Application of fluorescent probe in simultaneous imaging of mitochondria and nucleolus

Abstract

The invention discloses an application of a fluorescent probe in preparation of a reagent for simultaneously imaging mitochondria and nucleolus. The fluorescent probe is (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2) vinyl) pyridine-1-iodised salt. In addition, the probe and a mitochondrial red probe can be used for mitochondrial positioning at the same time. Mitochondria and nucleolus are closely related to diagnosis of various diseases (cancers and the like) and drug research and development. Therefore, the fluorescent compound provided by the invention can provide a more accurate and effective tool for research and application in the fields of chemical industry, biological medicine and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to an application of a fluorescent probe in simultaneously imaging mitochondria and nucleoli. Background Art

[0002] Mitochondria are the power plants of cells and are extremely important in living organisms. Mitochondria contain a large number of oxidases and are the main place for cells to carry out aerobic respiration. They can oxidize and decompose organic matter and release energy. This energy can be converted into ATP (adenosine triphosphate) to provide power for the life activities of cells. Mitochondria contain a large number of acidic substances such as citric acid and malic acid, which can promote the acid-base balance in the body and help maintain the stability of the internal environment in the body. Mitochondria can participate in the metabolic process of fat, protein, sugar and other substances in the body, and play an important role in maintaining the normal physiological functions of organisms. Mitochondria can maintain cell functions. If mitochondria are abnormal, it may cause damage to cell functions, resulting in the inability of cells to grow and divide normally.

[0003] The nucleolus is the most prominent structure in the interphase nucleus of eukaryotic cells. The nucleolus usually appears as a single or multiple homogeneous spherical bodies located in the cell nucleus. There are a variety of ribosomal proteins and rRNAs in the nucleolus, which are assembled in the nucleolus to form ribosomal subunits. Ribosomes are important sites for protein synthesis. The size, shape and number of nucleoli vary with the species, cell type and cell metabolic state of the organism. Abnormal nucleolar function may lead to uncontrolled cell proliferation and thus induce tumorigenesis. Nucleolar dysfunction may cause abnormal RNA structure or function, leading to errors in protein synthesis. Therefore, the nucleolus is of great significance in the fields of biology and medicine, and plays an important role in cell cycle regulation, RNA processing, ribosomal subunit synthesis and assembly, signal transduction, and growth factor-mediated biosynthesis.

[0004] Fluorescent probes have attracted widespread attention due to their ability to observe cells in situ and in real time. However, there are relatively few fluorescent probes that can simultaneously image nucleoli and mitochondria. Currently, there are relatively few such probes, and it is particularly important to develop a probe that can simultaneously image mitochondria and nucleoli. This will provide a more accurate and effective tool for research and application in the fields of chemical industry, biomedicine, etc. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an application of a fluorescent probe in simultaneous imaging of mitochondria and nucleoli.

[0006] Technical solution: The present invention provides an application of a fluorescent probe in simultaneous imaging of mitochondria and nucleoli, wherein the fluorescent probe is (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt, and the structural formula is:

[0007]

[0008] Furthermore, the probe concentration is 15-25 μM.

[0009] A dual-color co-localization mitochondrial reagent, wherein the mitochondrial green fluorescent probe in the reagent is (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt.

[0010] Furthermore, the excitation wavelength of the mitochondrial green fluorescent probe is 488 nm, and the fluorescence collection wavelength is 500-600 nm.

[0011] Furthermore, the reagent also includes a mitochondrial red fluorescent probe.

[0012] Furthermore, the mitochondrial red fluorescent probe is MTDR.

[0013] Furthermore, the excitation wavelength of the mitochondrial red fluorescent probe is 635 nm, and the fluorescence collection wavelength is 640-740 nm.

[0014] A two-color co-localization mitochondrial method is provided, wherein the cells to be tested are incubated once in a culture medium containing (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt, and then a mitochondrial red fluorescent probe is added for a second incubation.

[0015] Furthermore, the incubation time is 30 minutes.

[0016] Furthermore, the secondary incubation time is 30 minutes.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention discloses (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt as a fluorescent probe that can simultaneously image mitochondria and nucleoli, and can also be used for mitochondrial localization together with the mitochondrial red probe. Since mitochondria and nucleoli are closely related to the diagnosis and drug development of various diseases (cancer, etc.), the fluorescent compounds of the present invention can provide more accurate and effective tools for research and application in the fields of chemical industry, biomedicine, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The absorption and fluorescence spectra of PP-1 (10 μM) in different solvents.

[0019] Figure 2 The absorption and fluorescence spectra of PP-1 (10 μM) in solutions with different pH values.

[0020] Figure 3 Confocal fluorescence images of normal HeLa cells stained with PP-1 (5 μM, 30 min) and PP-1 (20 μM, 30 min), respectively.

[0021] Figure 4 Confocal fluorescence images of active HeLa cells co-stained with PP-1 (20 μM, 30 min) and MTDR (2 μM, 30 min). DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0023] The absorption spectrum test instrument used in the examples is a Hitachi U-2910 spectrophotometer; the fluorescence spectrum instrument is a Hitachi F-2700 spectrophotometer, and the cell imaging instrument is a Lecia confocal microscope.

[0024] Example 1 Synthesis of fluorescent compounds

[0025] (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt (i.e. compound PP-1)

[0026] The reaction route is as follows:

[0027]

[0028] Compound 1 (1 mmol) and compound 2 (1 mmol) were dissolved in 25 mL of methanol, stirred in a flask for 1 h, and 6 drops of piperidine were added. After stirring, the mixture was refluxed at 90 ° C for 10 h, cooled to room temperature, and washed with petroleum ether. A CH2Cl2 / CH3OH mixture (12:1-7:1, v / v) was used as the eluent for column chromatography separation and purification to obtain a yellow solid, which was compound PP-1 (yield: 60%).

[0029] 1H NMR (400MHz, DMSO-d6) δ (ppm): 8.69 (d, J = 8.0Hz, 2H), 8.11 (d, J = 8.0Hz, 2H), 7.91 (d, J = 16.0H z,1H),7.05-7.10(m,2H),6.88-6.89(m,1H),6.22(q,J=1.3Hz,1H),4.18(s,3H),3.81(s,3H). 13 C NMR (400MHz, DMSO-d6), δ (ppm): 153.75, 144.72, 130.94, 129.94, 129.14, 122.59, 117.91, 112.82, 110.38, 46.84, 34.55.

[0030] Example 2 Photophysical property test experiment

[0031] Different solvents (DMF, DMSO, ethanol (EtOH), 1,4-dioxane (1,4-Dioxane), Gly and H2O) were used to prepare test solutions containing 10 μM PP-1. The absorption spectra of the above solutions were measured by UV-visible spectrophotometer, and the fluorescence emission spectra were measured by fluorescence spectrometer. The results are shown in Figure 1 .

[0032] It can be seen from the figure that the fluorescent compound PP-1 has an absorption peak at 430nm, and the absorption peak range is 350-640nm (see Figure 1 A). It has a fluorescence peak in the range of 450-600nm (see Figure 1 B). The above results show that the fluorescent compound can be excited by light of 350-640nm and its emission spectrum ranges from 450-600nm.

[0033] Example 3 Fluorescence test experiment of fluorescent compound PP-1 in solutions with different pH values

[0034] (1) Take the organic fluorescent compound PP-1 prepared in Example 1 and prepare a probe stock solution with a concentration of 1 mM using DMSO;

[0035] (2) preparing PBS solutions with different pH values ​​(pH = 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0);

[0036] (3) adding the probe mother solution prepared in step (1) to the solutions of different pH values ​​prepared in step (2) to prepare test solutions, so that the final concentration of the fluorescent probe PP-1 in each solution is 10 μM;

[0037] (4) The above solution was tested for its absorption spectrum and fluorescence emission spectrum using a UV-visible spectrophotometer and a fluorescence spectrometer to obtain corresponding curves. PP-1 has absorption in the range of 350-510nm, and the fluorescence emission spectrum range is 420-650nm.

[0038] The absorption spectrum of the above solution was measured by UV spectrophotometer. Figure 2 A. Use a fluorescence spectrometer to test its fluorescence emission spectrum and obtain the corresponding curve, see Figure 2 B.

[0039] As can be seen from the figure, in PBS solutions with different pH values, its absorption peak is around 430nm, and the fluorescence intensity does not change with the change of pH value. This shows that the probe is not affected by pH, which facilitates more accurate cell imaging.

[0040] Example 4 Cell Imaging Experiment of Probe PP-1

[0041] 1. HeLa cells were cultured in high-glucose H-DMEM medium containing 10% fetal bovine serum in a saturated humidity incubator at 37°C and 5% CO2. The medium was replaced every 2-3 days and subcultured.

[0042] 2. When the cells grow to the logarithmic phase, carry out grafting culture:

[0043] ① Soak the cover glass in anhydrous ethanol for 30 minutes, dry it with an alcohol lamp, and place it in a disposable 35mm culture dish for later use;

[0044] ② Wash the HeLa cells in the 100mL cell bottle three times with PBS, digest with 1mL 0.25% trypsin for 5 minutes, carefully pour out the trypsin, add fresh culture medium, blow evenly and count the cells. Control the cell density by adding culture medium so that the final cell concentration is 1×10 per mL. 5 Then, the cells were inoculated into the culture dish containing the cover glass and placed in a 5% CO2 incubator to allow the cells to grow close to the culture dish. After the HeLa cells grew on the plate and covered the cover glass, they were used for cell experiments.

[0045] 3. Prepare a probe stock solution with a concentration of 1mM using DMSO. Incubate the cultured active HeLa cells in a culture solution containing 5μM PP-1 for 30 minutes, then wash with PBS and image using a laser confocal microscope. The experimental group was incubated in a culture solution containing 20μM PP-1 for 30 minutes. Then wash with PBS and record the coloring site, fluorescence distribution and brightness changes in the cells. The results are shown in Figure 3 .

[0046] in, Figure 3These are confocal microscopy images of normal HeLa cells treated with probe PP-1 (5 μM, 30 min) and stained with PP-1 (20 μM, 30 min). The excitation wavelength of PP-1 in the green light channel is 488 nm, and the fluorescence collection wavelength is 500-600 nm. It can be seen from the figure that the fluorescence intensity is weak when stained with 5 μM. When stained with 20 μM, the probe presents a typical mitochondrial image in the cytoplasm, indicating that the probe stains mitochondria. In addition, it can be seen from the overlapping pictures that the probe can light up the nucleolus in the cell nucleus, indicating that the probe can image the nucleolus and mitochondria at the same time.

[0047] Example 5 Cell co-localization experiment of probe PP-1

[0048] The steps of HeLa cell culture and patching were the same as those in Example 3. A probe stock solution with a concentration of 1 mM was prepared with DMSO. The cultured active HeLa cells were incubated in a culture solution containing 20 μM PP-1 for 30 min, then stained with 2 μM MTDR (mitochondrial tracking deep red fluorescent probe) for 30 min, washed twice with PBS, and then imaged with a confocal microscope.

[0049] in, Figure 4 This is a confocal image of active HeLa cells co-stained with PP-1 and MTDR. The excitation wavelength of PP-1 in the green light channel is 488nm, and the fluorescence collection wavelength is 500-600nm; the excitation wavelength of MTDR in the red light channel is 635nm, and the fluorescence collection wavelength is 640-740nm. As can be seen from the figure, the cytoplasmic part stained by PP-1 has a large overlap with the part stained by the commercial mitochondrial fluorescent probe, and the colocalization coefficient is 0.85, indicating that the probe can image mitochondria.

[0050] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. Use of a fluorescent probe in preparing a reagent for simultaneous imaging of mitochondria and nucleoli, characterized in that: The fluorescent probe is (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt, and the structural formula is:

2. The use according to claim 1, characterized in that: The probe concentration is 15-25 μM.

3. A dual-color co-localization mitochondrial reagent, characterized in that: The mitochondrial green fluorescent probe in the reagent is (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt.

4. The dual-color co-localization mitochondrial reagent according to claim 3, characterized in that: The excitation wavelength of the mitochondrial green fluorescent probe is 488 nm, and the fluorescence collection wavelength is 500-600 nm.

5. The dual-color co-localization mitochondrial reagent according to claim 3, characterized in that: The reagent also includes a mitochondrial red fluorescent probe.

6. The dual-color co-localization mitochondrial reagent according to claim 5, characterized in that: The mitochondrial red fluorescent probe is MTDR.

7. The dual-color co-localization mitochondrial reagent according to claim 5, characterized in that: The excitation wavelength of the mitochondrial red fluorescent probe is 635nm, and the fluorescence collection wavelength is 640-740nm.

8. A two-color co-localization mitochondrial method, characterized in that: The cells to be tested are incubated once in a culture medium containing (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt, and then a mitochondrial red fluorescent probe is added for a second incubation.

9. The dual-color co-localization mitochondrial method according to claim 8, characterized in that: The incubation time is 30 minutes.

10. The dual-color co-localization mitochondrial method according to claim 8, characterized in that: The secondary incubation time is 30 minutes.

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