Application of apoptotic bodies in neutralizing toxins

Abstract

The present application discloses an application of an apoptotic body in neutralizing toxins. The apoptotic body is an apoptotic body produced by staurosporine-induced cell apoptosis, and the toxin is a pore-forming toxin; the preparation method of the apoptotic body includes: extracting and culturing macrophages; stimulating and inducing macrophages with staurosporine; and extracting the apoptotic body. The apoptotic body produced by staurosporine-induced cell apoptosis has significantly upregulated expression of ADAM10, which can effectively counteract pore-forming toxins and may become an effective agent against bacteria and other pathogens during infection.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to the application of an apoptotic body in neutralizing toxins. Background Art

[0002] A common infection strategy of bacterial pathogens is to produce pore-forming toxins to destroy the cell membrane of host cells. Bacterial pore-forming toxins are a type of toxins that are secreted by pore-forming proteins, directly invade the cell membrane and form pore structures on the cell membrane, causing ions and other toxic cytokines that cannot enter the cell under normal circumstances to enter the cell, causing cell swelling and cell lysis. They are also called cytolytic toxins. Since red blood cells are usually used in such experiments, this type of toxin is also named hemolysin. It is a toxin that directly destroys the cell membrane and is an important pathogenic factor produced by Gram-negative bacteria and Gram-positive bacteria.

[0003] A series of studies published in the international authoritative journal Nature proves that ADAM10 (ADisintegrin And Metalloprotease 10) plays an important role in fighting against α-toxin (a pore-forming toxin that can bind to cell membranes and tissues). It proves that ATG16L1 and other ATG proteins can fight against α-toxin by releasing exosomes carrying ADAM10.

[0004] Extracellular vesicles (EVs) refer to tiny vesicles that are secreted into the extracellular environment through cell membrane fusion after cells in the body form multivesicular bodies through endocytosis under physiological and pathological conditions. According to the size of the vesicle diameter, EVs can be divided into three categories: apoptotic bodies (about 1000nm), microvesicles (100-1000nm), and exosomes (30-100nm). EVs were originally regarded as "garbage bags" of cells, used to remove unnecessary macromolecules, but now they are considered to be carriers of intercellular signals and can be used for intercellular communication. EVs surface protein signaling molecules can recognize target cells and take in EVs through receptor ligand binding or endocytosis, thereby changing the physiological and pathological state of target cells. A series of studies have shown that EVs are widely involved in the physiological and pathological processes of the human body. Summary of the invention

[0005] The present application provides an application of apoptotic bodies in neutralizing toxins. The apoptotic bodies produced under the stimulation of staurosporine can effectively resist pore-forming toxins and can become an effective agent against bacteria and other pathogens during infection.

[0006] The present application provides an application of an apoptotic body in neutralizing toxins.

[0007] Optionally, in some embodiments of the present application, the apoptotic body is an apoptotic body produced by staurosporine-induced cell apoptosis.

[0008] Optionally, in some embodiments of the present application, the apoptotic bodies are apoptotic bodies produced by staurosporine-induced macrophage apoptosis.

[0009] Optionally, in some embodiments of the present application, the toxin is a pore-forming toxin.

[0010] Optionally, in some embodiments of the present application, the method for preparing apoptotic bodies includes: extracting and culturing macrophages; stimulating and inducing the macrophages with staurosporine; and extracting apoptotic bodies.

[0011] Optionally, in some embodiments of the present application, the stimulation concentration of staurosporine may be 0.25-1.0 μM, 0.3-0.8 μM, or 0.5 μM.

[0012] Optionally, in some embodiments of the present application, the macrophages are mouse bone marrow-derived macrophages.

[0013] Optionally, in some embodiments of the present application, the step of extracting and culturing macrophages includes: injecting lipopolysaccharide intraperitoneally into mice to obtain mouse bone marrow and centrifuge; adding red blood cell lysis solution to lyse; adding culture medium to neutralize, filtering and centrifuging; resuspending the cells and culturing for 6 to 9 hours, taking the non-adherent cell suspension, centrifuging, resuspending and culturing with culture medium containing macrophage colony stimulating factor.

[0014] Optionally, in some embodiments of the present application, the culture medium is DMEM culture medium.

[0015] Optionally, in some embodiments of the present application, the step of extracting apoptotic bodies includes: centrifuging the macrophages after stimulation and induction, collecting the supernatant, and collecting the precipitate after centrifuging the supernatant to obtain apoptotic bodies.

[0016] Accordingly, the present application also provides a method for neutralizing toxins, comprising: extracting and culturing macrophages; stimulating and inducing the macrophages with staurosporine; extracting apoptotic bodies; and neutralizing toxins with apoptotic bodies.

[0017] The present application uses apoptotic bodies to neutralize toxins, which has the following beneficial effects:

[0018] Through protein expression analysis, the expression of ADAM10 in the apoptotic bodies produced after staurosporine stimulation of macrophages was significantly upregulated. ADAM10 plays an important role in combating the encoded alpha toxin (a pore-forming toxin produced by a bacterial pathogen that can bind to the host cell membrane). The present application found that the apoptotic bodies produced under staurosporine stimulation can effectively combat the alpha toxin produced by bacterial pathogens and can become an effective agent against bacteria and other pathogens during infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 is an electron microscopic image of the extracted apoptotic bodies;

[0021] Figure 2 is a graph showing the detection results of the extracted apoptotic body markers (C1q, Annexn V);

[0022] Figure 3 This is a graph showing the detection results of the extracted apoptotic body marker (Caspase-3);

[0023] Figure 4 This is the result of Western blot experiment of ADAM10 extracted apoptotic bodies;

[0024] Figure 5 This is a graph showing the results of a cytotoxicity assay. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application.

[0026] The present application provides an application of apoptotic bodies in neutralizing toxins. The following are detailed descriptions. It should be noted that the description order of the following embodiments is not intended to limit the preferred order of the embodiments.

[0027] The present application example provides an application of an apoptotic body in neutralizing toxins.

[0028] In some embodiments of the present application, the apoptotic body is an apoptotic body produced by staurosporine-induced apoptosis. The apoptotic body (>1000nm) produced by staurosporine-induced apoptosis has significantly upregulated expression of ADAM10, which can effectively resist pore-forming toxins and may become an effective agent against bacteria and other pathogens during infection.

[0029] In some embodiments of the present application, the apoptotic bodies are apoptotic bodies produced by staurosporine-induced macrophage apoptosis.

[0030] In some embodiments of the present application, the toxin is a pore-forming toxin.

[0031] In some embodiments of the present application, the toxin is alpha toxin; alpha toxin is a pore-forming toxin produced by a bacterial pathogen that can bind to the host cell membrane, wherein the bacterial pathogen mainly refers to methicillin-resistant Staphylococcus aureus.

[0032] In some embodiments of the present application, the method for preparing apoptotic bodies includes: extracting and culturing macrophages; stimulating and inducing the macrophages with staurosporine; and extracting apoptotic bodies.

[0033] In some embodiments of the present application, the stimulation concentration of staurosporine may be 0.25-1.0 μM, 0.3-0.8 μM, or 0.5 μM.

[0034] In some embodiments of the present application, the macrophages are mouse bone marrow-derived macrophages.

[0035] In some embodiments of the present application, the steps of extracting and culturing macrophages include: intraperitoneally injecting lipopolysaccharide (LPS) into mice to obtain mouse bone marrow and centrifuge; adding red blood cell lysis solution to lyse; adding culture medium to neutralize, filtering and centrifuging; resuspending the cells and culturing for 6 to 9 hours, taking the non-adherent cell suspension, centrifuging, resuspending and culturing with culture medium containing macrophage colony stimulating factor.

[0036] In some embodiments of the present application, the culture medium is DMEM culture medium; specifically, the culture medium is 10% DMEM high-glucose culture medium (containing 10% FBS).

[0037] In some embodiments of the present application, the steps of extracting and culturing macrophages include:

[0038] 1) LPS was intraperitoneally injected into 6- to 8-week-old C57 mice, with an injection volume of 240 to 260 g per mouse;

[0039] 2) After 2 to 4 hours, flush out the mouse bone marrow and centrifuge;

[0040] 3) Add 2-3 mL of red blood cell lysis buffer and lyse for 5 minutes;

[0041] 4) Add 3-5 ml of 10% DMEM high-glucose medium to neutralize, filter with a 70 μm filter, and centrifuge;

[0042] 5) Resuspend in 10% DMEM high-glucose medium, culture for 6 to 9 hours, take out the non-adherent cell suspension, centrifuge, resuspend and culture in 10% DMEM high-glucose medium containing macrophage colony stimulating factor (MCSF).

[0043] Among them, the amount of red blood cell lysis solution added in step 3) depends on the amount of cells, and the lysis time should not be too long, generally about 5 minutes.

[0044] The amount of 10% DMEM high-glucose medium added in step 4) needs to be greater than the amount of lysis solution added in step 3).

[0045] In some embodiments of the present application, the step of extracting apoptotic bodies includes: centrifuging the macrophages after stimulation and induction, collecting the supernatant, and collecting the precipitate after centrifuging the supernatant to obtain apoptotic bodies.

[0046] In some embodiments of the present application, the step of extracting apoptotic bodies comprises:

[0047] S1: Blow off the stimulated macrophages with a pipette and collect the mixture of macrophages and culture medium;

[0048] S2: centrifuge and collect the supernatant;

[0049] S3: After centrifugation of the supernatant, the precipitate was collected to obtain apoptotic bodies.

[0050] The embodiment of the present application also provides a method for neutralizing toxins, comprising: extracting and culturing macrophages; stimulating and inducing the macrophages with staurosporine; extracting apoptotic bodies; and neutralizing toxins with apoptotic bodies.

[0051] The following describes the invention in conjunction with specific embodiments.

[0052] Example 1: Preparation of apoptotic bodies

[0053] The method for preparing apoptotic bodies in this embodiment comprises the following steps:

[0054] 1) LPS (1 mg / mL) was intraperitoneally injected into 7-week-old C57 mice, with an injection volume of 250 g per mouse;

[0055] 2) 3 hours later, the mouse bone marrow was flushed out and centrifuged at 1000 rpm for 5 minutes;

[0056] 3) Add 2 mL of red blood cell lysis buffer and lyse for 5 minutes;

[0057] 4) Add 3 ml of 10% DMEM high glucose medium to neutralize, filter with a 70 μm filter, and centrifuge at 1000 rpm for 5 minutes;

[0058] 5) Resuspend in 10% DMEM high glucose medium and culture in a 100 mm cell culture dish. After 6-9 hours, take the non-adherent cell suspension, count, centrifuge at 1000 rpm for 5 minutes, and replace with 10% DMEM high glucose medium containing MCSF (30 ng / mL) at 2×10 6 / ml density and seeded on 12-well plates. After 3 days, the macrophages matured;

[0059] 6) The macrophages were stimulated with staurosporine at concentrations of 0.25 μM, 0.5 μM, and 1.0 μM;

[0060] 7) Blow off the stimulated and induced macrophages with a pipette to collect the mixture of macrophages and culture medium;

[0061] 8) Centrifuge at 50 g and collect the supernatant;

[0062] 9) The supernatant was centrifuged at 800 g and the precipitate was collected to obtain apoptotic bodies.

[0063] The basic morphology of the extracted apoptotic bodies was identified. Figure 1 As shown, Figure 1 This is the basic morphology of apoptotic bodies derived from mouse bone marrow macrophages observed under an electron microscope.

[0064] Example 2: Experimental Verification

[0065] The extracted apoptotic bodies were experimentally verified as follows:

[0066] 1) Detection of markers of apoptotic bodies (C1q, Annexn V) extracted: by immunofluorescence staining, cells were fixed with 4% paraformaldehyde, perforated with 0.1% Triton x-100, blocked with 5% bovine serum, and incubated with antibodies diluted with bovine serum as primary antibodies (anti-C1q, anti-Annexn V), and further incubated with secondary antibodies diluted with 2% bovine serum (the species should correspond to the primary antibody). Observed and photographed using a confocal microscope. The results are as follows Figure 2 As shown, it can be seen that the markers C1q and Annexn V are expressed in the extracted apoptotic bodies.

[0067] 2) Detection of the extracted apoptotic body marker (Caspase-3): A protein immunoblotting experiment was performed to separate proteins according to molecular weight by gel electrophoresis, and then the proteins were transferred to a blotting membrane made of PVDF (polyvinylidene fluoride). The probe was then detected using a primary antibody (Caspase3 antibody) specific for the target protein Caspase3. The transferred PVDF membrane was blocked with a blocking buffer to prevent the primary antibody from binding to the membrane itself. The primary antibody was further incubated with the transferred PVDF membrane to promote the binding of the primary antibody to the target protein and wash away the unbound primary antibody. A secondary antibody that can recognize and bind to the primary antibody was further added. This secondary antibody can be coupled to an enzyme that can detect the target protein, and the target protein is displayed as a band on the blot. Wash with a washing buffer to wash away the unbound secondary antibody. Depending on the secondary antibody used, the target protein is detected by luminescence. The results are as follows. Figure 3 As shown, Big-AB refers to apoptotic bodies. When induced by staurosporine stimulation, the extracted apoptotic bodies expressed Caspase-3, and when the stimulation concentration of staurosporine was 0.5 μM and 1.0 μM, the expression level of Caspase-3 was higher than that when the stimulation concentration was 0.25 μM.

[0068] 3) Detect the expression of ADAM10: Use protein immunoblotting experiment to separate proteins according to molecular weight by gel electrophoresis, then transfer the proteins to a blotting membrane made of PVDF (polyvinylidene fluoride), and then use a primary antibody (ADAM10 antibody) specific to the target protein ADAM10 for probe detection. The PVDF membrane after transfer is blocked with blocking buffer to prevent the primary antibody from binding to the membrane itself, and the primary antibody is further incubated with the PVDF membrane after transfer to promote the binding of the primary antibody to the target protein and wash away the unbound primary antibody. Further add a secondary antibody that can recognize and bind to the primary antibody. This secondary antibody can be coupled to an enzyme that can detect the target protein, and the target protein is displayed as a band on the blot. Wash with washing buffer to wash away the unbound secondary antibody. Depending on the secondary antibody used, the target protein is detected by luminescence. ADAM10 plays an important role in combating proteins that encode α-toxin (a pore-forming toxin that can bind to cell membranes and is widely present in tissues), such as Figure 4 As shown in the Western blotting experiment, compared with the negative control of macrophages not stimulated with staurosporine, the expression level of ADAM10 in the apoptotic bodies produced by macrophages stimulated with staurosporine was higher, and when the stimulation concentration of staurosporine was 1 μM, the expression level of ADAM10 was higher than that at the stimulation concentrations of 0.25 μM and 0.5 μM.

[0069] 4) Cytotoxicity detection: Using the thiazole blue colorimetric method (MTT method), the prepared MTT solution was added to the cell culture medium of A549 cells (human non-small cell lung cancer cell line), and the detection was performed by a microplate reader at a wavelength of A570. The cells were stimulated with apoptotic bodies extracted from macrophages stimulated with α-toxin and α-toxin and 0.5 μM staurosporine, respectively. NC was a negative control group that was not stimulated with α-toxin and apoptotic bodies. The results of the cytotoxicity test were as follows: Figure 5 As shown, apoptotic bodies can effectively reduce the cytotoxicity after toxin (α-toxin) stimulation, indicating that the apoptotic bodies of the present application can play a role in neutralizing toxins.

[0070] The apoptotic bodies produced by macrophages stimulated by staurosporine provided in the present application have a high expression level of ADAM10, and can effectively neutralize toxins and reduce cytotoxicity.

[0071] The above is a detailed introduction to the application of an apoptotic body provided by the present application in neutralizing toxins. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for technicians in this field, according to the ideas of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A use of an apoptotic body for neutralizing toxins for non-therapeutic purposes in vitro, characterized in that: The apoptotic bodies are apoptotic bodies produced by staurosporine-induced cell apoptosis; Wherein, the toxin is a pore-forming toxin, and the method for preparing the apoptotic body comprises: extracting and culturing macrophages, stimulating and inducing the macrophages with staurosporine, extracting the apoptotic body, and then using the apoptotic body to neutralize the toxin, wherein the macrophage is a macrophage derived from mouse bone marrow; Wherein, the stimulation concentration of staurosporine is 0.5 μM.

2. The use according to claim 1, characterized in that: The apoptotic bodies are apoptotic bodies produced by staurosporine-induced macrophage apoptosis.

3. The use according to claim 1, characterized in that: The steps of extracting and culturing macrophages include: intraperitoneally injecting lipopolysaccharide into mice to obtain mouse bone marrow and centrifuge it; Add red blood cell lysis buffer to lyse; add culture medium to neutralize, filter and centrifuge; resuspend the cells and culture for 6 to 9 hours, then take the non-adherent cell suspension, centrifuge, resuspend and culture with culture medium containing macrophage colony stimulating factor.

4. The use according to claim 3, characterized in that: The culture medium is DMEM culture medium.

5. The use according to claim 1, characterized in that: The step of extracting apoptotic bodies comprises: centrifuging the macrophages after stimulation and induction, collecting the supernatant, and collecting the precipitate after centrifuging the supernatant to obtain the apoptotic bodies.

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