人结直肠癌多肽探针的实验研究

【作者】 王瀚；

【导师】 侯颖春；

【作者基本信息】 陕西师范大学 ， 细胞生物学， 2013， 博士

【摘要】 背景：结直肠癌(Colorectal cancer)是一种临床上常见、高发、难于早期诊断、难治愈的消化道恶性肿瘤。早期诊断与治疗对于提高结直肠癌的患者的“五年生存率”具有重要的意义。在我国,随着人民生活水平的不断提高,生活方式及膳食结构的改变,这些年该病的发病率和致死率都有迅猛增高的趋势。由于缺乏有效的早期筛查手段,部分临床病例被延误诊断,丧失最佳的治疗时机。放化疗或者术后放疗及化疗毒性较大,效果不明显。绝大多数病人死于肿瘤的转移。致使手术后患者“五年生存率”降低。目前,通常采用CT(Computed tomography)扫描、纤维结肠镜(Fibrocolonoscopy)、超声波扫描(Ultrasonography)、CT引导下穿刺活检(CT-guided needle biopsy)或乙状结肠检查术(Sigmoidoscopy)等进行结直肠癌的早期筛查或术后效果评估。但部分检查手段分辨能力有限、灵敏性较差。检测的准确程度还取决于操作人员的技术水平。部分检测手段还具一定创伤性,会对患者造成二次伤害。所以如何寻找更灵敏、更安全的检测方法以便提高结直肠癌的早期诊断水平是一个亟待解决的问题。随着现代生物医学工程技术的发展,多种类型的以荧光为光源的高灵敏度、高清晰度的新型内镜相继被开发出来。结直肠癌由于其特殊的解剖特征而给全新的内窥可视影像技术及靶向化疗的有效实施提供了条件。这种诊疗策略实施的关键在于研发与肿瘤细胞及组织具有较高结合特异性和敏感性的靶向分子,进而展开分子探针及靶向化疗药物载体的开发。目前,一些通过噬菌体肽库筛选技术得到的特异性多肽分子以其较好的靶向性、较低的免疫原性以及较小的毒副作用,引起了人们的普遍关注。这些多肽分子一方面可有望用于靶向分子探针的创制。另一方面,通过偶联相应的化疗药物有望用于药物的靶向递送,从而有效改善化疗药物对患者正常组织或细胞所产生的毒副作用。目的：为了获得具有较好靶向性的多肽分子探针用以结直肠癌的早期诊断,本研究拟通过噬菌体展示技术筛选出能够与人结肠癌细胞Caco-2及人结直肠癌组织特异性结合的阳性噬菌体克隆,在此基础上获得特异性多肽分子,荧光标记后进一步研究其与细胞及组织的结合特异性,为进一步创制开发结肠癌特异性分子探针、后续用于纳米粒子耦联、药物耦联等具有广泛用途的研发奠定基础。方法：本研究利用噬菌体肽库筛选技术,以人结直肠癌细胞Caco-2细胞为靶细胞、人胚肾细胞HEK293细胞为阴性吸附细胞,进行四轮严格的消减筛选,随机挑选30个阳性克隆并分别进行扩增后进一步利用ELISA的方法进行阳性克隆的鉴定。然后将获得的阳性克隆进行DNA测序,根据噬菌体克隆基因中所插入的核酸序列推导出相应的多肽序列。对所得的四条多肽序列进行生物信息学检索。通过免疫荧光技术进一步鉴定所获得的阳性克隆,在此基础上进一步利用人结肠癌组织冰冻切片进行组织免疫荧光实验。获得了与细胞和组织结合能力较强的阳性克隆SP-2。采用FMOC固相合成法合成噬菌体克隆SP-2的展示肽序列,N端标记荧光素FITC后作为多肽分子探针Caco probe,以荧光显微镜及激光共聚焦扫描显微镜为主要手段对其与细胞及组织的结合特异性进一步表征。结果：1.我们通过优化改良的噬菌体肽库筛选技术获得了一个可与人结直肠癌细胞和临床肿瘤组织敏感、特异结合的噬菌体克隆SP-2。2.经多层次、多类型检测手段的检测,多肽分子探针Caco probe与人结直肠癌Caco-2细胞及组织的结合具有较好的特异性和敏感性。3.研究表明,多肽分子探针Caco probe能够特异结合于人结肠癌细胞Caco-2的细胞膜,但具体靶位尚有待深入研究。4.经组织芯片方法检测,多肽分子探针Caco probe与人结肠癌组织具有较好的结合特异性和敏感性,阳性率可达70%。结论：1.本研究通过优化改良的噬菌体肽库筛选技术成功地从噬菌体随机12肽库中筛选获得了与人结肠癌细胞和组织特异性结合的阳性噬菌体克隆。通过进一步鉴定,阳性噬菌体克隆SP-2(CVSVGMKPSPRP)为最佳克隆。2.荧光素FITC标记后的多肽分子探针Caco probe可特异结合于人结肠癌Caco-2细胞和人结肠癌组织。3.所获靶向多肽分子为结直肠癌的早期诊断、靶向化疗及抗肿瘤短肽的开发开辟了新的途径,具有十分重要的应用价值。更多还原

【Abstract】 1. BackgroundColorectal cancer is one of the most common malignant neoplasms in the digestive system. With the development of economy, people’s living standard, life-style and diet structure are changing. In recent years, the incidence and mortality of colorectal cancer is growing at a fast rate in China.At present, imaging examinations still play important roles in the early diagnosis of colorectal cancer, including computed tomography, fibrocolonoscopy, ultrasonography, CT-guided needle biopsy and sigmoidoscopy. However, limited sensitivity may occasionally lead to misdiagnosis. Cancer survival tends to be poorer in developing countries, most likely because of a combination of a late stage at diagnosis and limited access to timely and standard treatment. Thus, it is necessary to develop some more sensitive and safe methods for the early diagnosis of colorectal cancer.Advances have been made in the development of novel endoscopic instruments that are sensitive to fluorescence. Colon is a hollow organ so that it is convenient to be visualized directly by colonoscopy and performed targeted chemotherapy. Targeting imaging requires the detection of molecular targets. However, such targets are too small to be visualized directly by fluorescence colonoscopy, thus probes that are fluorescence labeled are needed to be developed.Currently, some targeting peptides selected from phage display have aroused wide concern due to their better properties, including better targeting, low immunogenicity and toxicity.2. PurposeTo select a novel peptide for the development of sensitive peptide probe, we used an improved subtractive phage display technology to identify positive phage clones that can bind specifically to Caco-2cells and colorectal cancer tissues. The synthetic peptide, when conjugated to fluorescein, has great potential to serve as a novel probe for the detection of CRC.3. MethodsA12-mer phage display library was used to select peptides that bind specifically to the human colorectal cancer cell line Caco-2. The human embryonic kidney cell line HEK293was used as negative cells. After four rounds of panning,30positive phage clones were selected and sequenced. Moreover, we performed various systematic approaches for the selection, characterization and validation of an affinity peptide that target Caco-2cells, including ELISA, immunofluorescent staining, competitive inhibition assay and fluorescence imaging. Positive phage clone SP-2was identified and its displayed peptide was deduced. A novel peptide probe named Caco probe was produced when combined with FITC. The specificity of peptide probe to Caco-2cells and colorectal cancer tissues was further studied by using fluorescence imaging.4. Results1. The novel phage clone SP-2that is sensitive and specific to Caco-2cells and colorectal cancer tissues has been selected via a modified assay.2. This peptide probe exhibited a high specificity to Caco-2cells and colorectal cancer tissues via multi-level and multi-type examinations.3. The peptide probe can specifically bind to the membrane of Caco-2cells.4. A tissue chip containing24cases tissue samples were performed to further evaluate its binding specificity. The results showed that the binding rate was approx.70%, which demonstrated this peptide probe had higher specificity to colorectal cancer tissues.5. Conclusions1. The phage clone SP-2was identified as the best positive candidate that specifically bind to Caco-2cells and colorectal cancer tissues.2. The FITC-labeled peptide probe can specifically bind to Caco-2cells and colorectal cancer tissues.3. These studies suggest that this peptide may be a promising lead candidate in the development of a useful colorectal cancer diagnostic and targeted drug delivery agent.更多还原