Application of pyrrole fluorescent compound in preparation of mitochondrial autophagy diagnostic reagent

Abstract

The invention discloses an application of a pyrrole fluorescent compound in preparation of a mitochondrial autophagy diagnostic reagent. The chemical name of the pyrrole fluorescent compound is (E)-4-(2-(1-methyl-1H-pyrrole-2) vinyl)-1-hexadecyl-1-iodised salt. The fluorescent compound provided by the invention can be used as a mitochondrial autophagy diagnosis fluorescent probe, can be used together with a commercial lysosome fluorescent probe, and can be used for observing the mitochondrial autophagy process through fluorescence resonance energy transfer. The fluorescent probe can be used for observing mitochondria for a long time. Therefore, the fluorescent compound disclosed by the invention can be used for observing the fields of screening of drugs related to mitochondria, vaccine development, cancer research and the like, and a new thought and method are provided for research in the fields.

Description

Technical Field

[0001] The invention relates to application of a pyrrole fluorescent compound in preparing a mitochondrial autophagy diagnostic reagent, and belongs to the technical field of fluorescent probes. Background Art

[0002] Mitochondrial autophagy is a selective autophagy process in cells that can remove damaged or redundant mitochondria, thereby maintaining the stability of the intracellular environment and the normal function of cells. Mitochondrial autophagy disorders are closely related to the occurrence and development of a variety of cellular diseases, such as Parkinson's disease, Alzheimer's disease, and tumors. These diseases are often accompanied by abnormal mitochondrial function and disordered mitochondrial autophagy.

[0003] At present, scanning electron microscopes, transmission electron microscopes, etc. are widely used to observe the autophagy process of mitochondria. However, these methods will destroy the cell structure and cannot observe the dynamic changes of mitochondrial autophagy in living cells in situ and in real time. Fluorescent probes have attracted widespread attention because of their simple operation and the ability to observe the dynamic changes of cells in situ, non-destructively, and in real time. Currently, people use two probes to co-stain to observe mitochondrial autophagy. However, many mitochondrial probes will fall off from mitochondria during cell autophagy, which is not conducive to observing the mitochondrial autophagy process. In addition, this method will produce interference with the fluorescence signal, which will produce false positive signals. Therefore, it is necessary to develop fluorescent probes that can observe mitochondrial autophagy over a long period of time and reduce interference with fluorescence signals. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an application of a pyrrole fluorescent compound in the preparation of a diagnostic reagent for mitochondrial autophagy.

[0005] Technical solution: The present invention provides an application of a pyrrole fluorescent compound in the preparation of a mitochondrial autophagy diagnostic reagent. The chemical name of the pyrrole fluorescent compound is: (E)-4-(2-(1-methyl-1H-pyrrole-2)vinyl)-1-hexadecyl-1-iodide, and the structural formula is:

[0006]

[0007] The present invention also provides a diagnostic kit for mitochondrial autophagy-related diseases, wherein the mitochondrial probe component in the kit is (E)-4-(2-(1-methyl-1H-pyrrole-2)vinyl)-1-hexadecyl-1-iodide salt.

[0008] Furthermore, the probe concentration is 1 to 15 μM.

[0009] Furthermore, the kit also includes a lysosome tracking red fluorescent probe LTDR.

[0010] Furthermore, the kit also includes a lysosomal red fluorescent probe LTR.

[0011] Furthermore, the mitochondrial autophagy-related diseases include Kinson's disease, Alzheimer's disease, and tumors.

[0012] Furthermore, the kit can be used to observe the mitochondrial autophagy process for a long time.

[0013] The present invention also provides a method for diagnosing mitochondrial autophagy, wherein the cells to be tested are incubated once in a culture solution containing (E)-4-(2-(1-methyl-1H-pyrrole-2)vinyl)-1-hexadecyl-1-iodine salt, and then a lysosome tracking red fluorescent probe is added for a second incubation.

[0014] Furthermore, the incubation time is 15 minutes.

[0015] Furthermore, the secondary incubation time is 10 minutes.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention discloses a fluorescent compound as a diagnostic fluorescent probe for mitochondrial autophagy, which can be used together with a commercial lysosomal fluorescent probe to jointly observe the mitochondrial autophagy process through fluorescence resonance energy transfer. The fluorescent probe can observe mitochondria for a long time. Therefore, the fluorescent compound of the present invention can be used to observe drug screening, vaccine development, cancer research and other fields related to mitochondria, providing new ideas and methods for research in these fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The absorption and fluorescence spectra of PP-16 (10 μM) in different solvents.

[0018] Figure 2 (A) is a confocal fluorescence image of HeLa cells co-stained with PP-16 (2 μM, 15 min) and MTDR (0.2 μM, 15 min); (B) is the fluorescence intensity of the corresponding positions of the green channel and the red channel in Figure A.

[0019] Figure 3 (A) Confocal fluorescence images of HeLa cells co-stained with PP-16 (2 μM, 15 min) and LTDR (0.2 μM, 15 min) after treatment with 20 μM CCCP and 5 μM pepstatin A for different time periods; (B) Colocalization coefficient values ​​of the two probes at different time points.

[0020] Figure 4(A) Confocal fluorescence images of HeLa cells treated with 20 μM CCCP and 5 μM pepstatin A for different time periods and co-stained with PP-16 (2 μM, 15 min) and LTR (0.2 μM, 15 min). (B) Average fluorescence intensity of the red channel. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0022] The absorption spectrum test instrument used in the following 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.

[0023] Example 1 Synthesis of fluorescent compounds

[0024] (E)-4-(2-(1-methyl-1H-pyrrole-2)vinyl)-1-hexadecyl-1-iodide (i.e. compound PP-16)

[0025] The reaction route is as follows:

[0026]

[0027] Synthesis of compound PP-16

[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 2 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 (13:1-7:1, v / v) was used as the eluent for column chromatography separation and purification to obtain a yellow solid, which was compound PP-16 (yield: 60%).

[0029] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.78 (d, J = 8.0Hz, 2H), 8.13 (d, J = 4.0Hz, 2H), 7.93 (d, J = 16.0Hz, 1H), 7.05-7.11 (m, 2H), 6.88 (d, J = 4.0Hz,1H),6.22(t,J=4.0Hz,1H),4.41(t,J=8.0Hz,2H),3.81(s,3H),1.88(t,J=6.0Hz,2H),1.23(s,26H),0.86(t,J=6.0Hz,3H). 13C NMR (400MHz, DMSO-d6), δ (ppm): 154.11, 142.86, 130.58, 130.34, 129.25, 123.08, 116.07, 113.93, 110.7 4,80.53,35.48,31.92,31.52,29.70,19.66,19.61,19.52,29.37,29.07,26.11,22.69,14.14.HRMS:m / z calculated for[C 28 H 45 N2] + 409.3627([MI] + );found:409.3708.

[0030] Example 2 Photophysical property test experiment

[0031] Different solvents (DCM, DMSO, ethanol (EtOH), 1,4-dioxane (1,4-Diox), Gly, H2O, MeCN and THF) were used to prepare test solutions containing 10 μM PP-16. 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] from Figure 1 It can be seen that the fluorescent compound PP-16 has an absorption peak at 430nm, and the absorption peak range is 320-520nm (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 320-520 nm and its emission spectrum ranges from 450-600 nm.

[0033] Example 3 Probe PP-16 observes mitochondria in cells

[0034] 1. Culture HeLa cells in high-glucose H-DMEM medium containing 10% fetal bovine serum at 37°C in a saturated humidity incubator with 5% CO2. Replace the medium every 2-3 days and perform subculture. 2. When the cells grow to the logarithmic phase, perform grafting culture:

[0035] 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;

[0036] 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.

[0037] 3. Prepare a probe stock solution with a concentration of 1mM using DMSO, and prepare a PP-16 solution with a concentration of 2μM before use. Incubate the cultured active HeLa cells in a culture solution containing 2μM PP-16 for 15min, then add 0.2μM MTDR (mitochondrial tracking deep red fluorescent probe) for staining for 10min. Record the coloring site in the cell, fluorescence distribution and brightness changes, co-localization information, etc. The results are shown in Figure 2 .

[0038] in, Figure 2 A is a confocal microscopy image of normal HeLa cells stained with probes PP-16 (2 μM, 15 min) and MTDR (0.2 μM, 10 min). Figure 2 B is the fluorescence intensity of the two channels corresponding to the straight line in Figure A. The excitation wavelength of PP-16 in the green channel is 488nm, and the fluorescence collection wavelength is 500-600nm; the excitation wavelength of MTDR in the red channel is 635nm, and the fluorescence collection wavelength is 650-750nm. It can be seen from the figure that in normal HeLa cells, the green channel and the red channel have a large overlap, and their colocalization coefficients are 0.86. In addition, the fluorescence intensity changes in the two channels are consistent, which indicates that the probe stains mitochondria.

[0039] Example 4 Probe PP-16 and LTDR tracking of mitochondrial autophagy experiment

[0040] The steps of HeLa cell culture and patching are the same as those in Example 3. Use DMSO to prepare a probe stock solution with a concentration of 1 mM, and prepare a PP-16 solution with a concentration of 2 μM before use. Incubate the cultured active HeLa cells in a culture solution containing 2 μM PP-16 for 15 minutes, and then add 0.2 μM LTDR (lysosome tracking deep red fluorescent probe) for staining for 10 minutes. Then use 20 μM CCCP and 5 μM inhibitor A to induce mitochondrial autophagy, and record the coloring sites in the cells during the staining period of 0 to 2.5 hours, fluorescence distribution and brightness changes, co-localization information, etc. The results are shown in Figure 3 .

[0041] in Figure 3(A) is a confocal fluorescence image of HeLa cells co-stained with CCCP by PP-16 (2μM, 15min) and LTDR (0.2μM, 15min) at different times; (B) is the colocalization coefficient of the two probes at different times. The excitation wavelength of PP-16 in the green light channel is 488nm, and the fluorescence collection wavelength is 500-600nm; the excitation wavelength of LTDR in the red light channel is 635nm, and the fluorescence collection wavelength is 650-750nm. As can be seen from the figure, as time goes on, the overlap between the green light channel and the red light channel gradually increases, and the colocalization coefficient value also gradually increases ( Figure 3 B), which indicates the occurrence of mitophagy.

[0042] Example 5 Probe PP-16 and LTR tracking mitochondrial autophagy experiment

[0043] The steps of HeLa cell culture and splicing were the same as those in Example 3. After splicing, the cultured active HeLa cells were incubated in a culture medium containing 2μM PP-16 for 15 min, and then 0.2μM LTR (lysosomal red fluorescent probe) was added for staining for 10 min. Then, 20μM CCCP and 5μM pepstatin A were used to induce mitochondrial autophagy, and the coloring sites in the cells, fluorescence distribution and brightness changes, co-localization information, etc. were recorded. The results are shown in Figure 4 .

[0044] in Figure 4 (A) is a confocal fluorescence image of HeLa cells co-stained with CCCP and treated for different time periods by PP-16 (2μM, 15min) and LTR (0.2μM, 15min); (B) is the fluorescence intensity of the red light channel. The excitation wavelength of PP-16 in the green light channel is 488nm, and the fluorescence collection wavelength is 500-600nm; the excitation wavelength of LTR is 488nm, and the fluorescence collection wavelength is 600-700nm. As can be seen from the figure, the fluorescence intensity of the red light channel gradually increases with time, which indicates that during the occurrence of mitophagy, as mitochondria and lysosomes fuse, the fluorescence resonance energy transfer process also occurs, proving the occurrence of mitophagy. Since this method only has one excitation light excitation, the interference of fluorescence signals generated by multiple laser excitations can be avoided.

[0045] 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 pyrrole fluorescent compound in the preparation of a diagnostic reagent for mitochondrial autophagy, characterized in that: The chemical name of the pyrrole fluorescent compound is: (E)-4-(2-(1-methyl-1H-pyrrole-2)vinyl)-1-hexadecyl-1-iodine salt, and the structural formula is:

2. A diagnostic kit for mitochondrial autophagy-related diseases, characterized in that: The mitochondrial probe component in the kit is (E)-4-(2-(1-methyl-1H-pyrrole-2)vinyl)-1-hexadecyl-1-iodide.

3. The diagnostic kit for mitochondrial autophagy-related diseases according to claim 2, characterized in that: The probe concentration is 1-15 μM.

4. The diagnostic kit for mitochondrial autophagy-related diseases according to claim 2, characterized in that: The kit also includes a lysosome tracking red fluorescent probe LTDR.

5. The diagnostic kit for mitochondrial autophagy-related diseases according to claim 2, characterized in that: The kit also includes a lysosomal red fluorescent probe LTR.

6. The diagnostic kit for mitochondrial autophagy-related diseases according to claim 2, characterized in that: The mitochondrial autophagy-related diseases include Kinson's disease, Alzheimer's disease, and tumors.

7. The diagnostic kit for mitochondrial autophagy-related diseases according to claim 2, characterized in that: The kit can be used to observe the mitochondrial autophagy process for a long time.

8. A method for diagnosing mitochondrial autophagy, characterized in that: The cells to be tested were incubated once in a culture medium containing (E)-4-(2-(1-methyl-1H-pyrrole-2)vinyl)-1-hexadecyl-1-iodide, and then a lysosome tracking red fluorescent probe was added for a second incubation.

9. The method for diagnosing mitophagy according to claim 8, characterized in that: The incubation time is 15 minutes.

10. The method for diagnosing mitophagy according to claim 8, characterized in that: The secondary incubation time is 10 minutes.

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