Method for extracting MRSA-phagocytizing pulmonary alveolar macrophage-derived extracellular vesicles

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a method for extracting extracellular vesicles (EVs) from alveolar macrophages for phagocytosing methicillin-resistant staphylococcus aureus (MRSA). According to the invention, firstly, an in-vitro model of MRSA infected alveolar macrophages is optimized, and an in-vivo pathophysiological process is simulated; the method comprises the following steps: extracting EVs in an alveolar macrophage culture supernatant swallowing MRSA through ultracentrifugation, and further purifying the EVs through iodixanol density gradient centrifugation. Finally, the functions and effects of the EVs can be studied in vivo and in vitro, and the pathogenesis of MRSA pneumonia can be explored and potential therapeutic targets can be screened through multi-omics analysis.

Description

Technical Field

[0001] The invention belongs to the technical field of cell biology, and in particular relates to a method for extracting extracellular vesicles derived from alveolar macrophages that phagocytize MRSA. Background Art

[0002] The high incidence and mortality of MRSA pneumonia are still a difficult problem that the current medical and health system needs to solve urgently. Further clarifying the pathogenesis of MRSA pneumonia is the key to finding new therapeutic targets. Extracellular vesicles (EVs) are nanoscale particles of lipid bilayers that can be secreted by most cells. They carry a variety of bioactive substances inside and play an important role in infectious diseases. Alveolar macrophages are important executors of innate immunity in the lungs. In MRSA pneumonia, alveolar macrophages will first recognize and engulf MRSA and release more EVs. The role of these EVs in the occurrence and development of MRSA pneumonia is still unknown, and whether they carry pathogenic proteins or nucleic acids needs to be further clarified. At present, too many technologies for extracting EVs come from a single cell line in vitro, which is quite different from the actual infection situation of MRSA pneumonia. There is still a lack of a stable and efficient method for extracting EVs from alveolar macrophages that engulf MRSA that is close to the pathophysiology of the disease. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for extracting extracellular vesicles derived from alveolar macrophages that phagocytize MRSA, which is closer to the actual situation of MRSA pneumonia infection.

[0004] The technical solution adopted by the present invention is as follows: A method for extracting extracellular vesicles derived from alveolar macrophages that phagocytize MRSA, comprising the following steps:

[0005] (1) Bacterial preparation for MRSA;

[0006] (2) Infecting MH-S cells, an alveolar macrophage cell line, with MRSA in vitro;

[0007] (3) collecting EVs released by MH-S cells that have engulfed MRSA;

[0008] (4) EVs were purified using iodixanol density gradient centrifugation;

[0009] (5) Characterization of EVs.

[0010] Preferably, in step (1), the preparation process of the MRSA bacterial solution comprises the following steps: taking 10 uL of the MRSA bacterial solution, performing continuous gradient dilution, plating and counting colony forming units (CFU), thereby calculating the concentration of the MRSA bacterial solution.

[0011] Preferably, step (2) comprises the following steps: infecting alveolar macrophages with MRSA at a multiplicity of infection of 100:1, and detecting the proportion of alveolar macrophages that phagocytize MRSA by flow cytometry and immunofluorescence.

[0012] Preferably, in step (3), the culture supernatant of MH-S cells that have engulfed MRSA is collected, and EVs therein are extracted by gradient high-speed centrifugation and ultracentrifugation, and the extraction process is as follows:

[0013] (3a) Centrifuge the cell culture supernatant at 300 g for 10 min at 4°C to remove residual cells;

[0014] (3b) Centrifuge the supernatant at 2000 g for 20 min at 4°C to remove cell debris;

[0015] (3c) The supernatant was centrifuged at 10,000 g for 30 min at 4°C to remove large vesicles.

[0016] (3d) filtering the collected supernatant using a 0.22 μm filter;

[0017] (3e) The filtered supernatant was ultracentrifuged at 100,000 g for 70 min at 4°C to precipitate EVs;

[0018] (3f) Discard the supernatant, wash the EVs with pre-cooled sterile PBS, and then centrifuge at 100,000 g for 70 min;

[0019] (3g) Discard the supernatant and resuspend the precipitate with pre-cooled sterile PBS to obtain the original EVs derived from alveolar macrophages that phagocytize MRSA.

[0020] Preferably, in step (4), the original EVs are centrifuged using iodixanol density gradient, and the marker proteins of each component EVs are detected by Western blotting to find the layer where the EVs are located, thereby purifying the EVs.

[0021] Preferably, in step (5), the characteristics of the purified EVs are identified by transmission electron microscopy, nano-tracing analysis and Western blotting, and multi-omics analysis is performed to explore the pathogenesis of MRSA pneumonia and potential therapeutic targets.

[0022] The beneficial effects of the present invention are as follows: from the perspective of simulating the pathophysiology of the disease, the present invention optimizes the in vitro model of MRSA infection of alveolar macrophages, simulates the real pathophysiological conditions in vivo during MRSA pneumonia infection, separates and extracts EVs derived from alveolar macrophages that phagocytize MRSA, studies the functions and effects of these EVs in vivo and in vitro, and explores the pathogenesis of MRSA pneumonia and screens potential therapeutic targets through multi-omics analysis, thereby providing technical support and scientific basis for further exploring the pathogenesis of MRSA pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some implementation cases of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0024] Figure 1 The preparation process of MRSA bacterial solution; (A) Rapidly heating the frozen bacterial solution; (B) Inoculating the MRSA bacterial solution with an inoculation loop; (C) Cultivating the MRSA solid plate in an incubator; (D) Picking MRSA monoclonal colonies; (E) Cultivating the MRSA bacterial solution on a shaker; (F) Measuring the concentration of the MRSA bacterial solution by gradient dilution;

[0025] Figure 2 The process of MRSA infecting alveolar macrophages in vitro; (A) Flow cytometry was used to detect the proportion of alveolar macrophages that phagocytized MRSA; (B) Immunofluorescence was used to verify the phagocytosis of MRSA by alveolar macrophages; (C) The bactericidal concentration of vancomycin against MRSA was measured; (D) The bactericidal effect of vancomycin on extracellular MRSA was verified;

[0026] Figure 3 The separation and purification process of EVs; (A) Schematic diagram of the separation and purification process of EVs; (B) Schematic diagram of the process of iodixanol density gradient centrifugation to purify EVs; (C) Western blotting to detect the marker proteins of each component EVs.

[0027] Figure 4 Characterization of EVs; (A) Transmission electron microscopy to observe the morphological characteristics of EVs; (B) Nano-tracing analysis of the diameter distribution of EVs; (C) Western blotting to detect the marker proteins of purified EVs;

[0028] Figure 5This is the operating flow of the present invention. Note: AMs: alveolar macrophages; MRSA: methicillin-resistant Staphylococcus aureus; EV: extracellular vesicle; Multi-omics analysis: multi-omics analysis. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0030] Example 1

[0031] 1. Bacterial preparation, such as Figure 1 As shown, the following steps are included:

[0032] 1. Heat the frozen MRSA in a 37°C water bath for 1-2 minutes to quickly thaw the bacterial solution ( Figure 1 A).

[0033] 2. Use a sterile inoculation loop to take 20 μL of bacterial solution and apply it on a sterile, non-resistant LB solid plate in a clean bench ( Figure 1 B).

[0034] a. Turn on the ultraviolet lamp of the clean bench for 30 minutes in advance for disinfection.

[0035] b. Spread the MRSA bacterial solution on a sterile LB solid plate by the partitioning and streaking method.

[0036] c. Place the LB solid plate in a 37°C incubator and let it stand for 1 hour until the liquid dries up, then flip the plate over and continue incubating for 12 hours ( Figure 1 C).

[0037] d. Pick a MRSA monoclonal colony and place it in 4 mL LB liquid medium. Shake the culture on a shaker at 220 rpm for 12 hours ( Figure 1 DE).

[0038] 3. Take 10uL of MRSA bacterial solution and use sterile LB medium at 10 2 , 10 4 , 10 6 and 10 8 Then take 10uL of the diluted bacterial solution and drop it on the LB solid plate. Incubate it in a 37°C incubator for 12 hours. Then calculate the corresponding colony forming units (CFU). Finally, multiply it by the corresponding dilution factor to get the concentration of the original MRSA bacterial solution ( Figure 1 F).

[0039] Note: When diluting MRSA bacterial solution with LB medium, the solution needs to be mixed thoroughly. Count each concentration 3 times to reduce experimental errors.

[0040] 2. In vitro MRSA infection of alveolar macrophages, such as Figure 2 As shown, the following steps are included:

[0041] 1. Flow cytometry was used to detect the phagocytic rate of MRSA by alveolar macrophage cell line MH-S cells at different multiplicity of infection (MOI);

[0042] Note: In order to detect the phagocytosis rate of MRSA by MH-S cells using flow cytometry, we constructed a MRSA strain expressing green fluorescent protein (GFP). GFP is a fluorescent protein widely used in the field of biological sciences, which can make MRSA emit green fluorescence. Other dyes can also be used to label MRSA, such as PKH26 (a membrane dye). If other dyes are selected to label MRSA, the corresponding fluorescent channel should be selected for flow cytometry detection.

[0043] a. At 8 pm one day in advance, plate 2 × 10 5 MH-S cells;

[0044] b. Take MH-S cells from one well at 8 am the next day for cell counting to obtain the accurate cell number for that day;

[0045] Note: Because MH-S cells can proliferate, it is important to re-count the cells the next day.

[0046] c. Take 1 mL of the MRSA-GFP bacterial solution with measured bacterial concentration, centrifuge at 6000 rpm for 5 minutes at room temperature, wash twice with sterile PBS, and finally resuspend with 1 mL of sterile PBS;

[0047] d. Calculate the dosage of MRSA based on the results of cell counting on the day, add MRSA to MH-S cells at an MOI of 100:1, and incubate in a 37°C incubator for 1 hour, with 3 replicate wells in each group.

[0048] e. The proportion of FITC-positive MH-S cells detected by flow cytometry is the proportion of MH-S cells that phagocytize MRSA. Figure 2 (A) is the test result of an implementation case of the present invention.

[0049] 2. Immunofluorescence verification of MH-S cells phagocytosis of MRSA;

[0050] a. According to the manufacturer's instructions, MH-S cells were stained with DAPI (4',6-diamidino-2-phenylindole, a fluorescent dye that can strongly bind to DNA and stains the cell nucleus in blue), GFP (belongs to the cyanine dye family, emits bright green fluorescence, and marks MRSA), and PKH26 (a lipophilic fluorescent dye used to mark the cell membrane, which is red);

[0051] b. Place the cell slides in a 24-well plate and plate 1×10 5 The MH-S cells stained with PKH26 were incubated in a 37°C incubator for 4 hours until the cells adhered to the wall;

[0052] c. MRSA-GFP was added to each well at an MOI of 100:1 and incubated with MH-S cells for 1 hour, with PBS added as a control;

[0053] d. Discard the cell culture supernatant, wash the cells twice with 1 mL of sterile PBS, and then fix them with 500 uL of pre-cooled methanol in a -20°C refrigerator for 20 minutes;

[0054] e. Wash the cells once with 1 mL of sterile PBS and permeabilize the membrane with 500 μL of 0.1% Triton X-100 at room temperature for 5 minutes;

[0055] f. Wash the cells once with 1 mL of sterile PBS, add 1 μg / mL DAPI, and let stand for 5 minutes to stain the cell nuclei.

[0056] g. The slides were sealed with an anti-fading agent, and then the phagocytosis of MRSA by MH-S cells was observed using a fluorescence microscope. Figure 2 (B) is the test result of an implementation case of the present invention.

[0057] 3. Measure the bactericidal ability of vancomycin against MRSA;

[0058] a. Figure 2 As shown in (C), 100 μL of vancomycin was added to a 96-well cell culture plate at concentrations of 0 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, and 16 μg / mL.

[0059] Note: Set up three replicate wells for each concentration.

[0060] b. Add 100 μL of 1×10 6 CFU / mL MRSA bacterial solution, and the blank control group was added with 200μL LB culture medium.

[0061] c. Incubate the 96-well plate in a 37°C incubator for 24 hours.

[0062] d. Use a microplate reader to measure the OD600 to measure the bactericidal ability of vancomycin against MRSA. When the measured OD is consistent with that of the blank control group, it is the minimum bactericidal concentration of vancomycin against MRSA. In this experiment, 4μg / mL vancomycin can effectively kill MRSA ( Figure 2 C).

[0063] 4. Verify the bactericidal effect of vancomycin on extracellular MRSA;

[0064] a. In a 12-well plate, add MRSA to each well at an MOI of 100:1 and incubate with MH-S cells for 1 hour.

[0065] b. Discard the cell culture supernatant and wash the cells once with 1 mL of sterile PBS.

[0066] c. Add 1 mL of 1640 medium containing 4 μg / ml vancomycin and incubate in a 37°C incubator for 1 hour

[0067] d. Discard the cell culture supernatant and wash the cells once with 1 mL of sterile PBS.

[0068] e. Change to 1640 complete medium and culture the MH-S cells that have engulfed MRSA for 24 hours

[0069] f. Collect the cell culture supernatant, spread it on LB plates, incubate in a 37°C incubator for 12 hours, and observe the colony-forming units on the plates to verify the bactericidal effect of vancomycin on extracellular MRSA ( Figure 2 D).

[0070] Note: It is important to choose the appropriate multiplicity of infection (MOI) and duration of infection. Insufficient MOI will result in most macrophages not engulfing pathogens, and too long an infection time will also lead to uncontrolled inflammatory response, resulting in cell death. Based on the test results of this example, it was found that the best effect was achieved when the MOI was set to 100:1 and the duration of infection was set to 1h.

[0071] When using vancomycin to kill extracellular MRSA, the concentration of vancomycin is also very important. If the concentration of vancomycin is too low, it cannot reach an effective bactericidal concentration. If the concentration of vancomycin is too high, it will affect the secretion of EVs by macrophages. Based on the test results of this embodiment, it was found that the vancomycin concentration was set to 4 μg / mL, which had the best effect.

[0072] Example 2 Isolation and purification of EVs:

[0073] 1. Collect the culture supernatant of MH-S cells that have engulfed MRSA and extract EVs from them

[0074] a. MH-S cells were cultured at 5×10 6 The density was spread in a 10 cm culture dish;

[0075] b. After MH-S cells adhered to the wall, MRSA was added at an MOI of 100:1 and incubated for 1 hour;

[0076] c. Discard the supernatant and wash twice with 5 mL of sterile PBS;

[0077] d. Change to 1640 medium containing 4 μg / mL vancomycin and incubate for 1 hour to kill extracellular MRSA;

[0078] e. Discard the supernatant and wash twice with 5 mL of sterile PBS;

[0079] f. Change to EVs-free 1640 complete culture medium and continue culturing in a 37°C incubator for 24 hours;

[0080] g. Take 1 mL of cell culture supernatant and apply it on LB plate. Observe whether there are colonies growing on the plate the next day to verify whether the extracellular MRSA is completely removed.

[0081] h. subjecting the collected cell culture supernatant to gradient high-speed centrifugation;

[0082] i. Centrifuge the cell supernatant at 300 g for 10 min at 4°C to remove residual cells;

[0083] j. Centrifuge the supernatant at 2000 g for 20 min at 4°C to remove cell debris;

[0084] k. Centrifuge the supernatant at 10,000 g for 30 min at 4°C to remove large vesicles.

[0085] l. The collected supernatant was filtered through a 0.22 μm filter;

[0086] Note: When filtering, be sure to replace the filter to ensure filtering efficiency.

[0087] m. Ultracentrifuge the supernatant at 100 000 g for 70 min at 4°C to precipitate EVs.

[0088] n. Discard the supernatant, wash EVs with pre-cooled sterile PBS, and continue ultracentrifugation at 100 000g for 70 min;

[0089] o. Discard the supernatant and resuspend the precipitate with 200 μL pre-cooled sterile PBS to obtain the original EVs ( Figure 3 A).

[0090] 2. Purification of EVs using iodixanol density gradient centrifugation:

[0091] We extracted original EVs from the culture supernatant of alveolar macrophages that engulfed MRSA. Since MRSA infection may cause cell death, resulting in apoptotic bodies and cell debris, in order to obtain purer EVs, we then used iodixanol density gradient centrifugation to purify EVs. The specific purification process is as follows Figure 3 (B) includes the following process:

[0092] a. Prepare a discontinuous gradient of iodixanol solution (12%, 18%, 24%, 30%, 36%) in pre-cooled PBS in advance;

[0093] b. Gently add 2.5 mL of 36% iodixanol solution to the bottom of the SW41 Ti ultracentrifuge tube, then add 2 mL of 30% iodixanol solution, 2 mL of 24% iodixanol solution, 2 mL of 18% iodixanol solution and 2 mL of 12% iodixanol solution;

[0094] Note: During the addition of the iodixanol solution, be careful to avoid the formation of bubbles and vibration.

[0095] c. Dissolve the raw EVs in 1.5 mL of sterile PBS and add it to the top of the centrifuge tube;

[0096] d. After accurate balancing, ultracentrifuge the sample at 120,000 g for 15 hours at 4°C;

[0097] Note: Since the original EVs may contain other non-EV components, it is necessary to purify EVs by iodixanol density gradient centrifugation.

[0098] e. Collect 12 1 mL fractions from the top to the bottom of the ultracentrifuge tube and transfer each fraction to a new SW41 Ti ultracentrifuge tube.

[0099] f. Add 10 mL of pre-cooled sterile PBS to each ultracentrifuge tube, accurately adjust the amount, and ultracentrifuge the individual components at 120,000 g for 2 hours at 4°C;

[0100] g. Discard the supernatant and resuspend the precipitate with 100 μL pre-cooled sterile PBS at the bottom of the centrifuge tube ( Figure 3

[0101] B);

[0102] h. Use Western blotting to detect specific protein markers of EVs in each component to determine the purification

[0103] The density gradient where EVs are located ( Figure 3 C);

[0104] i. Mix the components containing EVs and determine the protein concentration of EVs using the BCA method;

[0105] j. Store the purified EVs in a -80°C freezer.

[0106] 3. The purified EVs were characterized by transmission electron microscopy, nano-tracing analysis, and Western blotting. The analysis results were as follows: Figure 4 As shown. It can be determined that the purified EVs obtained in this example are inwardly concave cup-shaped or disc-shaped structures under electron microscopy ( Figure 4 A), whose diameter is mainly distributed between 30-150nm ( Figure 4 B), and expressed EV-specific biomarkers such as TSG-101, CD9, and CD81, but did not express non-EV biomarkers such as ApoA1 ( Figure 4 C). The entire invention operation process is shown in Figure 5 .

[0107] Macrophages are important executors of innate immunity in the body. Under stress, such as infection, they release more EVs. Exploring the functions and effects of these EVs is an important part of uncovering the occurrence and development of infectious diseases. Previous research methods are often single in vitro models, which are quite different from the actual infection situation in vivo. Using the method of the present invention, we can extract EVs secreted under conditions that are very close to the pathophysiology of the disease, further study the biological effects of these EVs in vivo and in vitro, and through multi-omics analysis, we can effectively explore the pathogenesis of MRSA pneumonia and screen potential therapeutic targets.

[0108] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for extracting extracellular vesicles derived from alveolar macrophages that engulf MRSA, which mainly comprises the following steps: (1) MRSA bacterial culture; (2) construct an in vitro cell model of MRSA-infected alveolar macrophages; (3) collecting EVs from the culture supernatant of alveolar macrophages that have phagocytosed MRSA; (4) EVs were purified using iodixanol density gradient centrifugation; (5) Characterization and application analysis of EVs.

2. The method for extracting extracellular vesicles derived from alveolar macrophages that phagocytize MRSA according to claim 1, characterized in that: In step (1), the frozen MRSA bacterial solution is thawed and then the concentration of the MRSA bacterial solution is calculated by continuous gradient dilution.

3. The method for extracting extracellular vesicles derived from alveolar macrophages that phagocytize MRSA according to claim 1, characterized in that: In step (2), alveolar macrophages are infected in vitro with MRSA at different infection multiplicities, and the proportion of alveolar macrophages that phagocytize MRSA is detected by flow cytometry and immunofluorescence, thereby optimizing the in vitro cell model of MRSA infection of alveolar macrophages.

4. The method for extracting extracellular vesicles derived from alveolar macrophages that phagocytize MRSA according to claim 1, characterized in that: In step (3), EVs in the culture supernatant of alveolar macrophages that have phagocytosed MRSA are extracted by using differential high-speed centrifugation and ultracentrifugation.

5. The method for extracting extracellular vesicles derived from alveolar macrophages that phagocytize MRSA according to claim 1, characterized in that: In step (4), EVs are centrifuged using iodixanol density gradient, and the marker proteins of EVs are detected by Western blotting to find the components where EVs are located and purify EVs.

6. The method for extracting extracellular vesicles derived from alveolar macrophages that phagocytize MRSA according to claim 1, characterized in that: In step (5), the characteristics of purified EVs were identified by transmission electron microscopy, nanotracer analysis, and Western blotting, and multi-omics analysis was performed to explore the pathogenesis of MRSA pneumonia and potential therapeutic targets.

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