研究目的
To develop a near-infrared fluorogenic probe for specific detection and non-invasive imaging of neutrophil elastase (NE) in vitro and in vivo, enabling monitoring of NE activity in biological systems for understanding its role in inflammatory diseases and clinical diagnosis.
研究成果
NEP is a highly specific near-infrared fluorogenic probe for NE, enabling rapid detection in vitro and in vivo. It allows monitoring of NE trafficking, uptake, and up-regulation in cells, and successful imaging in ALI model mice, making it a valuable tool for clinical diagnosis and pathological studies of NE-related diseases.
研究不足
The probe may not have specificity for lung tissue and could accumulate in multiple organs; the free fluorophore might be quickly metabolized. Specificity against all possible interferents not fully tested, and in vivo distribution not fully characterized.
1：Experimental Design and Method Selection:
The study involved designing a small-molecule near-infrared fluorogenic probe (NEP) for NE detection, based on a hemicyanine dye fluorophore with pentafluoroethyl recognition group. Methods included organic synthesis, fluorescence spectroscopy, cell culture, and in vivo imaging.
2：Sample Selection and Data Sources:
Human non-small cell lung carcinoma cells (A549), rat basophilic leukemia cells (RBL-2H3), and C57 and nude mice were used. Biological samples included mouse serum and bronchoalveolar lavage (BAL) fluid.
3：List of Experimental Equipment and Materials:
Equipment included microplate reader (SpectraMax M5), fluorescence microscope (Olympus IX71), in vivo imaging system (IVIS Lumina XRMS III), NMR spectrometers (Varian Mercury 400/600 MHz), GC-MS, HRMS. Materials included silica gel, solvents, enzymes (e.g., human NE), inhibitors (e.g., sivelestat), and cell culture media.
4：Experimental Procedures and Operational Workflow:
Synthesis of NEP and intermediates, optimization of pH and temperature for NE detection, specificity tests with various enzymes, cell culture and imaging with NEP and inhibitors, in vivo imaging in ALI model mice induced by LPS.
5：Data Analysis Methods:
Fluorescence intensity measurements, statistical analysis using t-tests, image analysis with Image J software.
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fluorescence microscope
Olympus IX71
Olympus
Used for acquiring fluorescence images of cells.
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in vivo imaging system
IVIS Lumina XRMS III
PerkinElmer
Used for in vivo fluorescence imaging in mice.
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microplate reader
SpectraMax M5
Molecular Devices
Used to measure UV-Vis absorption and fluorescence spectra.
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cuvette
Hellma
Used for holding samples during spectral measurements.
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96-well microplate
Corning
Used for inhibitory studies and fluorescence measurements.
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GC-MS
Used for characterizing intermediates via mass spectrometry.
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NMR spectrometer
Varian Mercury 400 MHz Plus, Varian Mercury 600 MHz Plus
Varian
Used for 1H and 13C NMR characterization of compounds.
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HRMS
AB SCIEX TOF/TOF TM 5800 System
AB SCIEX
Used for high-resolution mass spectrometry characterization.
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cell culture dish
NEST
Used for cell culture and imaging experiments.
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Image J software
National Institutes of Health
Used for analyzing fluorescence image data.
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