Novel non-invasive fluorescent labeling method for biological collagen fiber

Abstract

To provide an analyzing method for collagen fibers that includes a process of bringing a sample and a fluorescent probe into contact with each other and a process of detecting the fluorescent probe.SOLUTION: There is provided an analyzing method for collagen fibers, which includes a process of bringing a sample and a fluorescent probe into contact with each other and a process of detecting the fluorescent probe. wherein the fluorescent probe is selected from the group of diamino fluorescein-FM (DAF-FM), diamino rhodamine-4M(DAR-4M), diamino fluorescein FM diacetate (DAF-FM DA), diamino rhodamine-4M acetoxymethyl ester (DAR-4M AM), derivatives thereof, and combinations thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a method for analyzing collagen fibers, comprising the steps of contacting a sample with a fluorescent probe and detecting the fluorescent probe. [Background technology]

[0002] Collagen is a protein that mainly constitutes the dermis, ligaments, tendons, bones, and cartilage of vertebrates, and is also the main component of the extracellular matrix (ECM) of multicellular animals. Collagen fibers are fibrous structures formed by the aggregation of collagen molecules. Collagen molecules form a triple helix structure, which then further aggregates to form fibers, forming a structure with strength and elasticity.

[0003] Known methods for analyzing collagen include a method using Sirius Red, a method using a collagen-specific antibody, a method using second harmonic generation (SHG) (Non-Patent Document 1), and a method using collagen hybridizing peptide (CHP) (Non-Patent Document 2), but a new method has been desired. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Campagnola, Paul J et al. "Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues." Biophysical journal vol. 82,1 Pt 1 (2002): 493-508. [Non-patent document 2] Hwang, Jeongmin et al. "In Situ Imaging of Tissue Remodeling with Collagen Hybridizing Peptides." ACS nano vol. 11,10 (2017): 9825-9835. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure provides a method for analyzing collagen fibers, comprising the steps of contacting a sample with a fluorescent probe and detecting the fluorescent probe. [Means for solving the problem]

[0006] The present disclosure includes: [Section 1] A method for analyzing collagen fibers, the method comprising the steps of contacting a sample with a fluorescent probe and detecting the fluorescent probe; The fluorescent probe is selected from the group consisting of diaminofluorescein-FM (DAF-FM), diaminorhodamine-4M (DAR-4M), diaminofluorescein FM diacetate (DAF-FM DA), diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM), and derivatives thereof, and combinations thereof; method. [Section 2] Item 10. The method of claim 1, wherein the sample is a living organism, a biological sample, or a biological sample. [Section 3] Item 2. The method according to Item 1, wherein when the fluorescent probe is a combination, the step of contacting the sample with the fluorescent probe is a step of contacting each of the combinations with the sample simultaneously or separately. [Section 4] Item 1. The method according to Item 1, wherein when the fluorescent probe is a combination, the combination is a combination of DAF-FM or DAF-FM DA and DAR-4M or DAR-4M AM. [Section 5] Item 1. The method according to Item 1, wherein the sample contains collagen fibers. [Section 6] A kit for analyzing collagen fibers, comprising: (i) a fluorescent probe, and (ii) instructions; Including, wherein the fluorescent probe is selected from the group consisting of DAF-FM, DAR-4M, DAF-FM DA, DAR-4M AM, and derivatives thereof, and combinations thereof; kit. [Effects of the Invention]

[0007] According to the present disclosure, there is provided a method for analyzing collagen fibers, comprising the steps of contacting a sample with a fluorescent probe and detecting the fluorescent probe. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 shows the results of staining collagen fibers in vivo with DAF-FM DA. [Figure 2] FIG. 2 shows the results of staining collagen fibers of cells with DAF-FM. [Figure 3] FIG. 3 shows the results of multicolor staining of collagen fibers in cells. [Figure 4] FIG. 4 shows the results of the number of proliferating cells and the percentage of proliferating cells in the living organisms and cells treated with DAF-FM and DAF-FM DA. [Figure 5] Figure 5 shows a comparison of DAF-FM DA and second harmonic generation (SHG) for the observation of collagen fibers. From the left, DAF-FM DA, SHG, and their combined results. [Figure 6] Figure 6 shows a comparison of the observation of collagen fibers between DAF-FM DA and Collagen Hybridizing Peptide (CHP). From the left, DAF-FM DA, CHP, and the combination of the two. [Figure 7]FIG. 7 shows the relationship between the staining of collagen fibers with DAF-FM DA and nitric oxide (NO). DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description of the Invention Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0010] In the present disclosure, the term "collagen fiber" refers to a structure in which collagen molecules form a triple helix structure and are primarily aggregated into a fibrous form. The molecular weight of collagen fibers can range from tens to hundreds of kilodaltons. Because collagen contains very long polypeptide chains, even when defined by molecular weight, this can vary depending on the measurement method and conditions. Known methods for analyzing collagen fibers include second harmonic generation (SHG), antibody staining, and histochemical staining methods such as picrosirius red or Masson's trichrome staining. Collagen is known to be classified into types I, II, III, and IV, and in the present disclosure, the term "collagen fiber" encompasses fibers of all known types of collagen unless otherwise specified. In the present disclosure, the collagen in collagen fibers may be type I, II, or III, preferably type I or II.

[0011] The "analysis method" for collagen fibers of the present disclosure can analyze the presence or absence of collagen fibers in a subject, and if collagen fibers are present, can analyze their distribution, structure, quantity, or other characteristics. In one embodiment, the "analysis method" for collagen fibers of the present disclosure includes a step of fluorescently labeling collagen fibers and detecting the fluorescence. The analysis method for collagen fibers of the present disclosure can be used in combination with known analysis methods for collagen fibers, and the analysis is performed by various techniques such as chemical analysis, physical property analysis, and biological analysis. The analysis method of the present disclosure can be performed invasively or non-invasively, but non-invasive methods are preferred in that they can analyze collagen fibers in a living body. The analysis method of the present disclosure is preferred in that it can also be used to analyze a living body, that collagen fibers can be analyzed by combining the method of the present disclosure or known methods, or that collagen fibers can be analyzed over time.

[0012] In this disclosure, the term "sample" refers to, but is not limited to, a living organism, a biological sample, or a biological sample, and includes not only natural samples but also artificially created samples. In this disclosure, a "sample" may also refer to a genetically modified living organism, a biological sample, or a biological sample. A living organism refers to a living organism itself, i.e., a living individual. A biological sample refers to tissues or samples taken directly from a living individual. Cells in a biological sample may be live or dead. A biological sample refers to any type of sample collected for biological research, diagnosis, or other purposes. Examples of living organisms include, but are not limited to, vertebrates and invertebrates, mammals (e.g., humans, monkeys, horses, dogs, cats, mice, rats, rabbits), birds (e.g., chickens, pigeons), reptiles or amphibians (e.g., crocodiles, frogs, snakes, axolotls, axolotls), fish (e.g., zebrafish), insects (e.g., bees, cockroaches, flies), and protists. In one embodiment, small aquatic vertebrates such as zebrafish or larval axolotls can be used as biological samples. Biological samples include, for example, biological tissues (liver tissue, muscle tissue, skin tissue, etc.), blood, serum, saliva, and urine. Biological samples include, for example, cell cultures (cell strains, cell lines, etc.), tissue sections (tissue samples for pathological analysis), cell samples (cancer cells, immune cells, fibroblasts, etc.), blood samples (samples for blood tests, etc.), DNA samples, and RNA samples.

[0013] In the present disclosure, the "step of contacting" a sample with a fluorescent probe refers to physically contacting the sample with the fluorescent probe, including, for example, mixing the sample with the fluorescent probe. Typically, the contact is carried out in an aqueous solution. Contact conditions can be appropriately selected and determined by those skilled in the art, taking into account the sample biological species, sample size, and the like. The contact time is not particularly limited and can be appropriately determined by those skilled in the art, but may be about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes (i.e., 1 hour), about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours (i.e., 1 day), about 36 hours, or any time in between. The contact time is preferably about 1 hour. The fluorescent probe to be contacted is not particularly limited and can be appropriately determined by one skilled in the art, but the final concentration can be about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 10 μM, or about 10 μM. The concentration may be 5 μM, about 11 μM, about 11.5 μM, about 12 μM, about 12.5 μM, about 13 μM, about 13.5 μM, about 14 μM, about 14.5 μM, about 15 μM, about 15.5 μM, about 16 μM, about 16.5 μM, about 17 μM, about 17.5 μM, about 18 μM, about 18.5 μM, about 19 μM, about 19.5 μM, about 20 μM, or any concentration therebetween. A preferred final concentration of the contacting fluorescent probe may be about 5 μM for DAF-FM or DAF-FM DA, and about 10 μM for DAR-4M or DAR-4M AM.

[0014] In the present disclosure, the "step of detecting" a fluorescent probe refers to detecting light emitted by the fluorescent probe. The detection method is not particularly limited, but the fluorescence may be detected using a microscope or a spectrophotometer. Observing fluorescence using a microscope is preferable because, for example, structure or localization can be visually observed, and it is also preferable because, for example, multiple fluorescent probes can be observed simultaneously. Detecting fluorescence using a spectrophotometer is preferable because, for example, fluorescence intensity can be quantitatively measured.

[0015] In the present disclosure, the term "fluorescent probe" refers to any substance capable of staining collagen fibers in a sample. Preferably, the fluorescent probe is selected from the group consisting of fluorescent substances such as diaminofluorescein-FM (DAF-FM), diaminorhodamine-4M (DAR-4M), diaminofluorescein FM diacetate (DAF-FM DA), and diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM), as well as their derivatives and combinations. In the present disclosure, a derivative of a compound refers to a derivative that is produced by metabolism, conversion, or degradation in vivo. The combination of probes in the present disclosure is not particularly limited, but is preferably one that allows fluorescence of different wavelengths to be observed. DAF-FM DA is DAF-FM that has been diacetylated. DAR-4M AM is DAR-4M that has been acetoxymethyl esterified. In one embodiment, DAF-FM DA or DAR-4M AM is used to label collagen fibers in living organisms with the fluorescent probe, and DAF-FM or DAR-4M is used to label collagen fibers in cultured cells or the like with the fluorescent probe. The excitation and fluorescence wavelengths are not particularly limited as long as they are within the range that allows detection of the fluorescent probe, and can be appropriately set by those skilled in the art within the range commonly used in the art. For example, for DAF-FM or DAF-FM DA, the excitation wavelength is 500 nm, and the fluorescence wavelength is 515 nm; for DAR-4M or DAR-4M AM, the excitation wavelength is 550 nm, and the fluorescence wavelength is 572 nm. The excitation or fluorescence wavelength may be ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, ±11, ±12, ±13, ±14, ±15, ±16, ±17, ±18, ±19, or ±20 from these values. "±" means that there is a range above and below a particular specified value; for example, ±20 nm refers to a range that includes 20 nm above (plus) and 20 nm below (minus) the specified wavelength.

[0016] In one embodiment of the present disclosure, when a combination of fluorescent probes is used, the step of contacting the sample with the fluorescent probes is a step of contacting each of the combination with the sample simultaneously or separately. For example, when a combination of fluorescent probes is used separately, a first fluorescent probe can be contacted with the sample and its fluorescence detected, and then a second fluorescent probe can be contacted with the sample and its fluorescence detected. In one embodiment, when a combination of fluorescent probes is used separately, collagen fibers in a sample can be analyzed over time.

[0017] In one embodiment of the present disclosure, the combination of fluorescent probes is DAF-FM or DAF-FM DA and DAR-4M or DAR-4M AM. DAF-FM or DAF-FM DA has a peak fluorescence wavelength of 515 nm (green fluorescence), while DAR-4M or DAR-4M AM has a peak fluorescence wavelength of 572 nm (red fluorescence), making them preferable because they can be recognized as different colors when observing changes over time. This allows for time-lapse observation, which is advantageous because it allows, for example, the growth of collagen fibers to be observed. There are no particular limitations on the order of staining for the combination of fluorescent probes; for example, DAF-FM staining may be followed by DAR-4M staining, or DAR-4M staining may be followed by DAF-FM staining.

[0018] In one embodiment of the present disclosure, if a sample contains collagen fibers, the collagen fibers can be analyzed by the method for analyzing collagen fibers of the present disclosure. In another embodiment, if a sample does not contain collagen fibers, the absence of collagen fibers can be recognized by the method for analyzing collagen fibers of the present disclosure.

[0019] In one embodiment of the present disclosure, multicolor observation may be performed by combining not only the fluorescent probe of the present disclosure but also antibody staining of collagen fibers, etc. Multicolor staining of other target organelles, nuclei, etc. may also be performed. The fluorescence of collagen fibers labeled with the fluorescent probe of the present disclosure does not fade or tends to fade less easily when exposed to room light, so light shielding is not required or is less necessary, which is convenient. Furthermore, for long-term storage, to prevent decay of the observation sample, the sample may be washed with PBS after fixation and stored at 4°C while immersed in PBS.

[0020] One embodiment of the present disclosure relates to a kit for analyzing collagen fibers. The kit of the present disclosure includes a fluorescent probe and instructions, and the instructions may include instructions on how to use the fluorescent probe. The analytical method of the present disclosure may be performed using the kit of the present disclosure. The kit of the present disclosure is preferably optimized for analyzing collagen fibers and may include reagents, solvents for the reagents, or other components.

[0021] The collagen fiber analysis method of the present disclosure uses a fluorescent probe that is relatively stable and inexpensive, and therefore can analyze collagen fibers more cheaply, efficiently, and simply than conventional methods.

[0022] As used herein, "about" means within a range of ±10%, preferably ±5%.

[0023] The present invention will be specifically and in detail explained below by showing examples. However, the examples are used to illustrate the present invention and are not intended to limit the present invention. [Example]

[0024] [Example 1] Analysis of collagen fibers in living organisms For fluorescent labeling of collagen fibers in living tissues, we used DAF-FM DA (Goryo Chemical SK1004-01, excitation wavelength: 500 nm, emission wavelength: 515 nm), a diacetylated form of DAF-FM. Zebrafish were immersed in a DAF-FM DA staining solution diluted to a concentration of 5 μM using water containing no food or feces and incubated overnight in the dark. After incubation, the staining solution was replaced with water. The fish were anesthetized using tricaine solution and placed on a glass base dish for fluorescence observation. The labeled specimens were then examined for intra-tissue fibrous structure using a confocal microscope. Specifically, we confirmed collagen fibers in the skull, skin, and fin tip epithelium (Figure 1).

[0025] [Example 2] Analysis of collagen fibers in cells DAF-FM (Goryo Chemical SK1003-01, excitation wavelength: 500 nm, emission wavelength: 515 nm) was used to fluorescently label collagen fibers produced by mouse fibroblasts in culture dishes. Fibroblasts were seeded on glass-based dishes coated with 0.1% gelatin and cultured in mouse cell-specific DMEM + 10% FBS in a 37°C CO2 incubator for one week to allow sufficient cell proliferation. After incubation, the medium was removed and replaced with a DAF-FM staining solution diluted to a concentration of 5 μM in medium. The cells were then incubated for one hour in a 37°C CO2 incubator. After incubation, the staining solution was replaced with medium, and fluorescence observation was performed under a confocal microscope (Figure 2). Observations 0, 2, and 4 days after labeling confirmed that the fluorescence did not fade even after 4 days of labeling.

[0026] [Example 3] Analysis of cellular collagen fibers by multicolor staining DAF-FM (Goryo Chemical SK1003-01, excitation wavelength: 500 nm, emission wavelength: 515 nm) and DAR-4M (Goryo Chemical SK1006-01, excitation wavelength: 550 nm, emission wavelength: 572 nm) were used to fluorescently label collagen fibers produced by mouse fibroblasts in culture dishes. Fibroblasts were seeded on glass-based dishes coated with 0.1% gelatin and cultured in mouse cell-specific DMEM + 10% FBS at 37°C in a CO2 incubator for one week to allow sufficient cell proliferation. After incubation, the medium was removed and replaced with DAF-FM staining solution diluted to 5 μM in medium and incubated for one hour in a 37°C CO2 incubator. After incubation, the DAF-FM staining solution was replaced with medium and the cells were cultured for four days. After culturing, the medium was removed and replaced with a DAR-4M staining solution diluted to a concentration of 10 μM in medium, and the cells were incubated for one hour in a 37°C CO2 incubator. After incubation, the staining solution was replaced with medium, and fluorescence observation was performed under a confocal microscope (Figure 3). As a result, fluorescence of DAF-FM and DAR-4M was observed, confirming how collagen fibers had grown.

[0027] [Example 4] Examination of effects on living organisms and cells The effects of DAF-FM and DAF-FM DA on living organisms and cells were investigated. DAF-FM staining was performed using the same procedures as in Examples 1 and 2 above. Cell proliferation was evaluated using the following procedure. After staining with DAF-FM DA / DAF-FM, BrdU (5-bromo-2'-deoxyuridine) (Abcam ab142567) was incorporated into cell nuclei for 1 hour, and then the samples were fixed overnight at 4°C in 4% PFA solution. The following day, the samples were washed with PBS, and all cell nuclei were stained with Hoechst (Dojindo 346-07951). Cell nuclei in the proliferative phase were also stained with an anti-BrdU antibody (Molecular Probes PRB-1). After staining, the percentage of proliferating cells was compared between samples treated with and without DAF-FM DA / DAF-FM. The results of the number of proliferating cells after vital staining and the percentage of proliferating cells after cell staining are shown in Figure 4. These results suggest that DAF-FM and DAF-FM DA staining can be performed on living cells, and that cells can continue to grow after staining.

[0028] [Example 5] Comparison with Second Harmonic Generation (SHG) and Collagen Hybridizing Peptide (CHP) To compare the results of DAF-FM DA staining with those of second harmonic generation (SHG), which has previously been used to visualize collagen fibers, axolotl finger tendons were observed and compared using a two-photon microscope (Figure 5). To compare the results of DAF-FM DA staining with those of collagen hybridizing peptide (CHP), which has previously been used to visualize collagen fibers, collagen fibers produced by mouse embryonic fibroblasts (MEFs) were observed and compared using a confocal microscope (Figure 6). These results demonstrate that the method disclosed herein can stain collagen fibers more clearly and facilitates collagen fiber analysis compared to conventional methods used to visualize collagen fibers.

[0029] [Example 6] Relationship with nitric oxide (NO) Because the fluorescent probe disclosed herein has been used as a nitric oxide indicator, we investigated the relationship between collagen fibers and nitric oxide. Specifically, collagen fibers purified from zebrafish fin tissue were classified into a control group, an NO synthase inhibition group, and an NO depletion group, and observed by fluorescent staining with DAF-FM DA (Figure 7). These results confirmed that the probe detected collagen fibers, not nitric oxide contained in collagen fibers. [Industrial Applicability]

[0030] According to the present disclosure, a method for analyzing collagen fibers can be provided, which includes the steps of contacting a sample with a fluorescent probe and detecting the fluorescent probe.

Claims

1. A method for analyzing collagen fibers, the method comprising the steps of contacting a sample with a fluorescent probe and detecting the fluorescent probe; The fluorescent probe is selected from the group consisting of diaminofluorescein-FM (DAF-FM), diaminorhodamine-4M (DAR-4M), diaminofluorescein FM diacetate (DAF-FM DA), diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM), and derivatives thereof, and combinations thereof; method.

2. 10. The method of claim 1, wherein the sample is a living organism, a biological sample, or a biological sample.

3. The method according to claim 1 , wherein when the fluorescent probes are a combination, the step of contacting the sample with the fluorescent probes is a step of contacting each of the combinations with the sample simultaneously or separately.

4. 2. The method of claim 1, wherein when the fluorescent probe is a combination, the combination is a combination of DAF-FM or DAF-FM DA and DAR-4M or DAR-4M AM.

5. The method of claim 1 , wherein the sample comprises collagen fibers.

6. A kit for analyzing collagen fibers, comprising: (i) a fluorescent probe, and (ii) instructions; Including, wherein the fluorescent probe is selected from the group consisting of DAF-FM, DAR-4M, DAF-FM DA, DAR-4M AM, and derivatives thereof, and combinations thereof; kit.

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