靶向性多肽联合多谱段分子内窥镜原位实时诊断胃癌的基础研究

The objective of this proposed research is to develop a real time endoscopic imaging strategy to visualize multiple molecular targets overexpressed by gastric cancer in vivo using highly specific fluorescent-labeled peptides. Specifically, we aim to 1) select a panel of peptides that bind specifically to tissue and protein targets from intestinal-type and diffuse-type gastric cancer by phage display technique; and 2) validate specific binding of this panel of peptides on gastric cancer specimens using a multi-spectral fiber endoscope, measure the apparent dissociation and association time constants, and identify the cell surface targets. Peptides will be selected using phage display technology and a subtractive biopanning strategy against tissue targets from normal and gastriccancer specimens and extra-cellular domain protein targets. The peptides will be labeled with a set of non-spectral overlapping fluorophores (AMCA, FITC, Cy3). The binding affinity of the candidate peptides will be measured by incubating gastric adenocarcinoma and normal epithelial cells with a range of peptide concentrations and measuring binding on flow cytometry. The apparent association time constant will be measured by assessing peptide binding on flow cytometry over various time points. The targets of the candidate peptides will be identified by crosslinking biotinylated peptides that bind to the cell surface associated proteins with either disuccinimidyl suberate or bis-sulfosuccinimidyl-suberate, purifying the products with an affinity column, and performing mass spectrometry analysis.

早期诊断是提高胃癌5年生存率最有效的手段，但是以普通光学胃镜检查为主的筛查方法，不但费时费力而且依从性差。本研究拟建立一种能在内窥镜下快速、实时、原位诊断胃癌的分子影像学方法，该方法应用系列荧光标记多肽使癌变和正常粘膜差异性显像，在多谱段纤维分子内窥镜下识别胃癌细胞的表面标记物，达到快速诊断的目的。我们既往已经应用噬菌体肽库技术筛选出了能与胃癌组织及胃癌标记物CD44分子特异性结合的系列多肽，本研究中我们拟将这些多肽标记上不同波长的的荧光素，应用多谱段纤维分子内窥镜系统，在体外以全胃或胃大部切除标本为研究对象，验证多肽与胃癌组织的结合，计算该方法模拟诊断胃癌的特异性和灵敏度。并用亲和柱层析的方法纯化与部分多肽结合的肿瘤细胞表面未知蛋白分子，最后应用质谱分析法鉴定该蛋白。这些多肽将用于获取胃癌细胞与慢性炎症组织的同期多谱段荧光图，达到在分子内窥镜下早期诊断胃癌的目的，并提高胃镜筛查的效率。

目前，诊断消化系统肿瘤最有效的方法仍是内窥镜直视下组织病理学检测，然而在疾病早期癌前病变的胃黏膜组织仅涉及很小面积的胃黏膜区域，并且无明显的组织形态学改变，普通胃镜很难从形态学角度确定早期病变的位置，因此通过普通光学内窥镜进行胃癌的早期诊断，其能力是有限的。本研究通过噬菌体展示技术筛选胃癌靶向性的多肽探针，有望实现内窥镜下实时诊断胃癌甚至预测胃癌患者的预后。在筛选出的3条多肽探针中，RP-1能够以胃癌细胞所表达的CD44分子特异性结合，具有分子量小、高特异性、高敏感性、高亲和力、高信噪比、低毒性、快速的体内清除速率等生物学特性，其诊断胃癌具有高敏感性及高特异性的特点，并且其阳性染色是患者不良预后的独立预测因子，因而具有实现辅助胃癌靶向诊断的同时预测患者预后的能力，有良好的临床应用前景。