筛选鉴定CD44多个结构域特异性多肽用于胃癌多光谱靶向内镜诊断的研究

The traditional endoscopy，which is based on morphology，has some limitations in early diagnosis of gastric cancer, while tumor specific molecular probes can make up the deficiency with targeted endoscopy. CD44 molecules with different regions encoded by different exons have different sensitivity and specificity in gastric cancer diagnosis。Probes targeting pan-CD44 has higher sensitivity of precancerous lesions while probes targeting CD44v has higher specificity of gastric cancer. Imaging of gastric caner using mixture of these probes and muti-spectral endoscopy can enhance the signal, as well as sensitivity and specificity. To increase the screening efficiency, we reconstitute CD44 into nanodiscs that were screened on with a phage display library for several peptides targeting different regions of CD44. Peptides were synthesized, and their affinity, specificity, pharmacokinetic properties and ability in evaluation of prognosis were tested on cell, tissues and xenotransplanted tumor models. An in vivo endoscopy simulation experiment is carried on orthotopic tumor models. Last, human fresh specimens of gastric cancer is investigated using mixture of probes targeting different regions of CD44 and muti-spectral endoscopy. We evaluate the diagnostic value of this method to establish an in situ, real-time, efficient endoscopy diagnostic approach with both high sensitivity and specificity.

肿瘤特异性分子探针结合靶向内镜对胃癌显像，相对传统形态学为基础的内镜更易早期、准确地发现肿瘤病灶。CD44是重要的胃癌标记物，对多种胃癌前病变具有高灵敏性，而包含可变型外显子编码区的CD44v则对胃癌组织有高度特异性，将二者探针联合应用，使用多光谱靶向内镜同时对病变成像，可以弥补单一探针信号弱、灵敏性特异性无法兼顾的问题。Nanodisc技术可以体外模拟膜蛋白的高级结构，本课题使用该技术包装CD44及CD44v蛋白，采用改良噬菌体展示技术筛选多个CD44结构域多肽，合成相应探针，并在细胞、组织、动物模型多水平验证多肽的亲和力、特异性、药代动力学特性及胃癌预后评估价值。采用原位移植瘤动物模型模拟内镜检查，并在离体胃癌切除标本中使用多条探针复合，联合多光谱共聚焦内镜实行检查，评估其实际意义。旨在建立一种实时、原位、高效，兼顾诊断灵敏性及特异性的内镜分子影像方法，提高胃癌早期筛查效率。

肿瘤特异性分子探针结合靶向内镜对胃癌显像，相对传统形态学为基础的内镜更易早期、准确地发现肿瘤病灶。CD44是重要的胃癌标记物，对多种胃癌前病变具有高灵敏性，而包含可变型外显子编码区的CD44v则对胃癌组织有高度特异性，将CD44及CD44v探针联合应用，使用多光谱内镜同时对病变成像，可以弥补单一探针信号弱、灵敏性特异性无法兼顾的问题。Nanodiscs技术可以体外模拟膜蛋白的高级结构，使用该技术包装CD44v进行噬菌体展示技术，可在模拟体内CD44v的高级结构的同时保障筛选的特异性。本研究成功组装CD44v3-v10至Nanodiscs，使用基于Nanodiscs的噬菌体展示技术筛选结合于CD44v6的亲和噬菌体。同时，采用过表达细胞同时进行了噬菌体展示筛选结合于CD44v6的亲和噬菌体。使用ELISA、噬菌体免疫荧光试验多方法选择最佳序列，合成多肽。蛋白水平验证多肽结合，计算解离常数。细胞水平从靶蛋白过表达HEK-293细胞、天然表达靶蛋白胃癌细胞多角度，采用免疫荧光、流式细胞多方法验证多肽结合及结合特异性。使用组织芯片及临床标本评估探针对胃癌的结合，评估前期筛选探针的诊断效力，对胃癌预后的评估作用，对不同胃及胃癌组织的结合情况，对胃癌血清的检出作用。收集胃癌切除标本，复合多标记探针，模拟多光谱下探针检测。构建胃癌动物模型，体内检测探针的结合、代谢及生物分布情况。本项目采取新的噬菌体展示方法，获取多条CD44特殊结构域结合肽，经临床前期验证，获取的多肽探针有体内外对胃癌的特异性结合能力，有靶向诊断治疗，评估预后的应用前景。经模拟内镜光学显像试验证明，可能借此研究建立一种实时、原位，兼顾诊断灵敏性及特异性的内镜分子影像方法，从而改善胃癌早期的筛查效率。