研究目的
To study the physiological process of Bcl-2 protein by designing and evaluating novel small molecule fluorescent probes for detecting and imaging Bcl-2 proteins in living systems.
研究成果
The designed fluorescent probes, especially probes 1 and 3, exhibit potent inhibitory activity, high selectivity, and excellent fluorescent properties for Bcl-2 proteins. They enable effective detection and imaging in cells and tissues, with potential applications in cancer diagnosis and drug screening. Future studies should explore in vivo use and clinical translation.
研究不足
The study may have limitations in probe specificity and sensitivity in complex biological environments, potential off-target effects, and the need for further validation in vivo and clinical settings. Optimization could focus on improving quantum yield and reducing background fluorescence.
1：Experimental Design and Method Selection:
The study involved designing small molecule fluorescent probes (1-3) based on structural modifications of a lead compound to improve binding affinity and utilize aggregation-induced emission (AIE) effects. Methods included fluorescence spectroscopy, binding assays, cytotoxicity tests, cell imaging, flow cytometry, and tissue section imaging.
2：Sample Selection and Data Sources:
Used recombinant Bcl-2, Mcl-1, and Bcl-xL proteins, bovine serum albumin (BSA), cell lines (K562, PC-3, HeLa, MDA-MB-231, HEK293), and mouse tumor and normal tissue sections.
3：List of Experimental Equipment and Materials:
Instruments included Bruker 400 MHz NMR spectrometer, Thermo Varioskan microplate reader, Zeiss Axio Observer A1 and LSM780 microscopes, Agilent 1260 HPLC system, Tecan Infinite M1000 microplate reader, BD FACSCalibur flow cytometer, Olympus Imager fluorescence microscopy. Materials included probes 1-3, AT-101 (positive control), SRB, MitoTracker Red CMXRos, Hoechst 33342, Bcl-2 antibody.
4：Experimental Procedures and Operational Workflow:
Synthesized probes via chemical routes; conducted fluorescence tests in PBS buffer; performed Bcl-2 inhibition assays using TR-FRET and fluorescence polarization; assessed cytotoxicity with SRB assay; imaged cells and tissues with probes and controls; analyzed flow cytometry; conducted immunohistochemistry.
5：Data Analysis Methods:
Analyzed data using statistical methods for IC50 calculation, fluorescence intensity measurements, and image analysis with specified software tools.
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NMR Spectrometer
400 MHz
Bruker
Recording 1H-NMR and 13C-NMR spectra of synthesized compounds.
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Microplate Reader
Varioskan
Thermo
Recording absorption and fluorescence spectra, and conducting fluorescence properties tests.
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Fluorescence Microscope
Axio Observer A1
Zeiss
Obtaining fluorescence imaging of cells.
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Confocal Fluorescence Microscope
LSM780
Zeiss
Obtaining confocal fluorescence imaging of cells.
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HPLC System
1260
Agilent
Performing HPLC examination of compounds.
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Fluorescence Microscopy
Imager
Olympus
Performing fluorescence imaging of tissue sections.
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Microplate Reader
Infinite M1000
Tecan
Used in Bcl-2 inhibition assay with TR-FRET technology.
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Flow Cytometer
FACSCalibur
BD
Analyzing flow cytometry tests on HeLa cells.
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MitoTracker Red CMXRos
Commercial dye
Co-staining for mitochondria in cell imaging.
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Hoechst 33342
Commercial dye
Co-staining for nuclear in cell imaging.
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