Method and kit for detecting activity of intracellular myeloperoxidase by using flow cytometer
The reaction substrate is allowed to enter the cell through surfactant pore punching technology, and fluorescent substrates are used to detect the intracellular MPO activity, solving the problem of difficulty in accurately detecting the intracellular MPO activity in the prior art, and achieving rapid and accurate detection in the state of living cells, which is of practicality and promotional value.
Patent Information
- Application Number
- CN202411760669.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-03
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art is difficult to accurately detect the activity of myeloperoxidase (MPO), especially in the living cell state, and flow cytometry detection methods have not been applied in this field.
Surfactant pore punching technology is used to enable reaction substrates such as chloride ions and hydrogen peroxide to enter the cells, and combine them with fluorescent substrates (such as APF or HKOCl-3) for enzyme-catalyzed reactions to generate fluorescent signals. The fluorescence intensity is detected by flow cytometry to reflect the activity of MPO in the cell.
It realizes rapid and accurate detection of the activity of MPO in the cell in viable cell state, avoids the loss of enzyme activity caused by cell fixation, and is of practicality and promotional value.
Smart Images
Figure CN119619091A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of immunological myeloperoxidase activity detection, and specifically relates to a method and a kit for detecting intracellular myeloperoxidase activity using a flow cytometer. Background Art
[0002] Myeloperoxidase (MPO) is mainly present in the azurophilic granules of neutrophils, accounting for about 5% of the total protein in neutrophils. In addition, MPO is also present in small amounts in macrophages and monocytes. The mature MPO protein is a tetramer, containing two large subunits (60kd, 467 amino acids) and two small subunits (15kd, 105 amino acids), with a total molecular weight of about 150kd. Each large subunit is connected to the small subunit by heme, and the two large subunits are connected by disulfide bonds. The biosynthesis steps of MPO are relatively complex and can be divided into the following steps: the first step is to synthesize preMPO in the ribosomes of the endoplasmic reticulum; the second step is to undergo glycosylation modification in the endoplasmic reticulum to form the glycoprotein apoproMPO; the third step is to bind to heme in the endoplasmic reticulum to form proMPO; the fourth step is that proMPO leaves the endoplasmic reticulum and enters the cytoplasm; the fifth step is to form hemi-MPO after being cleaved by proteolytic enzymes; the sixth step is that in the azurophilic granules, a disulfide bond is formed between the two large subunits at the Cys319 position, and at the same time, the peptide bond between each group of large and small subunits is opened, and a small peptide (ASFVTG) consisting of six amino acids is cut off. In the final tetramer, each group of large and small subunits is bound by heme, and the two large subunits are bound by disulfide bonds. Current studies have shown that MPO has two main catalytic activities: halogen oxidation (halogenation cycle) to form hypohalous acid (such as hypochlorous acid) and peroxidase activity. The human body contains a large amount of chloride ions as MPO substrates, which catalyze hypochlorous acid with bactericidal activity; a small amount of halogen-like substances, such as thiocyanate (the content in the blood is at the micromolar level), is more easily oxidized, followed by iodide and bromide; although chloride ions have weak reducing ability, the chloride ion content in the body is high, so MPO in the body mainly produces hypochlorous acid for bactericidal purposes. Although the halogenation cycle is considered an antibacterial pathway due to the production of hypohalous acid, MPO has more single-electron substrates that are metabolized through the peroxidase pathway, and many free radical molecules are produced through this pathway. After neutrophils phagocytize bacteria and other pathogenic microorganisms, MPO can quickly catalyze the reaction of hydrogen peroxide and chloride ions to produce hypochlorous acid, which has a strong bactericidal effect. Neutrophils can also form NETs through NETosis, release MPO, catalyze reactions outside the cell to form hypochlorous acid, and kill extracellular pathogenic microorganisms. MPO deficiency can make the body susceptible to microbial infection. Excessive MPO activity can cause tissue and organ damage. In cardiovascular and cerebrovascular diseases, the products produced by MPO catalysis lead to oxidative damage, which can cause instability of arteriosclerotic lesions, expose collagen tissue under the endothelium of blood vessels, further cause platelet aggregation and thrombosis, cause arterial blockage, and cause and aggravate cardiovascular and cerebrovascular diseases. In addition, abnormal MPO activation plays a promoting role in the occurrence and development of neurodegenerative diseases, liver damage, diabetes, tumors, and autoimmune diseases.
[0003] Regarding the detection of MPO activity, the catalytic activity of the enzyme is currently mainly used. The MPO activity in the solution state is detected by colorimetry and fluorescence using an enzyme marker. There is no literature reporting the method of using flow cytometry to detect the activity of MPO enzymes in cells. In theory, MPO is highly active in the microenvironment inside the cell. After being released into the solution, it leaves the microenvironment in the cell, and its catalytic activity may be affected. On the other hand, inside living cells, MPO activity is regulated by other factors. When the cell state changes (for example, after the cell engulfs bacteria), the cell will activate MPO by changing the microenvironment or changing protein modification. Analyzing the activity of intracellular MPO is more scientifically meaningful for evaluating the cell state and even the body's immune function and inflammatory state. Therefore, it is necessary to establish a method for detecting the activity of intracellular MPO using flow cytometry. Summary of the invention
[0004] The purpose of the present invention is to provide a method and kit for detecting intracellular myeloperoxidase activity using flow cytometry. Surfactant is used to make holes, reaction substrate enters cells, intracellular enzyme catalyzes reaction substrate to produce fluorescence, and flow cytometry detects fluorescence intensity to reflect intracellular myeloperoxidase activity.
[0005] Wherein, MPO catalyzes the reaction of hydrogen peroxide and chloride ion to generate hypochlorous acid, and hypochlorous acid reacts with aminophenylfluorescein APF (or fluorescent hypochlorous acid probe HKOCl-3) to produce fluorescence, which can be detected by flow cytometry. Chloride ions, hydrogen peroxide and APF (or HKOCl-3) need to enter cells through cell membranes during the detection process, so it is necessary to punch holes in cells with surfactants such as saponin, so as to facilitate various reaction substrates to enter cells. In the present invention, probenecid sodium can be used to prevent cells from transporting fluorescent substrates out of cells by active transport.
[0006] Note: APF, aminophenyl fluoresecein, CAS No. 359010-70-1; HKOCl-3, CAS No. 2031170-80-4.
[0007] In order to achieve the above object, the first aspect of the present invention is to provide a method for detecting intracellular myeloperoxidase activity using a flow cytometer, comprising the following steps:
[0008] 1) Taking cells containing myeloperoxidase;
[0009] 2) using a solution containing a surfactant to perforate the cells containing myeloperoxidase;
[0010] 3) incubating the punched cells obtained in step 2) in a detection buffer, wherein the detection buffer contains a fluorescent substrate transport inhibitor, chloride ions, hydrogen peroxide and a fluorescent substrate;
[0011] 4) Detect the cell fluorescence intensity by flow cytometry, and determine the relative activity of intracellular myeloperoxidase based on the cell fluorescence intensity.
[0012] In a preferred embodiment, in step 2), the surfactant is at least one of saponin, digitonin, Triton X-100, deoxycholic acid, and Tween-80, and the concentration of the surfactant in the solution containing the surfactant is 0.0001-0.1%; the cells containing myeloperoxidase are incubated in the solution containing the surfactant for 3-5 minutes.
[0013] In the prior art, when punching cells, the cells are usually fixed with a fixative (such as formaldehyde, ethanol, etc.) before punching. After the cells are fixed, they become dead cells, and the enzyme activity is lost, and the enzyme activity can no longer be evaluated. No method for punching living cells has been found in the prior art.
[0014] Furthermore, in step 2), the cells containing myeloperoxidase are incubated in a solution containing a surfactant for 3-5 minutes, and then the surfactant is removed to separate and obtain the perforated cells.
[0015] In a preferred embodiment, in step 3), the punched cells obtained in step 2) are incubated in a detection buffer for 10-30 minutes, and the detection buffer contains 0.2-1.5 mM fluorescent substrate transport inhibitor.
[0016] Furthermore, the detection buffer contains 0.2-1.5 mM fluorescent substrate transport inhibitor, 50-200 mM chloride ion, 20-1000 μM hydrogen peroxide and 15-50 μM fluorescent substrate; the fluorescent substrate transport inhibitor includes probenecid sodium.
[0017] In a preferred embodiment, the fluorescent substrate includes aminophenylfluorescein APF or fluorescent hypochlorous acid probe HKOCl-3.
[0018] Furthermore, the incubation in step 2) is carried out at room temperature, and the incubation in step 3) is carried out at 25-40°C.
[0019] The second aspect of the present invention discloses a kit for detecting intracellular myeloperoxidase activity, comprising an aqueous buffer solution containing a surfactant; and a detection buffer solution containing a fluorescent substrate transport inhibitor, chloride ions, hydrogen peroxide and a fluorescent substrate.
[0020] In a preferred embodiment, the detection buffer contains 0.2-1.5 mM probenecid sodium, 50-200 mM chloride ions, 20-1000 μM hydrogen peroxide, and 15-50 μM fluorescent substrate. If the concentrations of chloride ions, hydrogen peroxide, and fluorescent substrate are too low, the reaction is insufficient and the activity of the intracellular MPO enzyme cannot be accurately detected. If the concentrations of chloride ions, hydrogen peroxide, and fluorescent substrate are too high, cell function may be affected.
[0021] Furthermore, the surfactant is at least one of saponin, digitonin, TritonX-100, deoxycholic acid, and Tween-80, and the surfactant concentration in the solution containing the surfactant is 0.0001-0.1%.
[0022] Furthermore, for cells from different sources, the preferred ranges of hydrogen peroxide concentration and surfactant concentration are different. For example, for mouse cells, when the hydrogen peroxide concentration range is 20-200 μM and the surfactant concentration is 0.0001-0.01%, the fluorescence intensity is strong, and at least one of the hydrogen peroxide and the surfactant outside the concentration range will cause the fluorescence intensity to weaken; for human cells, the hydrogen peroxide concentration range is 100-1000 μM and the surfactant concentration is 0.005%-0.1%, and at least one of the hydrogen peroxide and the surfactant outside the concentration range will also cause the fluorescence intensity to weaken.
[0023] Compared with the prior art, the present invention has the following beneficial effects: using surfactant to make holes, reaction substrates to enter cells, intracellular enzymes catalyzing reaction substrates to produce fluorescence, flow cytometry to detect fluorescence intensity, and intracellular myeloperoxidase activity. The present invention is fast, accurate, simple, easy to implement, and convenient for popularization and use.
[0024] By punching holes, fluorescent substrates, hydrogen peroxide and chloride ions can enter the cells smoothly. Controlling the concentration of surfactant is very critical. Since we need to detect the relative activity of myeloperoxidase in living cells, the cells should still be living cells after punching. A surfactant concentration of 0.0001-0.1% is used to allow the fluorescent substrates, hydrogen peroxide and chloride ions to enter the cells smoothly while ensuring that the cells will not rupture and die due to the treatment of surfactants. The use of probenecid sodium can prevent cells from transporting fluorescent substrates out of cells by active transport. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 .The effect of saponin on the MPO activity of mouse neutrophils detected by flow cytometry;
[0026] Figure 2 . Using APF, flow cytometry was used to detect the inhibitory effect of 4-ABAH on MPO activity of HL-60 cells;
[0027] Figure 3 HKOCl-3 was used to detect the inhibitory effect of 4-ABAH on MPO activity of HL-60 cells by flow cytometry. DETAILED DESCRIPTION
[0028] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.
[0029] In the present invention, the method for detecting intracellular myeloperoxidase activity using flow cytometry can be:
[0030] (1) Mouse bone marrow cells (or mouse peripheral blood cells or human peripheral blood cells) are obtained, and neutrophils are separated using a granulocyte separation solution; or macrophages and monocytes are separated; or HL-60 cells are obtained; or other cells containing MPO are obtained.
[0031] (2) Take 300,000 to 1.5 million cells, add 200 μl of physiological saline (or PBS buffer) containing 0.0005-0.05% (mass fraction) surfactant (such as saponin, digitonin, Triton X-100, deoxycholic acid or Tween-80, etc.), gently resuspend and mix the cells, and incubate at room temperature for 3-5 minutes. Add 1-2 ml of physiological saline (or PBS buffer), centrifuge and discard the supernatant, wash once with physiological saline (or PBS buffer), centrifuge and discard the supernatant, and obtain the punched cells.
[0032] (3) preparing a detection buffer, comprising 0.2-1.5 mM probenecid sodium, 50-200 mM sodium chloride, 20-1000 μM hydrogen peroxide, and 15-50 μM APF or HKOCl-3 in normal saline (or PBS or other buffer);
[0033] (4) Take 200 μl of detection buffer, mix the punched cells in the detection buffer, and incubate at 37°C for 10-30 minutes.
[0034] (5) Detect cell fluorescence intensity using flow cytometry. Use 488 nm for excitation light and 530 nm for emission light (when using flow cytometry, use the classic FITC channel to collect fluorescence signals).
[0035] (6) Count the fluorescence intensity and analyze the activity of MPO enzyme.
[0036] Example 1
[0037] (1) Mouse bone marrow cells were obtained and neutrophils were isolated using a granulocyte separation solution.
[0038] (2) Take 500,000 neutrophils, add 200 μl of saline solution containing 0.005% (mass fraction) of surfactant saponin, resuspend and mix the cells, and incubate at room temperature for 3 minutes. Add 2 ml of saline solution, centrifuge and discard the supernatant, wash the cells with 2 ml of saline solution, centrifuge and discard the supernatant, and obtain the punched cells.
[0039] (3) Prepare the detection buffer, which contains 0.5 mM probenecid sodium, 150 mM sodium chloride, 50 μM hydrogen peroxide and 20 μM APF in normal saline.
[0040] (4) Take 200 μl of detection buffer, mix the punched cells in the detection buffer, and incubate at 37°C for 20 minutes.
[0041] (5) Detect cell fluorescence intensity using flow cytometry. Use 488 nm for excitation light and 530 nm for emission light (when using flow cytometry, use the classic FITC channel to collect fluorescence signals).
[0042] (6) Count the fluorescence intensity and analyze the activity of MPO enzyme.
[0043] The results are as follows Figure 1 As shown, the red color is the mouse neutrophils treated with saponin (fluorescence intensity 381395), and the blue color is the mouse neutrophils in the control group (fluorescence intensity 67079). The results show that the saponin group in the flow histogram shifts to the right and the fluorescence intensity increases. This shows that the method of the present invention improves the detection of MPO activity.
[0044] The cells in the control group and saponin group were from the same mouse and sampled at the same time. Saponin perforation allowed the reaction substrates (chloride ions, hydrogen peroxide) and fluorescent substrates to enter the cells smoothly, thereby realizing the detection of intracellular MPO enzyme activity.
[0045] Example 2
[0046] (1) Take 500,000 HL-60 cells (human acute promyelocytic leukemia cells), add 200 μl of saline solution containing 0.02% surfactant saponin, resuspend and mix the cells, and incubate at room temperature for 5 minutes. Add 2 ml of saline solution, centrifuge and discard the supernatant, wash the cells with 2 ml of saline solution, centrifuge and discard the supernatant, and obtain the punched cells.
[0047] (2) Prepare the detection buffer, which contains 0.5 mM probenecid sodium, 150 mM sodium chloride, 1 mM hydrogen peroxide and 20 μM APF in normal saline.
[0048] (3) Control group: Take 200 μl of detection buffer, mix the perforated cells of the control group with the detection buffer, and incubate at 37°C for 20 min; MPO inhibitor group: Add 100 μM 4-ABAH (4-aminobenzoic acid hydrazide) to the detection buffer to obtain the inhibitor group detection buffer, mix the perforated cells of the MPO inhibitor group with 200 μl of the inhibitor group detection buffer, and incubate at 37°C for 20 min.
[0049] (4) Detect cell fluorescence intensity using flow cytometry. Use 488 nm for excitation light and 530 nm for emission light (when using flow cytometry, use the classic FITC channel to collect fluorescence signals).
[0050] (5) Count the fluorescence intensity and analyze and evaluate the activity of MPO enzyme.
[0051] The control group and the MPO inhibitor group differed only in step (3), and the rest of the steps were the same.
[0052] The HL-60 cells used in the control group and the inhibitor group were from the same batch, so that the HL-60 cells used in the control group and the inhibitor group were consistent. Figure 2 As shown, the blue is the control group (4-ABAH is not contained in the detection buffer), and the MPO activity of HL-60 cells is detected by using the fluorescent substrate APF (fluorescence intensity 791701), and the red is the inhibitor group. After the MPO inhibitor 4-ABAH is additionally added to the detection buffer, the MPO activity of HL-60 cells is detected (fluorescence intensity 354663). The results show that the MPO inhibitor group shifts to the left in the flow histogram, and the fluorescence intensity is weakened. This shows that the method of the present invention is feasible.
[0053] Example 3
[0054] (1) Take 500,000 HL-60 cells, add 200 μl of saline solution containing 0.02% surfactant saponin, resuspend and mix the cells, and incubate at room temperature for 5 minutes. Add 2 ml of saline solution, centrifuge and discard the supernatant, wash the cells with 2 ml of saline solution, centrifuge and discard the supernatant, and obtain the punched cells.
[0055] (2) Prepare a detection buffer solution containing 0.5 mM probenecid sodium, 150 mM sodium chloride, 1 mM hydrogen peroxide and 20 μM HKOCl-3 in normal saline.
[0056] (3) Control group: Take 200 μl of detection buffer, mix the perforated cells of the control group with the detection buffer, and incubate at 37°C for 20 min; MPO inhibitor group: Add 100 μM 4-ABAH (4-aminobenzoic acid hydrazide) to the detection buffer to obtain the inhibitor group detection buffer, mix the perforated cells of the MPO inhibitor group with 200 μl of the inhibitor group detection buffer, and incubate at 37°C for 20 min.
[0057] (4) Detect cell fluorescence intensity using flow cytometry. Use 488 nm for excitation light and 530 nm for emission light (when using flow cytometry, use the classic FITC channel to collect fluorescence signals).
[0058] (5) Count the fluorescence intensity and detect the activity of MPO enzyme.
[0059] The control group and the MPO inhibitor group differed only in step (3), and the rest of the steps were the same.
[0060] The HL-60 cells used in the control group and the inhibitor group were from the same batch, so that the HL-60 cells used in the control group and the inhibitor group were consistent. Figure 3 As shown, the blue is the control group (4-ABAH is not contained in the detection buffer), and the fluorescence substrate HKOCl-3 is used to detect the MPO activity of HL-60 cells (fluorescence intensity 988876), and the red is the inhibitor group. After the MPO inhibitor 4-ABAH is additionally added to the detection buffer, the MPO activity of HL-60 cells is detected (fluorescence intensity 189975). The results show that the MPO inhibitor group shifts to the left in the flow histogram, and the fluorescence intensity decreases. This shows that the method of the present invention is feasible.
[0061] As can be seen from Example 1, the method of the present invention can determine the intracellular MPO enzyme activity in the living cell state; as can be seen from Examples 2 and 3, by comparing the control group and the MPO inhibitor group, the method of the present invention can detect the relative strength of the MPO enzyme activity. In actual use, the relative strength of the MPO activity in neutrophils (or macrophages, etc.) of different disease populations (or experimental animal disease models) and normal healthy populations (or normal animals) can be compared. This technology platform can also be used to screen new, efficient and safe MPO inhibitors as candidate drugs for disease treatment (such as myocardial infarction, etc.).
[0062] The above embodiments are only used to illustrate the technical solutions of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation shall fall within the protection scope of the present invention.
Claims
1. A method for detecting intracellular myeloperoxidase activity using flow cytometry, characterized in that: The steps include: 1) Taking cells containing myeloperoxidase; 2) using a solution containing a surfactant to perforate the cells containing myeloperoxidase; 3) incubating the punched cells obtained in step 2) in a detection buffer, wherein the detection buffer contains a fluorescent substrate transport inhibitor, chloride ions, hydrogen peroxide and a fluorescent substrate; 4) Detect the cell fluorescence intensity by flow cytometry, and determine the relative activity of intracellular myeloperoxidase based on the cell fluorescence intensity.
2. The method for detecting intracellular myeloperoxidase activity using flow cytometry according to claim 1, characterized in that: In step 2), the surfactant is at least one of saponin, digitonin, Triton X-100, deoxycholic acid, and Tween-80, and the surfactant concentration in the solution containing the surfactant is 0.0001-0.1%; the cells containing myeloperoxidase are incubated in the solution containing the surfactant for 3-5 minutes.
3. The method for detecting intracellular myeloperoxidase activity using flow cytometry according to claim 2, characterized in that: In step 2), the cells containing myeloperoxidase are incubated in a solution containing a surfactant for 3-5 minutes, and then the surfactant is removed to separate the perforated cells.
4. The method for detecting intracellular myeloperoxidase activity using flow cytometry according to claim 1, characterized in that: In step 3), the punched cells obtained in step 2) are incubated in a detection buffer for 10-30 minutes, wherein the detection buffer contains 0.2-1.5 mM fluorescent substrate transport inhibitor.
5. The method for detecting intracellular myeloperoxidase activity using flow cytometry according to claim 1, characterized in that: The detection buffer contains 0.2-1.5 mM fluorescent substrate transport inhibitor, 50-200 mM chloride ion, 20-1000 μM hydrogen peroxide and 15-50 μM fluorescent substrate; the fluorescent substrate transport inhibitor includes probenecid sodium.
6. The method for detecting intracellular myeloperoxidase activity using flow cytometry according to claim 1, characterized in that: The fluorescent substrate includes aminophenylfluorescein APF or fluorescent hypochlorous acid probe HKOCl-3.
7. The method for detecting intracellular myeloperoxidase activity using flow cytometry according to claim 1, characterized in that: The incubation in step 2) is carried out at room temperature, and the incubation in step 3) is carried out at 25-40°C.
8. A kit for detecting intracellular myeloperoxidase activity, characterized in that: It includes a solution containing a surfactant; a detection buffer containing a fluorescent substrate transport inhibitor, chloride ions, hydrogen peroxide and a fluorescent substrate.
9. A kit for detecting intracellular myeloperoxidase activity according to claim 8, characterized in that: The detection buffer contains 0.2-1.5 mM probenecid sodium, 50-200 mM chloride ion, 20-1000 μM hydrogen peroxide and 15-50 μM fluorescent substrate.
10. The kit for detecting intracellular myeloperoxidase activity according to claim 8, characterized in that: The surfactant is at least one of saponin, digitonin, TritonX-100, deoxycholic acid and Tween-80. The surfactant concentration in the solution containing the surfactant is 0.0001-0.1%.