Study On Application Of MMP-2 Molecular Probe In Detection Of Colorectal Cancer

Objective:A specific fluorescent molecular probe for matrix metalloproteinase-2(MMP-2)was designed and constructed,and the rapid and accurate detection of colorectal cancer tissues in vitro by molecular imaging was preliminarily developed.Methods:In this study,we had designed and synthesized a novel fluorescence probe Dab-GPLGVRGY-FITC for specific detection of matrix metalloproteinase MMP-2 activity.The probe was synthesized by a copper(I)-catalyzed "click" reaction of alkyne-functionalized MMP-2 peptide substrate(GPLGVRGY-Pra)with Fluorescein isothiocyanate azide(FITC-N3)followed by amidation with Dabcyl-NHS(DABCYL acid,SE).After the probereacted with recombinant MMP-2,Furin,DNase and other solutions,respectively,the fluorescence signals at 520 nm were detected to evaluate the specificity and detection sensitivity of the probe.The cytotoxicity of the molecular probe was preliminarily evaluated by MTT method.Then,human colon cancer cell LoVo and normal gastric mucosal epithelial cell GES-1 incubated with the molecular probe and differentiated by fluorescence confocal microscopy.Finally,15 cases of clinical colorectal cancer and adjacent tissues reacted with themolecular probe,differentiation between colorectal cancer and paracancerous tissue were observed by fluorescence spectroscopy and fluorescence confocal microscopy.Results: 1.The molecular probe Dab-PLGVRGY-FITC was prepared by coupling a series of chemical reactions from GPLGVRGY-Pra,FITC-N3 and DABCYL acid,and confirmed by high-resolution mass spectrometry.2.After the probe Dab-PLGVRGY-FITC reacted with recombinant MMP-2,the fluorescence signal was significantly enhanced,andthe fluorescence intensity increased linearly with the increase of MMP-2.When the molecular probe interacted with Furin,DNase,RNase,BSA and MMP-2,respectively,only the fluorescence signalof MMP-2changed obviously.3.The MTT results showed that the molecular probe had low toxicity to cells and could be applied to the research of living cell imaging.After incubation of LoVo and GES-1 cells with molecular probes,the cells were imaged by fluorescence confocal microscopy,Lo Vo showed strong green fluorescence,while GES-1 showed almost no fluorescence signal.4.Using fluorescence spectroscope detection and fluorescence confocal microscopy to observe the changes of fluorescence signalsafterclinical colorectal cancer and paracancerous tissue reacted with the molecular probe,and we foundthe resultsshowed that the fluorescence signals of colorectal cancer tissues were significantly stronger than the paracancerous tissues.Conclusion:In this study,a novel fluorescent molecular probe Dab-PLGVRGY-FITC,which can be specificallyidentified by MMP-2,was designed and constructed.This molecular probe presentedhigh specificity and sensitivity in the detection of MMP-2,andlow cytotoxicity.This molecular probecontributed to not only specific imaging of MMP-2 overexpression in human colon cancer cell LoVo,but also imaging differentiation between clinical colorectal cancer and paracancerous tissues.