ELISA results, one phage clone was selected to program further identifications.Results:（1）After 4 rounds of panning in vitro, phages binding to the HepG2 cells were enriched from 0.6×10⁴pfu at the first round to 5.856×10⁶pfu at the end of the fourth round of panning （with an increase of the 976-fold）; the output/input ratio was also increased from 0.3×10^-7 at the first round to 292.8×10^-7 at the end of the fourth round of panning. Binding of these phages to wild type L-02 cells were decreased.（2） After panning from hepatoma cells in vitro, 50 phage clones were randomly picked and sequenced. 7 sequences were obtained from these 50 clones. S1, S4, S13 were repeated found in the sequences. Among them, S1 was the one with most repeats. Low homology between the peptide sequences displayed by the phages and no known proteins was identified by BLAST analysis.（3） ELISA results suggested that S1, S4, S13, S8, S9, S14, S20 phages could bind differentially to hepatoma cells and control cells. S1 phage was chosen for further analysis.Chapter Two Identification of binding specificity of HCBP1 to Hepatoma cellsObjective : To identify the affinity of S1 phage and peptide HCBP1 to hepatoma carcinoma cells in order to make nano-drug carrier with high target to hepatoma in the future.Methods:（1） In vitro binding assay and immunocytochemical stain were performed to determine the specificity of the phages to hepatoma cells, and immunohistochemical staining was used to examine the binding specificity of the phages to hepatoma cells.（2）Peptides displayed on the phages were synthesized and competitive binding assay was performed to observe the competitive inhibition effect of the peptides with their phage counterparts.（3）Immunoflurescence microscopy was used to study the binding of synthesized peptides to hepatoma cells.Results:（1）In vitro binding assay, immunocytochemical staining and immunohistochemical staining suggested that S1 phage preferably binds to hepatoma cells rather than controls, and phage was also found to be able to bind to hepatoma tissue sections.（2）Competition binding assay revealed a significant competition between the synthesized peptide HCBP1 displayed on the S1 phage and the phage itself while binding to targets.（3）Using immunofluorescence microscopy, fluorescence labeled HCBP1 peptide was seen on the membrane and in the perinuclear cytoplasm of hepatoma cells.Conclusions:（1）7 phage clones binding preferably with hepatoma cells were identified. HCBP1 was binding site of the S1 phage with targets.（2）Synthesized peptide has specificity to hepatoma cells. It may become the targeting molecular to hepatoma and can be used to make nano-drug carriers with high specificity to hepatome in the future.更多还原