新型Plectin-1荧光、MRI靶向分子探针对胰腺癌早期诊断的实验研究

Molecular imaging make the early detection of molecular abnormalities in the process of cancer possible in vivo. Recent studies display that Plectin-1 is overexpressed in human pancreatic cancer and metastasis,but undetectable in normal pancreas. The expression of Plectin-1 associated closely with pancreatic cancer, so we choose Plectin-1 for imaging targets. Firstly, we established dual-mode targeted probe with fluorescent and magnetic Nano-MR imaging (Cy5.5-anti- Plectin-1-PEG/SPIONs). The morphology and functions of the probe were tested by transmission electron microscope, X-ray diffraction, vibration magnetometer, Fourier transform infrared,et al.And the vitro toxicity and pharmacokinetic test were performed with pancreatic cancer cells PANC-1 ,as well as in normal islet cells and fibroblast for comparison. Secondly,the nontargeted and targeted molecular contrasts were injected intravenously into animal models of pancreatic cancer. Finally,we collected the specimen of tumors,surrounding tissues and other major organs,and detected the pathological types and distribution of probe arroud cells by HE dyeing, Prussian blue staining and Western blotting, and then, the results of targeted enhancement in animal models of pancreatic cancer were compared.The research aims to achieve early diagnosis at the molecular level in pancreatic cancer,which is based on the Plectin-1 targeted molecular imaging in vivo, dynamic monitoring molecular abnormalities of pancreatic cancer.

分子影像使活体、早期发现肿瘤癌变过程中的分子异常成为可能。研究发现Plectin-1在胰腺癌及转移灶中高表达，在正常胰腺中不表达，表明Plectin-1与胰腺癌关系密切。本项目选择Plectin-1为成像靶点，首先构建荧光、纳米粒双模态靶向探针，采用透射电镜、X线电子衍射、振动样品磁强计、傅立叶红外等方法，对该探针进行性能检测；并将该探针和胰腺癌细胞PANC-1进行系列体外毒性试验和药代动力学试验，并和正常胰岛、成纤维细胞等对照；其次，将未标记和Plectin-1标记的探针注入胰腺癌鼠模型体内，进行活体荧光及MR显像；最后，使用HE染色、普鲁士蓝染色和Western印迹法等技术，检测离体标本肿瘤、瘤周组织的病理类型及探针分布状况，并与鼠模型的靶向成像结果进行对照分析。本研究通过Plectin-1靶向分子成像，活体、动态监测胰腺癌早期分子水平的异常，达到实现对胰腺癌早期诊断之目的。

针对目前胰腺癌仍是临床诊治最为困难的实体肿瘤，早期诊断较为罕见，60%患者在确诊时已经发生远处转移，20%为局部晚期，不能行根治性切除术，中位生存期为6-9个月。因此，重视胰腺癌的早期诊断，可以改善胰腺癌的治疗和预后，使胰腺癌的总体诊疗状况得到改观。.本项目基于可以活体早期发现肿瘤癌变过程分子异常的分子影像，选择在胰腺癌及转移灶中高表达、在正常胰腺中不表达的Plectin-1为靶标。构建了靶向Plectin-1的荧光/磁共振双模态靶向分子诊断探针，探针以氧化铁纳米颗粒（10nm）为磁共振成像核心，修饰可体内长循环高分子化合物聚乙二醇（PEG），表面偶联Cy5.5荧光试剂和靶向分子anti-Plectin-1。 采用透射电子显微镜、动态光散射仪、振动样品磁强计等进行探针物化性质和结构性能的表征，探针呈均匀球状，水动力尺寸在100nm以下，不同pH和长时储存条件下探针样品稳定，临床磁共振扫描仪检测探针r2弛豫率为116mM-1s-1。.由于增生、瘤变到癌变的过程Plectin-1的表达逐步升高，因此利用Plectin-1靶向可以实现胰腺癌组织靶向和主动靶向胰腺癌细胞，达到诊断胰腺癌的目的。通过体外细胞实验（包括细胞靶向检测、普鲁士蓝染色，细胞透射电子显微镜、细胞毒性检测、细胞凋亡流式细胞仪检测等）验证了探针的体外靶向性和促胰腺癌细胞凋亡的作用。.首先验证了探针在活体正常小鼠体内中毒性较小，然后通过构建体内原位荷BxPC-3胰腺癌裸鼠模型，荧光/磁共振成像从活体水平发现探针在体内具有良好的胰腺癌靶向性，最后通过免疫组化等检测探讨了胰腺癌组织与探针作用后的病理改变和探针分布情况，与成像结果进行对照分析。本课题的顺利实施，为早期诊断胰腺癌、动态监测胰腺癌早期分子水平异常提供了新的思路与实验依据。