Application of apoptotic bodies in the preparation of products for the treatment of sepsis

Abstract

The present application discloses an application of an apoptotic body in the preparation of a product for treating sepsis. The apoptotic body is an apoptotic body produced by stimulating cell apoptosis with staurosporine; 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 stimulating cell apoptosis with staurosporine can effectively prolong the survival time of mice with sepsis, indicating the application potential of the apoptotic body in the preparation of a product for treating sepsis.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to the use of an apoptotic body in the preparation of a product for treating sepsis. Background Art

[0002] Pathogenic bacteria or conditional pathogens invade the blood circulation, grow and reproduce, produce toxins and inflammatory molecules, and cause acute systemic infection. For the elderly, children, those with chronic diseases or low immune function, those who are not treated in time, and those with complications, it may develop into sepsis or septicemia. If not treated, the mortality rate is very high. However, for inexperienced doctors, unreasonable and irregular prescriptions of antibiotics are associated with high mortality rates. Therefore, it is urgent to find a new and effective solution that can effectively inhibit the growth of bacteria in addition to antibiotic treatment.

[0003] The cell membrane shrinks and invaginates, dividing and wrapping the cytoplasm, containing DNA materials and organelles, forming a bubble-like body called apoptosis body (AB). AB contains nucleic acids, proteins and lipids. Compared with other types of extracellular vesicles, the underlying mechanism of AB formation is poorly understood. Recent studies have shown that it is a highly regulated process mediated by membrane protrusions controlled by actin contraction. More and more evidence supports the importance of AB in immune regulation and related disease environments. Therefore, AB is not just a byproduct of cell apoptosis or apoptosis, but should be regarded as a key mechanism for communication between apoptotic cells and surrounding cells. As a bridge for transmitting functional substances between adjacent cells, AB has received increasing attention in the microenvironment of cell-to-cell interactions, and it has been found that they can deliver useful substances to healthy receptor cells. Summary of the invention

[0004] The present application provides an application of apoptotic bodies in the preparation of a product for treating sepsis. The apoptotic bodies produced under the stimulation of staurosporine can effectively prolong the survival time of mice with sepsis, indicating the application potential of apoptotic bodies in the preparation of a product for treating sepsis.

[0005] The present application provides an application of an apoptotic body in the preparation of a product for treating sepsis.

[0006] Optionally, in some embodiments of the present application, the apoptotic bodies are apoptotic bodies produced by staurosporine-stimulated cell apoptosis.

[0007] Optionally, in some embodiments of the present application, the apoptotic bodies are apoptotic bodies produced by staurosporine-stimulated macrophage apoptosis.

[0008] Optionally, in some embodiments of the present application, the method for preparing apoptotic bodies includes: extracting and culturing macrophages; stimulating and inducing macrophages with staurosporine; and extracting apoptotic bodies.

[0009] Optionally, in some embodiments of the present application, the stimulation concentration of staurosporine may be 0.3-0.7 μM, 0.4-0.6 μM, or 0.5 μM.

[0010] Optionally, in some embodiments of the present application, the macrophages are mouse bone marrow-derived macrophages.

[0011] 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.

[0012] Optionally, in some embodiments of the present application, the culture medium is DMEM culture medium.

[0013] 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.

[0014] Optionally, in some embodiments of the present application, the product is a pharmaceutical preparation, and the active ingredient of the pharmaceutical preparation is apoptotic bodies.

[0015] Optionally, in some embodiments of the present application, the dosage form of the drug preparation is an injection, a lyophilized agent or a sustained-release agent.

[0016] The present application uses apoptotic bodies to neutralize toxins, which has the following beneficial effects:

[0017] Through protein expression analysis, the expression of ADAM10 (A Disintegrin And Metalloprotease 10) in the apoptotic bodies produced after staurosporine stimulated macrophages was significantly upregulated. ADAM10 plays an important role in combating the alpha toxin (a pore-forming toxin produced by a bacterial pathogen that can bind to the host cell membrane), which can cause systemic infection and develop into sepsis. The present application found that the apoptotic bodies produced under the stimulation of staurosporine can effectively prolong the survival time of mice with sepsis, indicating the potential application of apoptotic bodies in the preparation of products for the treatment of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 is an electron microscopic image of the extracted apoptotic bodies;

[0020] Figure 2 This is a survival curve of mice with sepsis. DETAILED DESCRIPTION

[0021] 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.

[0022] The present application provides an application of apoptotic bodies in the preparation of a product for treating sepsis. 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.

[0023] The present application embodiment provides an application of an apoptotic body in the preparation of a product for treating sepsis.

[0024] In some embodiments of the present application, the apoptotic bodies are apoptotic bodies produced by staurosporine-stimulated cell apoptosis. The apoptotic bodies produced under staurosporine stimulation can effectively prolong the survival time of mice with sepsis, indicating the potential application of apoptotic bodies in the preparation of products for treating sepsis.

[0025] In some embodiments of the present application, the apoptotic bodies are apoptotic bodies produced by staurosporine-induced macrophage apoptosis.

[0026] In some embodiments of the present application, the method for preparing apoptotic bodies comprises: extracting and culturing macrophages; stimulating and inducing the macrophages with staurosporine; and extracting apoptotic bodies.

[0027] In some embodiments of the present application, the stimulation concentration of staurosporine may be 0.3-0.7 μM, 0.4-0.6 μM, or 0.5 μM.

[0028] In some embodiments of the present application, the macrophages are mouse bone marrow-derived macrophages.

[0029] 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.

[0030] 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).

[0031] In some embodiments of the present application, the steps of extracting and culturing macrophages include:

[0032] 1) LPS was intraperitoneally injected into 6- to 8-week-old C57 mice, with each mouse receiving an injection volume of 240 to 260 g;

[0033] 2) After 2 to 4 hours, flush out the mouse bone marrow and centrifuge;

[0034] 3) Add 2-3 mL of red blood cell lysis buffer and lyse for 5 minutes;

[0035] 4) Add 3-5 ml of 10% DMEM high-glucose medium to neutralize, filter with a 70 μm filter, and centrifuge;

[0036] 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).

[0037] 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.

[0038] 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).

[0039] 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.

[0040] In some embodiments of the present application, the step of extracting apoptotic bodies comprises:

[0041] S1: Blow off the stimulated macrophages with a pipette and collect the mixture of macrophages and culture medium;

[0042] S2: centrifuge and collect the supernatant;

[0043] S3: After centrifugation of the supernatant, the precipitate was collected to obtain apoptotic bodies.

[0044] In some embodiments of the present application, the product is a pharmaceutical preparation, and the active ingredient of the pharmaceutical preparation is the apoptotic body produced by staurosporine-induced macrophage apoptosis.

[0045] In some embodiments of the present application, the dosage form of the pharmaceutical preparation is an injection, a lyophilized preparation or a sustained-release preparation.

[0046] In some embodiments of the present application, when the dosage form of the pharmaceutical preparation is an injection, the pharmaceutical preparation comprises the apoptotic bodies produced by the above-mentioned staurosporine-induced macrophage apoptosis and an injection solution, and the injection solution is selected from physiological saline or PBS buffer.

[0047] In some embodiments of the present application, when the dosage form of the pharmaceutical preparation is a lyophilized preparation, the pharmaceutical preparation is lyophilized from the apoptotic bodies produced by the above-mentioned staurosporine-induced macrophage apoptosis.

[0048] In some embodiments of the present application, when the dosage form of the drug preparation is a sustained-release preparation, the drug preparation comprises the apoptotic bodies produced by the above-mentioned staurosporine-induced macrophage apoptosis and a sustained-release carrier, and the material of the sustained-release carrier is selected from microcapsules, microspheres, microparticles, liposomes or nano-microspheres.

[0049] The following describes the invention in conjunction with specific embodiments.

[0050] Example 1: Preparation of apoptotic bodies

[0051] The method for preparing apoptotic bodies in this embodiment comprises the following steps:

[0052] 1) LPS (1 mg / mL) was intraperitoneally injected into 7-week-old C57 mice, with an injection volume of 250 g per mouse;

[0053] 2) 3 hours later, the mouse bone marrow was flushed out and centrifuged at 1000 rpm for 5 minutes;

[0054] 3) Add 3 mL of red blood cell lysis buffer and lyse for 5 minutes;

[0055] 4) Add 5 ml of 10% DMEM high glucose medium to neutralize, filter with a 70 μm filter, and centrifuge at 1000 rpm for 5 minutes;

[0056] 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;

[0057] 6) The macrophages were stimulated with staurosporine at a concentration of 0.5 μM;

[0058] 7) Blow off the stimulated and induced macrophages with a pipette to collect the mixture of macrophages and culture medium;

[0059] 8) Centrifuge at 50 g and collect the supernatant;

[0060] 9) The supernatant was centrifuged at 800 g and the precipitate was collected to obtain apoptotic bodies.

[0061] 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.

[0062] Example 2: Validation of mouse sepsis model

[0063] The validation method and results of the mouse sepsis model of this example are as follows:

[0064] 1) Establishment of mouse sepsis model: 8-week-old C57BL / 6J mice weighing 18-20 g were selected and injected with 3x10 7 CFU of heat-killed Staphylococcus aureus. Within 12-24 days after inoculation, mice will be less active but will not die. After 24 hours, the liver of mice will have cysts or spots, and the spleen will have no obvious changes.

[0065] 2) A small dose (10 μg / each) and a large dose (30 μg / each) of apoptotic bodies were injected into mice, and the survival status of the mice was tracked and recorded.

[0066] Through the survival curve analysis, it was found that Figure 2 As shown in the figure, S.au represents Staphylococcus aureus, STS represents staurosporine, AB represents apoptotic bodies, STS AB refers to apoptotic bodies produced under the stimulation of staurosporine, S.au NC is the group injected with Staphylococcus aureus alone, S.au+high-dose STS AB is the group injected with Staphylococcus aureus and then injected with high-dose apoptotic bodies, and S.au+low-dose STS AB is the group injected with Staphylococcus aureus and then injected with low-dose apoptotic bodies. The survival time of mice stimulated by apoptotic bodies after injection of staurosporine was significantly prolonged, and most of the control group (sepsis model group) died within 5 days. For the group injected with apoptotic bodies, most of the mice survived for more than two weeks, and the survival probability of mice in the S.au+low-dose STS AB group was higher than that in the S.au+high-dose STS AB group. It can be said that apoptotic bodies produced by macrophages stimulated by staurosporine can effectively prolong the survival time of mice with sepsis.

[0067] The apoptotic bodies produced by macrophages stimulated by staurosporine provided in the present application can effectively prolong the survival time of mice with sepsis, indicating the application potential of apoptotic bodies in the preparation of products for the treatment of sepsis.

[0068] The above is a detailed introduction to the application of an apoptotic body provided by the present application in the preparation of a product for treating sepsis. Specific examples are used herein 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 those skilled in the art, according to the idea 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 limiting the present application.

Claims

1. Use of apoptotic bodies in the preparation of drugs for treating sepsis, wherein the apoptotic bodies are apoptotic bodies produced by staurosporine-stimulated macrophage apoptosis, and the macrophages are mouse bone marrow-derived macrophages.

2. The use according to claim 1, characterized in that: The method for preparing the apoptotic body comprises: extracting and culturing the macrophages; using the staurosporine to stimulate and induce the macrophages; and extracting the apoptotic body.

3. The use according to claim 2, characterized in that: The stimulation concentration of staurosporine is 0.3-0.7 μM.

4. The use according to claim 2, characterized in that: The steps of extracting and culturing macrophages include: intraperitoneally injecting lipopolysaccharide into mice to obtain mouse bone marrow and centrifuging it; adding red blood cell lysis solution to lyse it; adding culture medium to neutralize it, filtering it and centrifuging it; resuspending the cells and culturing them for 6 to 9 hours, taking the non-adherent cell suspension, centrifuging it, and resuspending and culturing it with a culture medium containing macrophage colony stimulating factor.

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

6. The use according to claim 2, 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.

7. The use according to claim 1, characterized in that: The medicine is a pharmaceutical preparation, and the active ingredient of the pharmaceutical preparation is the apoptotic body.

8. The use according to claim 7, characterized in that: The dosage form of the pharmaceutical preparation is injection, lyophilized preparation or sustained release preparation.

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