Method for obtaining immune cell extracellular trap and application

Abstract

The invention provides a method for obtaining an immune cell extracellular trap and application thereof. According to the method disclosed by the invention, the generation of extracellular traps of macrophages is efficiently induced by utilizing nano piezoelectric particles and cooperating with ultrasonic treatment. The method provided by the invention is simple to operate, effectively induces the generation of extracellular traps of macrophages in a short time, and can effectively avoid the use of pure pathogenic stimulating substances or chemical substances to induce immune cells to continuously activate the extracellular traps and avoid the damage of bacteria or chemical stimulating substance residues to organisms.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology, and in particular relates to a method for obtaining extracellular traps of immune cells and an application thereof. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Brinkmann et al. found that neutrophils can release a mesh structure called extracellular traps (ETs) after exposure to a series of pathogenic and pro-inflammatory stimuli. ETs are composed of DNA skeletons, proteins and hydrolases, including myeloperoxidase (MPO), matrix metalloproteinase 12 (MMP12), histones, etc. The composition of ETs may be affected by the cell type and the local environment where the traps are released. ETs are released by a special cell death pathway called "ETosis" that is different from apoptosis and necrosis, and can neutralize and kill pathogenic microorganisms. Therefore, the release of extracellular traps provides a means to eliminate and destroy invading pathogens.

[0004] Like neutrophils, macrophages can also release extracellular traps (METs) after exposure to a range of pathogenic and pro-inflammatory stimuli (e.g., bacteria, LPS). Recent studies have shown that METs can not only capture and immobilize bacteria very quickly (within 30 seconds) with low energy consumption, but also fully engage with microbial pathogens and kill them with high efficiency. Recently, it has been reported that chemical stimuli can induce pro-inflammatory responses in bone marrow-derived macrophages (BMDM), leading to the production of reactive oxygen species (ROS) and pro-inflammatory cytokines (e.g., TNFα and IL-8). Subsequently, PAD enzymes (PAD2 or PAD4) are activated and transported to the nucleus and citrullinated histones, thereby promoting chromatin decondensation and the release structure of METs. In addition, it has been reported that chemical substances (gold nanoparticles, silver nanoparticles, nanopowders, etc., NPs) can induce the release of ETs from immune cells. When NPs contact the neutrophil membrane, cell activation and / or cell membrane damage can lead to lysosomal damage and ROS generation. NP stimulation can induce histone degradation and chromatin condensation, promote the formation of NETs, ​​and neutrophils may also form aggregated NETs and capture NPs to eliminate and fix inflammatory particles. However, there are few reports on macrophages. For example, phorbolmyristate acetate (PMA) is used as a common extracellular trap inducer to induce METs. In addition, mineral nanoparticles formed by combining calcium phosphate-based biomaterials with human platelet lysate can drive BMDM to release extracellular traps. However, the duration of chemical stimulation is uncontrollable. If cells are continuously stimulated, it may cause chemical contamination of cells, persistent inflammatory response or other uncontrollable cell responses. Summary of the invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a method and application for obtaining macrophage extracellular traps. Specifically, the present invention utilizes nano-piezoelectric particles and cooperates with ultrasonic treatment to controllably induce the generation of immune cell extracellular traps. The method provided by the present invention is simple to operate, effectively induces the generation of immune cell extracellular traps in a short time, and can effectively avoid the induction of continuous stimulation of immune cells by simple pathogenic stimuli or chemical substances, and avoids the residual bacteria or chemical stimuli that affect biosafety.

[0006] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions:

[0007] In a first aspect, the present invention provides a method for obtaining extracellular traps of immune cells, wherein the immune cells are treated by mechanical stimulation in combination with nano-piezoelectric particle stimulation;

[0008] The mechanical stimulation is ultrasonic treatment; the nano piezoelectric particles are nanoparticles with the ability of piezoelectric catalysis to produce ROS.

[0009] In a specific embodiment of the present invention, the immune cells include macrophages.

[0010] In a specific embodiment of the present invention, the nano piezoelectric particles include barium titanate, surface-loaded metal nanoparticles barium titanate, lithium niobate, bismuth iodate, hydroxyapatite, polylactic acid, collagen, two-dimensional MoS2, etc.

[0011] Further, the nano-piezoelectric particles are surface-loaded metal nano-particles of barium titanate. The metal nano-particles are Au, Pt, Pd, Al or Ni nano-particles. Preferably, the nano-piezoelectric particles are BaT&Au, which are obtained by depositing Au on the surface of BaTiO3.

[0012] In a specific embodiment of the present invention, the particle size of the nano piezoelectric material is 30-300nm.

[0013] In a specific embodiment of the present invention, the amount of the nano piezoelectric particles added is 0.2-0.5 mg / 10 6 Preferably, the amount of nano piezoelectric particles added is 0.35 mg / 10 6 cells.

[0014] In a specific embodiment of the present invention, the method is: first, nano-piezoelectric particles are co-incubated with immune cells to allow the immune cells to internalize the nano-piezoelectric particles, excess nano-piezoelectric particles are cleaned, and then ultrasonic treatment of the immune cells produces METs, and finally centrifugation purification is performed to obtain immune cell extracellular traps.

[0015] In a specific embodiment of the present invention, the co-incubation time is 0.5-1.5h.

[0016] In a specific embodiment of the present invention, the ultrasonic treatment time is 5-20s.

[0017] In a specific embodiment of the present invention, the power of the ultrasonic treatment is 0.1 to 1 W / cm 2 ;

[0018] Preferably, the ultrasonic power is 0.1 W / cm 2 .

[0019] Macrophages phagocytized nanoparticles, and ultrasound-treated cells increased intracellular ROS levels, pro-inflammatory cytokines (such as TNFα and IL-6), and Ca 2+Subsequently, PAD enzymes (PAD2 or PAD4) are activated and transported to the nucleus and citrullinated histones, thereby promoting chromatin condensation and the release of METs. Ultrasonic treatment and nanopiezoelectric particles synergistically treat macrophages to promote the massive production of macrophage extracellular traps while avoiding continuous activation of macrophages. Subsequent centrifugal purification avoids the residual nanopiezoelectric particles.

[0020] The second aspect of the present invention provides immune cell extracellular traps prepared by the method described in the first aspect.

[0021] The third aspect of the present invention provides the use of the preparation method described in the first aspect or the immune cell extracellular trap described in the second aspect in the preparation of a product for preventing, treating and / or improving bacterial inflammation;

[0022] Preferably, the product comprises an antimicrobial material.

[0023] A fourth aspect of the present invention provides an anti-inflammatory drug, which includes the immune cell extracellular trap described in the second aspect.

[0024] One or more of the above technical solutions have the following beneficial effects:

[0025] The method provided by the present invention can effectively induce macrophages, and the induced macrophages can generate ETs on their own without stimulation. The operation is simple, and can effectively avoid the continuous activation of extracellular traps by immune cells induced by simple pathogenic stimuli or chemical substances, and avoid the damage to the body caused by residual bacteria or chemical stimuli.

[0026] The METs active substance produced by the method of the present invention can effectively capture and kill bacteria, and can achieve a 98% sterilization efficiency against Staphylococcus aureus.

[0027] Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1 This is a diagram showing the mechanism by which mechanical stimulation ultrasound combined with nano-piezoelectric particles promotes the release of extracellular traps of macrophages and BMDM.

[0030] Figure 2This is a comparison of Hoechst33342 and SYTOX Green fluorescence imaging of macrophages in Experimental Example 1 of the present invention, among the BMDM extracellular traps, the control group, the ultrasound group, the nano piezoelectric particles (PNPs) group and the method of the present invention (PNPs+US group) at different culture times.

[0031] Figure 3 This is the WB-protein expression of BMDM extracellular traps in Experimental Example 1 of the present invention.

[0032] Figure 4 This is the Hoechst33342 and SYTOX Green fluorescence imaging of BMDM extracellular traps in Example 1 of the present invention.

[0033] Figure 5 This is a scanning electron microscope observation image of BMDM extracellular traps according to Example 1 of the present invention.

[0034] Figure 6 The inhibition of METs on S. aureus obtained at different culture times in Example 2 of the present invention is shown. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. Experimental steps not described in detail in the present invention are performed according to conventional experimental operations or according to product instructions.

[0037] BaT&Au were dispersed into 1640 culture medium.

[0038] Experimental Example 1

[0039] Control group: BMDM were seeded in CellAttach six-well plates, 5×10 6 Cells (825 μL 1640 medium) were cultured in 1640 medium containing 20% ​​FBS at 37°C and 5% CO2 until adhered. Cultured in a 37°C and 5% CO2 cell culture incubator for 4 h;

[0040] Ultrasound group: BMDM were seeded in CellAttach six-well plates, 5×10 61640 medium containing 20% ​​FBS was used to culture cells (825 μL 1640 medium) in a 37°C, 5% CO2 cell culture incubator until they adhered to the wall. Ultrasound was applied after 1 hour of culture: P = 0.1 W / cm 2 10s, placed in a 37°C, 5% CO2 cell culture incubator for 4h;

[0041] PNPs group: BMDM were seeded in CellAttach six-well plates, with 5×10 6 Each well was added with 175 μL of 2 mg / mL BaT@Au, incubated for 1 hour, and then washed away the unphagocytosed BaT&Au, and then placed in a 37°C, 5% CO2 cell culture incubator for 4 hours.

[0042] PNPs+US group: BMDM were seeded in CellAttach six-well plates, with 5×10 6 Each well was added with 175 μL of 2 mg / mL BaT&Au, and the unphagocytosed BaT&Au was washed off after 1 hour of incubation. Ultrasound was applied: P = 0.1 W / cm 2 10s, and culture in a 37°C, 5% CO2 cell culture incubator for 4h.

[0043] The cells obtained from the above four groups were rinsed twice with PBS, 200 μL μM Hoechst 33342 (Biosharp, BL803A) was added to each well, and incubated in a cell culture incubator at 37°C and 5% CO2 for 20 min in the dark, rinsed twice with PBS, and then 200 μL, 1 μM SYTOX Green (ThermoFisher, S7020) was added, and incubated in a cell culture incubator at 37°C and 5% CO2 for 10 min in the dark. After rinsing with PBS three times, the cells were observed using a laser scanning confocal microscope (CLSM).

[0044] Alternatively, the cells obtained from the above four groups were rinsed with PBS, 200 μL of lysis buffer (RIPA:PMSF=100:1) was added to each well, and SDS-PAGE electrophoresis was performed after protein denaturation.

[0045] Laser scanning confocal microscopy results Figure 2 As shown, 4 h after treatment, there was no or almost no extracellular traps produced in the control group, ultrasound group and PNPs group, and 2 mg / mL BaT&Au induced the production of BMDM extracellular traps under ultrasound conditions, proving that mechanical stimulation can be used to induce BMDM and obtain BMDM with extracellular traps.

[0046] MPO, PAD4 and cit-H3 proteins are related proteins produced by extracellular traps. Western Blot results are shown in Figure 3 As shown, 2 mg / mL BaT&Au synergistic ultrasound treatment (PNPs+US) effectively induced the increase of MPO, PAD4 and cit-H3 protein expression in BMDM, further proving that mechanical stimulation synergistically induces nanopiezoelectric particles to induce BMDM and obtain BMDM with extracellular traps.

[0047] Embodiment 1

[0048] A method for obtaining macrophage extracellular traps, specifically, (1) macrophage BMDM were seeded in a 24-well plate, with 1×10 6 The cells were cultured normally in 20% FBS1640 medium at 37°C and 5% CO2 in a cell culture incubator until they adhered to the wall.

[0049] Nanopiezoelectric BaT&Au particles were dispersed into 1640 culture medium, 175 μL of 2 mg / mL BaT&Au was added to each well, and the final volume of 1640 culture medium was 1 mL. After incubation for 1 h, PBS was used to wash away the unphagocytosed BaT@Au.

[0050] The cells after washing were treated with a power of P = 0.1 W / cm 2 , 1MHz, 10s ultrasonic sonication, after which 1640 medium containing 20% ​​FBS was added. The cells were cultured in a 37°C, 5% CO2 cell culture incubator, and samples were taken at 0h, 2h, 4h, 8h, and 12h for observation.

[0051] The cells obtained in step (1) at different times were rinsed twice with PBS, 200 μL Hoechst 33342 (Biosharp, BL803A) was added to each well, incubated at 37°C in the dark for 20 min, rinsed twice with PBS, and then 200 μL 1 μM SYTOX Green (ThermoFisher, S7020) was added, incubated at 37°C in the dark for 10 min, rinsed three times with PBS, and observed with a laser scanning confocal microscope (CLSM). The results are shown in Figure 2. Figure 3 As shown, the generation of macrophage extracellular traps can be clearly observed 2 hours after the piezoelectric material is washed away and ultrasonic treatment is performed, and a large number of macrophage extracellular traps can still be observed after 12 hours, indicating that the method of the present invention can induce macrophage extracellular traps for a long time.

[0052] The cells obtained at different times in step (1) were rinsed with PBS twice, added with 500 μL 2.5% glutaraldehyde (pH 7.2-7.4), fixed at 4°C for 1 h, washed with PBS three times, dehydrated with ethanol gradient (30%, 50%, 70%, 90%), dehydrated with anhydrous ethanol three times, each for 15 min, replaced with tert-butyl alcohol twice, and freeze-dried. After gold plating, SEM (PhenomProx) observation was performed.

[0053] Figure 4 , Figure 5 It can be seen that 2 mg / mL BaT@Au induced the extracellular trap phenomenon of BMDM producing fibrous network structures under ultrasonic conditions (PNPs+US).

[0054] Embodiment 2

[0055] Verification of the antibacterial effect of the macrophage extracellular traps obtained in Example 1

[0056] Step 1: Preparation of bacterial suspension

[0057] S. aureus was cultured in BHI medium, and the bacteria were collected by centrifugation at 8000r / min for 5 minutes. The bacteria were washed twice with PBS, and the bacterial concentration was quantified in PBS to 10 7 CFU / mL.

[0058] Step 2: Detection of antibacterial effect

[0059] Take 270 μL of BMDM cultured at 0 h, 2 h, 4 h, 8 h, and 12 h in Example 1 and add 30 μL of 10 7 CFU / mL S.aureus, incubate on ice for 15 min, dilute 100 times, apply 100 μL of the mixture on BHI agar plate, incubate at 37°C for 12 h, and use the plate count method to calculate the killing of S.aureus by METs. Figure 6 Marked as PNPs+US.

[0060] The same treatment conditions were set up as the control group (only nano-piezoelectric particles BaT@Au without ultrasonic treatment, Figure 6 In the control group, BMDM were seeded in a Cell Attach six-well plate, with 5×10 6 Cells were cultured normally until adhered. 175 μL of 2 mg / mL BaT&Au was added to each well, and the unphagocytosed BaT&Au was washed away after incubation for 1 hour. The induced BMDMs were obtained after culturing for 0 hours, 2 hours, 4 hours, 8 hours, and 12 hours.

[0061] Figure 6The results showed that BMDM treated with BaT@Au without ultrasound had no bactericidal effect, and the bactericidal efficiency of extracellular traps against S. aureus reached 98% after culturing BMDM for 0h, 2h, 4h, 8h, and 12h after ultrasound induction with BaT&Au. The macrophages of the present invention can significantly inhibit the growth of S. aureus after ultrasound induction with BaT&Au.

[0062] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A method for obtaining immune cell extracellular traps, characterized in that: Treat immune cells by using mechanical stimulation in conjunction with nano-piezoelectric particle stimulation; The mechanical stimulation is ultrasonic treatment; The nano piezoelectric particles are nanoparticles with the ability of piezoelectrically catalyzing ROS generation.

2. The method for obtaining immune cell extracellular traps according to claim 1, characterized in that: The immune cells include macrophages; Or, the nano piezoelectric particles include barium titanate, metal-loaded barium titanate, lithium niobate, bismuth iodate, hydroxyapatite, polylactic acid, collagen, and two-dimensional MoS2.

3. The method for obtaining immune cell extracellular traps according to claim 1, characterized in that: The method comprises the following steps: firstly, nano-piezoelectric particles are co-incubated with immune cells to allow the immune cells to internalize the nano-piezoelectric particles, then excess nano-piezoelectric particles are cleaned, then ultrasonic treatment of the immune cells is used to generate extracellular traps, and finally centrifugation and purification are performed to obtain the immune cell extracellular traps.

4. The method for obtaining immune cell extracellular traps according to claim 3, characterized in that: The amount of the nano piezoelectric particles added is 0.2-0.5 mg / 10 6 Preferably, the amount of nano piezoelectric particles added is 0.35 mg / 10 6 cells.

5. The method for obtaining immune cell extracellular traps according to claim 3, characterized in that: The co-incubation time is 0.5-1.5h.

6. The method for obtaining immune cell extracellular traps according to claim 3, characterized in that: The ultrasonic treatment time is 5-20s.

7. The method for obtaining immune cell extracellular traps according to claim 3, characterized in that: The power of the ultrasonic treatment is 0.1-1 W / cm 2 ; Preferably, the ultrasonic power is 0.1 W / cm 2 .

8. The method according to any one of claims 1 to 7 for preparing immune cell extracellular traps.

9. Use of the preparation method according to any one of claims 1 to 7 or the immune cell extracellular trap according to claim 8 in the preparation of a drug for preventing, treating and / or ameliorating bacterial inflammation.

10. An anti-inflammatory drug, characterized in that: Comprising the immune cell extracellular trap described in claim 8.

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