共聚焦激光显微内镜对胃癌血管特异性结合短肽GX1体内分子成像的研究

In vivo endomicroscopic molecular imaging represents a powerful tool for the immediate diagnosis and targeted treatment of early gastrointestinal cancer. This study was designed to explore the feasibility of confocal laser endomicroscopy (CLE)for in vivo molecular imaging of gastric cancer by tergeting a specific short peptide GX1 in live human gastric cancer cell lines, and then to evaluate in vivo molecular imaging in tumor-bearing mice and live human tissues.① In order to observe whether specific combination of GX1 peptide can occur, confocal endomicroscopic images are obtained for live Human Umbilical Vein Endothelial Cells (HUVEC), human gastric cancer cell line SGC7901, Co-HUVEC cells and normal gastric cell line GES-1 using the probe confocal endomicroscopic system FIVE1; ② Perform in vivo molecular imaging in tumor-bearing mice and surgical orthotopic implantations of gastric cancer to assess in vivo molecular imaging of GX1 using the probe confocal endomicroscopic system FIVE1; ③ Spray fluorescein labelled GX1 peptide on fresh human tissue of gastric cancer, and observe molecular imaging of GX1 receptor using probe confocal endomicroscopic system FIVE1 and CLE. In short, this is the first study using CLE for in vivo targeted molecular imaging of vascular endothelial cells in gastric cancer, which could contribute to enhanced diagnosis of gastric cancer and accurate selection of GX1 receptor positive patients.

显微内镜分子成像技术作为在体诊断和治疗消化道早期肿瘤的新途径，近年来 开始得到了广泛关注。本研究拟通过细胞、动物模型及人体新鲜胃癌组织3个方面逐步深入探讨利用共聚焦激光显微内镜（CLE）对胃癌血管特异性短肽GX1体内分子成像的可行性：①在探头式共聚焦激光显微内镜FIVE1 系统协助下体外实时观察人脐静脉内皮细胞（HUVEC）、人源性胃癌细胞系SGC7901、Co-HUVEC细胞及正常胃黏膜上皮细胞系的GX1特异性结合情况；②利用探头式共聚焦显微内镜FIVE1 系统，实现对胃癌动物模型GX1特异性结合情况的活体内分子成像；③在体外对人体新鲜胃癌组织及非癌组织表面喷洒荧光标记的GX1探针，利用探头式共聚焦显微内镜FIVE1 和共聚焦激光显微内镜对其GX1受体的表达情况进行分子成像。本研究首次提出应用CLE对胃癌血管内皮细胞进行活体内靶向分子成像，对胃癌的实时诊断与将来的靶向治疗具有重要意义

显微内镜分子成像技术是在体诊断和治疗消化道早期肿瘤的新途径。本研究通过细胞、动物模型及人体新鲜胃癌组织3个方面逐步深入探讨了利用共聚焦激光显微内镜（CLE）对胃癌血管特异性短肽GX1体内分子成像的可行性，现已顺利完成了预期研究计划，并取得了如下研究结果：⑴流式细胞术检测结果显示本研究中所使用的BGC823和SGC7901胃癌细胞系均存在GX1特异性受体的表达。CLE观察与fluorescein-GX1共孵育后的活的胃癌细胞，发现细胞膜和胞浆存在特异性荧光信号。荧光显微镜和免疫组织化学方法与CLE成像结果基本一致。⑵荷瘤鼠整体动态荧光成像观察结果显示，尾静脉注射fluorescein-GX1多肽（1μg/g体重）避光饲养24小时后可见肿瘤部位特异性的荧光信号，且在48小时达最强。CLE在体观察肿瘤表面均可见特异度荧光信号。体外免疫组化结果和台式荧光显微镜观察冰冻切片的图像与体内CLE图像相似。⑶尾静脉注射fluorescein标记的重组融合蛋白GX1-rmhTNFα的荷瘤鼠的肿瘤体积显著小于fluorescein-rmhTNFα，fluorescein-GX1-rmhTNFα组荷瘤鼠体重显著大于fluorescein-rmhTNFα组。于造模后第20天应用CLE观察荷瘤鼠肿瘤部位，fluorescein-GX1-rmhTNFα组肿瘤部位有特异性结合的荧光信号，fluorescein-rmhTNFα组肿瘤部位有较弱的特异性结合的荧光信号；fluorescein-GX1组亦有特异性结合的荧光信号。⑷ 人体活组织体外研究：共纳入6例低分化胃腺癌和4例高分化胃腺癌患者，CLE观察上述胃癌患者手术切除的新鲜肿瘤组织结果表明，胃癌组织有特异性结合的荧光信号，非肿瘤组织表面无特异性结合的荧光信号。本研究首次应用CLE对胃癌血管内皮细胞进行活体内靶向分子成像，对胃癌的实时诊断与将来的靶向治疗具有重要意义。此外，在本项目资助下，我们还建立了探头式CLE（pCLE）的胃小凹及微血管分型标准，为实时诊断胃黏膜癌前病变及早期胃癌提供了新的有力工具。