Preparation Of Aptamer Fluorescent Probes For The Identification Of MUC-1 And Its Preliminary Application In Pancreatic Cancer

Objective: Traditional antigen-antibody reaction plays an important role in modern biomedical detection technology because of its outstanding sensitivity and specificity. But as a probe molecule, the antibody is easily affected by pH, temperature and other environmental factors, and the production cost is high. Aptamer is a single stranded DNA or RNA oligonucleotide that has the same specificity and sensitivity with the antibody, and smaller than the antibody molecules, synthesis easier, better stability. In the near future, aptamer as a molecular probe will be widely used in biomedical in vivo, in vitro testing and treatment. In this study, we designed and constructed a aptamer fluorescent probe that can specifically recognize MUC-1 to detect the cell lines expressing of MUC-1. The study of this aptamer probe for fluorescence staining of pancreatic cancer tissue sections and in vivo targeted diagnosis of pancreatic cancer were also performed.Materials and Methods:1. The synthesis and charaterization of anti-MUC-1 aptamer probe: the 3’- azide modified DNA and alkynyl-modified Rhodamine B were covalently linked by “click reaction” high productively. The crude product was purified by size exclusion chromatography(SEC) monitored byγ-counter and UV-vis.2. Application of anti-MUC-1 fluorescently labeled aptamer in pancreatic cancer:(1) Anti-MUC-1 fluorescently labeled aptamer for identifying pancreatic cancer cell lines:Anti-MUC-1 fluorescently labeled aptamers were incubated with CFPAC-1, BXPC-3, A549, U251 and HPDE6-C7, and the results were compared with the experimental results of anti MUC-1 monoclonal antibody to verify the specificity of the probe.(2) Anti-MUC-1 fluorescently labeled aptamers were used for fluorescence staining of pancreatic carcinoma tissue sections:The nude mice model of pancreatic cancer(BXPC-3, CFPAC-1) was established. The tumor tissue was taken out when the tumor grew to a diameter of 1-1.5cm then paraffin-embedded and sliced. Anti-MUC-1 fluorescently labeled aptamers and anti-MUC-1 monoclonal antibodies were used for fluorescence staining and immunohistochemical analysis of tumor tissue sections. At the same time, the effects of probe concentration, incubation temperature and time, sealing fluid on the fluorescence staining were also observed. We also study Aptamer fluorescent probes and anti MUC-1 monoclonal antibodies for histopathological analysis of tissue sections from 72 cases of pancreatic cancer.(3). Anti-MUC-1 fluorescently labeled aptamers for in vivo imaging diagnosis of pancreatic cancer:The nude mice model of pancreatic cancer(CFPAC-1) was established. The aptamer fluorescent probe(150 ul, about 1.5 nmol) was injected into the nude mice by tail vein injection. Then the fluorescence imaging effect of the tumor was detected by vivo in small animal in vivo imaging instrument.Results:1. The Chracterazation of Anti-MUC-1 fluorescently labeled aptamers: the intermediate and final product was characterized with NMR and mass spectrum: 1H-NMR、13C-NMR、MALDI-ESI.2. Application of anti-MUC-1 fluorescently labeled aptamers in pancreatic cancer(1) Anti-MUC-1 fluorescently labeled aptamers for identifying pancreatic cancer cell lines:The labeling results of aptamer fluorescent probes were consistent with the anti MUC-1 monoclonal antibody: CFPAC-1 was strongly positive, BXPC3 was positive, HPDE6-C7 was weak positive, and U251 was negative(2) Anti-MUC-1 fluorescently labeled aptamers were used for fluorescence staining of pancreatic carcinoma tissue sections. Aptamer probe fluorescence analysis of tumor tissue sections from the nude mice model of pancreatic cancer was consistent with the anti MUC-1 monoclonal antibody: CFPAC-1 cancer was strongly positive, and BXPC-3 was positive. However, aptamers substituting for antibodies in histopathological protocols showed non-specific staining which can be reduced with heparin. Results of fluorescence staining and immunohistochemical analysis of tumor tissue sections : With 72 pancreatic cancer patients, Positive identification of Aptamer fluorescent probes 64(90%); positive identification of anti MUC-1 monoclonal antibodies 60(85%); 56(79%) of all positive cases.(3) Anti-MUC-1 fluorescently labeled aptamers for imaging diagnosis of pancreatic cancer in vivo:Obvious fluorescence signal was visible in the nude mice, but no significant fluorescence signal was found in the tumor site.Conclusions:1. In our study, an Aptamer fluorescent probe which can specifically recognize the MUC-1 was succesfully established.2. The MUC-1 specific Aptamer fluorescent probe can specifically identify the cell lines expressing MUC-1. The positive rate of fluorescence staining of aptamer for pancreatic cancer tissue sections was higher than that of anti MUC-1 monoclonal antibody.3. Aptamer probe histologic diagnosis of pancreatic cancer, experimental conditions with 4 ℃, incubation 12 hours and 20 nm, heparin sodium closed for good, and heparin can significantly reduce the fluorescent dye non-specific background of the organization.4. The MUC-1 specific Aptamer probes need to be further modified to be used for the diagnosis of pancreatic cancer in vivo...