Application of MSC-Exo in preparation of medicine for treating necrotic apoptosis of dendritic cells

Abstract

The invention relates to the technical field of biomedicine, in particular to application of mesenchymal stem cell exosomes (MSC-Exo) in preparation of drugs for treating necrotic apoptosis of sepsis dendritic cells, through regulation and control research of the exosomes on the dendritic cells, immune activation and inhibition performance can be more accurately and effectively regulated, immune response efficiency is improved, and the application of the exosomes in preparation of drugs for treating necrotic apoptosis of the sepsis dendritic cells is promoted. And a new thought and a new method are provided for the treatment of immune-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of MSC-Exo in preparing a drug for treating dendritic cell necroptosis. Background Art

[0002] Sepsis is usually caused by microbial infection, which triggers a systemic inflammatory response, leading to the activation of multiple cells and molecules and the release of inflammatory mediators. These mediators are originally used to fight infection, but in sepsis, the inflammatory response may be out of control, triggering fatal organ dysfunction. In my country, about 1 million patients die from sepsis each year, and the mortality rate of sepsis patients in intensive care units is as high as 35.5%. With the intensification of the aging trend of the population, the incidence and mortality of sepsis are increasing year by year, causing huge medical burden and social pressure.

[0003] Prior art, such as the invention patent with patent application number 2016102794739, provides an exosome, a method for preparing exosomes, and their use in preparing drugs or preparations for treating sepsis, specifically involving optimizing the treatment of human umbilical cord mesenchymal stem cells with recombinant human IL-1β to enhance their immunosuppressive function, and extracting the exosomes produced for the treatment of sepsis. The exosomes can effectively alleviate sepsis symptoms and increase the survival rate of septic mice by inhibiting T cells and macrophages.

[0004] However, studies have found that dendritic cells (DC) play an important role in both immune activation and immune tolerance, and one DC can activate 100 to 3,000 T cells, which is 100 to 1,000 times the ability of macrophages and B cells to activate T cells. Dendritic cells (DC) play a central role in activating and regulating immune responses. The role of dendritic cells in immune system diseases and neurological diseases also makes them a potential target for the treatment of these diseases. Therefore, through the study of the regulation of dendritic cells by exosomes, we can more accurately and effectively regulate immune activation and inhibition, improve the efficiency of immune response, and provide new ideas and methods for the treatment of immune-related diseases. Summary of the invention

[0005] In order to solve the problems in the above-mentioned background technology, the first object of the present invention is to provide the use of mesenchymal stem cell exosomes in the preparation of a drug for treating sepsis dendritic cell necroptosis, so as to clarify that MSC-Exo can inhibit DC necroptosis under sepsis conditions.

[0006] The second object of the present invention is to provide the use of mesenchymal stem cell exosomes in the preparation of a drug for reducing the level of necroptosis-related proteins in splenic dendritic cells.

[0007] The third object of the present invention is to provide the use of mesenchymal stem cell exosomes in the preparation of drugs for reducing the level of molecular patterns associated with dendritic cell necroptosis.

[0008] The fourth object of the present invention is to provide the use of mesenchymal stem cell exosomes in the preparation of a drug for reducing the level of cytokines related to dendritic cell necroptosis.

[0009] In summary, the beneficial effects of the present invention are: MSC-Exo can inhibit DC necroptosis under sepsis conditions, and can reduce the levels of necroptosis-related proteins, molecular patterns and cytokines in splenic dendritic cells. The above verification facilitates us to more accurately and effectively regulate immune activation and inhibition performance, and provide new ideas and methods for the treatment of immune-related diseases.

[0010] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following specifically cites a preferred embodiment and describes it in detail with the accompanying drawings as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Figure 1 shows the identification of mesenchymal stem cell differentiation: (A) osteogenic differentiation of mesenchymal stem cells (MSC) stained with Alizarin Red, scale bar 200 μm; (B) adipogenic differentiation of MSC stained with Oil Red O, scale bar 200 μm; (C) chondrogenic differentiation of MSC stained with Toluidine Blue, scale bar 20 μm;

[0012] Figure 2 This is an identification diagram of surface markers of human umbilical cord mesenchymal stem cells (hUCMSC);

[0013] Figure 3 Figure 1 shows the isolation and identification of exosomes derived from mesenchymal stem cells: (A) Western Blot identification of exosome positive protein markers (TSG101, CD9, CD63) and negative protein markers (Calnexin); (B) Particle size density distribution analysis to identify exosome diameter and distribution; (C) Transmission electron microscopy to identify exosome morphology;

[0014] Figure 4 Flow cytometry was used to detect the proportion of DC necroptosis (parents) in the treatment groups of different doses (Dose) of mesenchymal stem cell exosomes (MSC-Exo) under sepsis conditions;

[0015] Figure 5 SYTOX-Green staining was used to detect the necroptotic cell ratio in different doses of MSC-Exo treatment groups under sepsis conditions;

[0016] Figure 6 ELISA was used to detect the levels of necroptosis-related cytokines in the culture supernatant of dendritic cells (DCs) under sepsis conditions;

[0017] Figure 7 Flow cytometry was used to detect the proportion of DC necroptosis at different times in the in vitro stimulation group and the treatment group (parents);

[0018] Figure 8 SYTOX-Green staining was used to detect the proportion of DC necrosis and apoptosis cells in the in vitro stimulation group and the treatment group at different times;

[0019] Fig. 9 Flow cytometry was used to detect the proportion of necroptosis of DC in the spleen of mice in the sepsis group and the treatment group at different times (parents);

[0020] Fig.10 Western Blot was used to detect the expression levels of DC necroptosis-related proteins in the in vitro stimulation group and the treatment group at different times;

[0021] Fig.11 Laser confocal microscopy was used to observe the p-MLKL fluorescence intensity and positive cell ratio in DCs at different times in the in vitro stimulation group and treatment group;

[0022] Fig.12 Laser confocal microscopy was used to observe the p-RIPK3 fluorescence intensity and positive cell ratio in DCs at different times in the in vitro stimulation group and treatment group;

[0023] Fig.13 Western Blot was used to detect the expression levels of necroptosis-related proteins in spleen DCs of mice in the sepsis group and the treatment group at different times;

[0024] Fig.14 Laser confocal microscopy was used to observe the p-MLKL fluorescence intensity and positive cell ratio in the spleen DC of mice in the sepsis group and the treatment group at different time points;

[0025] Fig.15 Laser confocal microscopy was used to observe the fluorescence intensity and positive cell ratio of p-RIPK3 in spleen DC of mice in sepsis group and treatment group at different time points;

[0026] Fig.16 ELISA was used to detect the levels of necroptosis-related cytokines in the DC culture supernatant at different times in the in vitro stimulation group and the treatment group;

[0027] Fig.17 ELISA was used to detect the levels of necroptosis-related cytokines in the serum of mice in the sepsis group and the treatment group at different times. DETAILED DESCRIPTION

[0028] In order to make the content of the present invention more clearly understood, the present invention is further described below based on specific embodiments in conjunction with the accompanying drawings.

[0029] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0030] WT male C57BL / 6J mice were used in the following experiments, each weighing about 20-25 g, provided by Huafukang Biotechnology Co., Ltd. The mice were kept in an SPF animal room with an ambient temperature of 22°C and an ambient humidity of 65%, with good ventilation, drinking water at any time, and adaptive feeding for 1 week.

[0031] Before the experiment, 64 C57BL / 6J mice were randomly divided into 8 groups according to the random number table method. 4 groups were subjected to CLP modeling, which was the model group; 4 groups were injected with MSC-exo through the tail vein immediately after the CLP modeling was completed, with a dose of 100 μg / mouse, which was the treatment group. The model group and the treatment group were sampled at 0h, 6h, 12h and 24h after modeling, and the eyeballs were removed after anesthesia to collect blood. After the blood collection was completed, the mice were killed by cervical dislocation for sampling.

[0032] Experiment 1: Culture and identification of mesenchymal stem cells

[0033] 1. Mesenchymal stem cell culture

[0034] The present application uses human umbilical cord mesenchymal stem cells (hUCMSC), which were purchased from Beijing Aiyi Biotechnology Co., Ltd. and are second-generation hUCMSCs. They were cultured in a T75 culture flask using MEM medium containing 10% mesenchymal stem cell nutrient additives and passaged using 0.25% trypsin, with each passage being counted as one generation.

[0035] 2. Identification of Mesenchymal Stem Cell Differentiation

[0036] (1) Directed induction of hUCMSC osteogenic differentiation

[0037] S1, culture the third generation hUCMSCs, seed the third generation hUCMSCs with good growth in a six-well plate, and ensure that the number of cells seeded in each well is the same;

[0038] S2, when the growth density of the third generation hUCMSCs is about 70%, replace the complete medium, add 2 ml of growth medium to each well, and continue to incubate in the cell culture incubator;

[0039] S3, when the cell density grows to 90%, replace the culture medium, add 2 ml of complete osteogenic induction medium to each well, continue to incubate in the cell culture incubator, and replace the culture medium every 3 days until the formation of bone nodules can be observed under a microscope;

[0040] S4, after the bone nodule structure was formed, the culture medium was removed, and then the samples were rinsed three times with sterile phosphate-buffered saline (PBS), and then fixed with 4% paraformaldehyde at room temperature for 30 min;

[0041] S5, Alizarin red staining for 1 h, rinsed three times with PBS;

[0042] S6, counterstain with nuclear fast red for 5 min, rinse three times with PBS, and observe under a microscope.

[0043] The results are as follows Figure 1 As shown in A.

[0044] (2) Directed induction of hUCMSC adipogenic differentiation

[0045] S1, seed the third-generation hUCMSCs with good growth into a six-well plate, ensuring that the number of cells seeded in each well is the same;

[0046] S2, when the growth density of the third generation hUCMSCs is about 70%, replace the complete medium, add 2 ml of growth medium to each well, and continue to incubate in the cell culture incubator;

[0047] S3, when the cell density grows to 90%, replace the culture medium, add 2 ml of complete adipogenic induction medium to each well, continue to incubate in the cell culture incubator, and replace the culture medium every 3 days until lipid droplets can be observed under a microscope;

[0048] S4, after lipid droplets were formed, the culture medium was removed, the cells were rinsed with PBS, and fixed with 4% paraformaldehyde at room temperature for 30 min;

[0049] S5, stain with Oil Red O for 10 min, wash off the stain with 60% isopropanol, rinse three times with PBS, and observe under a microscope.

[0050] The results are as follows Figure 1 As shown in B.

[0051] (3) Directed induction of hUCMSC into chondrogenic differentiation

[0052] S1, seed the third-generation hUCMSCs with good growth into a six-well plate, ensuring that the number of cells seeded in each well is the same;

[0053] S2, when the growth density of the third generation hUCMSCs is about 70%, replace the complete medium, add 2 ml of growth medium to each well, and continue to incubate in the cell culture incubator;

[0054] S3, when the cell density grows to 90%, replace the culture medium, add 2 ml of complete chondrogenic induction medium to each well, continue to incubate in the cell culture incubator, and replace the culture medium every 3 days until the formation of cartilage nodules can be observed under a microscope;

[0055] S4, after the cartilage nodules were formed, the culture medium was removed, the cells were rinsed with PBS, and the cells were fixed with 4% paraformaldehyde at room temperature for 30 min;

[0056] S5, stain with toluidine blue for 30 min at room temperature, rinse three times with PBS, and observe under a microscope.

[0057] The results are as follows Figure 1 As shown in C.

[0058] 3. Identification of hUCMSC surface markers

[0059] S1, take well-grown third-generation hUCMSCs, digest them with 0.25% trypsin for 3 min, centrifuge at 1000 rpm for 10 min, and discard the supernatant;

[0060] S2, wash the cells twice with pre-cooled PBS, resuspend the cells, and dispense into different flow tubes, with a cell volume of about 105 / ul, 100ul per tube;

[0061] S3, add CD11b, CD34, CD44, CD45, CD90, CD105 antibodies and isotype control antibodies to the cells in the flow tube, and then vortex on a vortex oscillator for 10 seconds to mix them thoroughly. Take another flow tube as a blank control (no antibody is added);

[0062] S4, after adding the antibody, incubate at room temperature in the dark for 30 min, then centrifuge at 1000 rpm for 10 min, and discard the supernatant;

[0063] S5, add 1 ml PBS to resuspend the cells, centrifuge at 1000 rpm for 10 min, discard the supernatant, and repeat the centrifugation once;

[0064] S6, then add 200ul PBS to resuspend the cells, blow thoroughly and then test on the instrument.

[0065] The results are as follows Figure 2 As shown, flow cytometry identified MSC-specific positive surface antigens CD44, CD90 and CD105, with an expression rate greater than 95%; flow cytometry identified MSC-specific negative surface antigens CD11b, CD34 and CD45, with an expression rate no more than 2%.

[0066] Experiment 2: Isolation and identification of exosomes from mesenchymal stem cells

[0067] 1. Isolation of Mesenchymal Stem Cell Exosomes (MSC-Exo) by Ultracentrifugation

[0068] S1, culture the fourth-generation hUCMSCs in complete medium until the cell density reaches more than 65%, discard the complete medium, wash once with PBS, replace with serum-free medium, and continue culturing;

[0069] S2, when the cell density reaches more than 90% after rapid growth, collect the culture supernatant, and then freeze the culture medium in a -80°C refrigerator for subsequent exosome isolation and use;

[0070] S3, the supernatant collected in step S2 was restored to room temperature, mixed evenly and poured into a 50 mL centrifuge tube, centrifuged at 3000 g for 10 min at 4°C, and then filtered out cell debris using a 0.45 μm vacuum filter, and then filtered out cell debris using a 0.22 μm vacuum filter;

[0071] S4, using a tangential flow filtration automatic concentrator to concentrate the supernatant treated in S3 to obtain a clarified culture supernatant, and then dispensing the clarified culture supernatant into ultracentrifuge tubes, using a balance to balance, to ensure that the weight difference between every two ultracentrifuge tubes does not exceed 1 g;

[0072] S5, placing the balanced supernatant in an ultracentrifuge for centrifugation at a centrifugal force of 100,000 g, a centrifugal temperature of 4° C., and a centrifugal time of 2 h to obtain a sample;

[0073] S6, remove the sample supernatant and resuspend all the precipitates in pre-cooled PBS to obtain exosomes;

[0074] S7, BCA method was used to detect the concentration of exosome protein. After dilution, the samples were packaged at 200 μg / tube and then frozen in a -80°C refrigerator.

[0075] 2. MSC-Exo Identification

[0076] (1) Detection of exosome particle size by nanoparticle tracking analysis (NTA):

[0077] In this application, ZetaView PMX 110 was used to measure the concentration of the isolated MSC-Exo, wherein the emission wavelength was 405 nm, and the MSC-Exo suspension was diluted to 1×10 7 Particles / mL-1×10 9 After the particles / mL were measured, the size and distribution of MSC-Exo particles were determined using the corresponding software according to the instrument manual. The results are as follows Figure 3 As shown in B.

[0078] (2) Electron microscopy observation of exosome morphology:

[0079] Take 10 μL of the separated MSC-Exo solution and drop it on the copper grid, incubate it at room temperature for 10 minutes; after the incubation is completed, wash it with sterile deionized water, and then absorb the excess liquid with absorbent paper; drop 10 μL of 2% uranyl acetate on the copper grid and negatively stain it for 1 minute; after the negative staining is completed, use absorbent paper to absorb the floating liquid and dry it for 2 minutes; place the copper grid under a transmission electron microscope, use 80kv imaging to observe the morphology of exosomes and take pictures and record the results. Figure 3 As shown in C.

[0080] (3) Detection of exosome-specific proteins:

[0081] Step 1: Obtain denatured protein:

[0082] According to the protein quantification results of the BCA method, the loading amount of 20 μg of exosome protein was calculated, and the protein loading buffer was added to the exosomes. Then, the mixture was vortexed and mixed, and then placed in a 100°C metal bath for 10 min to obtain denatured protein.

[0083] Step 2, SDS-PAGE gel preparation:

[0084] Before the experiment, clean the utensils used for preparing glue with distilled water, install the glass plates in order on the glue tank and clamp them, fill the glass plates with distilled water and test for leaks for 15-20 minutes;

[0085] Add separation gel buffer A and separation buffer B in a ratio of 1:1 into a centrifuge tube to prepare 9 ml of separation gel / block, then add 90 μl of 10% ammonium persulfate and mix thoroughly with a disposable pipette to obtain separation gel;

[0086] Meanwhile, in a centrifuge tube, mix the stacking gel buffer A and the stacking gel buffer B at a ratio of 1:1 to prepare 3 ml of stacking gel / block, and add 30 μl of 10% ammonium persulfate, and mix thoroughly with a disposable pipette to obtain stacking gel;

[0087] Add separation gel to the gap of the leak-tested glass plate to 4 / 5 of the height, then slowly add concentrated gel on top of the separation gel along the edge (no need to wait for the separation gel to solidify), then quickly insert a 15mm thick comb, make sure there are no obvious bubbles, and let it stand at room temperature for 20-25 minutes. After solidification, obtain SDS-PAGE gel;

[0088] Step 3, electrophoresis:

[0089] Place the SDS-PAGE gel in an electrophoresis tank, add the prepared 1× electrophoresis buffer (1× electrophoresis buffer is a mixture of 50 ml of 10× electrophoresis buffer and 450 ml of distilled water), remove the comb teeth and check the integrity of the gel; ensure that the electrophoresis buffer in the gel tank exceeds the upper edge of the gel, and continue to add electrophoresis buffer to the outside to half the height of the separation gel; use a pipette to add the denatured protein into the well, and connect the electrophoresis tank to the power supply; use a constant voltage of 180V for electrophoresis for about 45 minutes; keep the electrophoresis tank in a low temperature environment during the electrophoresis process; after the electrophoresis is completed, remove the glass plates on both sides, take out the gel and soak it in the prepared 1× electrotransfer buffer (the 1× electrotransfer buffer is a mixture of 50 ml of 10× electrotransfer buffer, 350 ml of distilled water and 100 ml of anhydrous methanol added in sequence).

[0090] Step 4: Electroporation:

[0091] Use the wet transfer method, prepare filter paper according to the size of the gel and soak it in 1× electrotransfer buffer. Take a PVDF membrane of appropriate size and soak it in anhydrous methanol for more than 5 minutes to charge the PVDF membrane. Place filter paper-SDS-PAGE gel-PVDF membrane-filter paper in order, remove bubbles and press tightly, and put it into the electrotransfer tank of 1× electrotransfer buffer. Transfer the membrane at a constant current of 200-300mA for 2-2.5h. Adjust the current size and transfer time according to the molecular weight of the target protein.

[0092] Step 5: Closing:

[0093] The PVDF membrane was blocked in a rapid blocking solution for 10 min, and then washed with the prepared 1×TBS-T (1×TBS-T is prepared by mixing 50 ml of 10×TBS-T with 450 ml of distilled water) for 3 times, each time for 15 min.

[0094] Step 6, incubate with antibodies:

[0095] Prepare the primary antibody with a dilution ratio of 1:1000, obtain the antibody diluent, place the PVDF membrane in the antibody diluent, and incubate overnight at 4°C; on the second day, wash the PVDF membrane three times with 1×TBS-T, incubate with anti-mouse or anti-rabbit HRP-coupled secondary antibody (secondary antibody dilution ratio of 1:5000) at room temperature for 60 minutes, and use 1×TBS-T for development after washing.

[0096] Step 7, protein blot development:

[0097] Prepare ECL developer and protect from light for subsequent use. Use ECL gel imaging system to detect protein expression, use β-actin expression as an internal reference, and use Image J software to analyze the gray value of the target band and perform statistical analysis.

[0098] The positive protein markers for exosome identification include CD9, CD63 and TSG101, and the negative protein marker is Calnexin. After incubation with the primary antibody of exosome protein marker and the secondary antibody of the corresponding species, the image was developed, exposed and recorded. The results are as follows: Figure 3 As shown in A, MSC is the mesenchymal stem cell group, MSC-Exo is the mesenchymal stem cell exosome group, and EFs is the blank control group.

[0099] Experiment 3: Isolation and culture of mouse spleen DC

[0100] 1. Isolation of mouse spleen DCs using the STEMCELL EasySep method

[0101] S1, mice were killed by cervical dislocation, the abdominal skin was cut open after alcohol disinfection, and the spleen was bluntly separated and placed in a sterile centrifuge tube containing pre-cooled sterile phosphate-buffered saline (PBS);

[0102] S2, process spleen tissue in a clean bench, use forceps to remove connective tissue such as spleen capsule, carefully tear the spleen into pieces, and carefully transfer it to a cell filter (75 μm) to fully grind the spleen tissue. During the grinding process, rinse the filter with PBS intermittently. After the grinding is completed, mix the filtrate and transfer it to a centrifuge tube to obtain a spleen cell suspension;

[0103] S3, based on STEMCELL CD11c + Magnetic beads instructions, 1×10 8 Resuspend cells in 1 mL PBS; 8 The cells were blocked with 50 μL of rat serum, mixed by oscillation and incubated at room temperature for 5 min. The dosage of solution A and solution B was 25 μL / 10 8 For each cell, mix solution A and solution B in a ratio of 1:1, let stand at room temperature for 5 min after fully mixing, then add to the blocked spleen cell suspension, mix fully and incubate at room temperature for 5 min;

[0104] S4, after incubation, use a vortex mixer to fully shake the CD11c + Magnetic beads (RapidSpheres) 30s, 60μL / 10 8 The ratio of 10 cells was added to the cell suspension and incubated at room temperature for 3 minutes;

[0105] S5, transfer the incubated cell suspension to a 14 mL separation tube and resuspend it to the corresponding volume using PBS containing 2% fetal bovine serum (FBS) (if the cell suspension volume is <2 mL, the total volume after resuspension is 5 mL; if the cell suspension volume is ≥2 mL, the total volume after resuspension is 10 mL), place the separation tube in the EsaySep magnetic column, let it stand at room temperature for 3 minutes, then discard the liquid in the tube, take out the separation tube, and use PBS containing 2% FBS to carefully blow off the cells adsorbed on the tube wall and mix them, finally resuspend the total volume to 10 mL, repeat this step 3 times; after discarding the liquid in the separation tube for the last time, use 5 mL PBS to resuspend the cells in the tube, which is the mouse spleen CD11c + DC, counted and then processed.

[0106] 2. Isolation of mouse spleen DCs using Miltenyi CD11c magnetic beads

[0107] According to the instructions of Miltenyi CD11c magnetic beads, 1×10 7 Resuspend cells in 40 μL MACS buffer; adjust the volume to 10 μL / 10 7 CD11c magnetic beads were added to the spleen cell suspension and incubated at 4°C for 15 min;

[0108] After incubation, PBS was added to the spleen cell suspension for re-suspending, and then placed in a centrifuge for centrifugal separation at a speed of 1500 rpm for 5 min. After discarding the supernatant, the cells were resuspended in MACS buffer (the resuspension volume for MS separation column was 500 μL, and the resuspension volume for LS separation column was 1 mL). The separation column of the corresponding specification was selected according to the number of cells (the maximum number of cells separated by the MS separation column is 2×10 8 The maximum number of cells separated by the LS separation column is 2×10 9 ), put the separation column into the Miltenyi MiniMACS magnetic rack, rinse the separation column with a specified amount of PBS (MS: 500 μL; LS: 1 mL) before adding the cell suspension, add the spleen cell suspension when the PBS is about to drip off, add PBS for washing (MS: 500 μL; LS: 1 mL) when the spleen cell suspension is about to drip off, and repeat this step 3 times; after washing, carefully remove the separation column and place it in a 15 mL centrifuge tube, quickly add a specified amount of PBS (MS: 1 mL; LS: 3 mL), and then use the piston to push the liquid in the separation column out at a uniform speed, and the centrifuge tube is the mouse spleen CD11c + DC cell suspension, counted and ready for subsequent processing.

[0109] Experiment 4: Exploration of the dose-effect relationship of MSC-Exo inhibiting DC necroptosis under sepsis conditions

[0110] The present application isolated wild-type (WT) mouse spleen DCs for in vitro experiments. Mouse spleen DCs were obtained through experiment three. After LPS and z-VAD (LPS+z-VAD) co-stimulated mouse spleen DCs, they were treated with 0.1 ml MSC-Exo at concentrations of 0.4 μg / mL, 2 μg / mL, 10 μg / mL and 50 μg / mL, respectively. After 24 hours, flow cytometry, SYTOX-Green staining and ELISA were used to evaluate the necroptosis of DCs in each group.

[0111] The method of establishing an in vitro sepsis model by LPS stimulation in the present application is as follows: 2 hours after the cells are inoculated in the cell culture flask, an in vitro sepsis model is established by LPS stimulation at a concentration of 1 μg / mL. The cells are pre-stimulated with the broad-spectrum caspase kinase inhibitor z-VAD-FMK (z-VAD) at a working concentration of 20 μM, and the cells are pre-stimulated with z-VAD for 30 minutes and then combined with LPS stimulation to induce necroptosis in the cells.

[0112] 1Flow cytometry

[0113] S1, collect cells from each group and count them according to 2.5×10 5 Place the cells / tube in a flow tube, add 1 ml PBS to allow the cells to be evenly distributed in the flow tube, and then centrifuge at a speed of 1500 rpm for 5 min.

[0114] S2, discard the supernatant and resuspend the cell pellet with 1 mL Binding Buffer, centrifuge again, the speed of the centrifuge is 1500 rpm, the centrifugation time is 5 min, discard the supernatant and resuspend the cell pellet with 100 μL Binding Buffer;

[0115] S3, add 5 μL Annexin V and 2.5 μL 7-AAD dye to each flow tube, tap the tube wall to evenly disperse the cells after adding the reagents, and incubate at room temperature in the dark for 15 min;

[0116] S4, set up double-negative tubes (without Annexin V and 7-AAD), single-stained tubes (only Annexin V was added), and single-stained tubes (only 7-AAD was added) as controls;

[0117] S5. After incubation, add 200 μL Binding Buffer to each tube, flick the tube wall to mix, and then detect and analyze necrosis and apoptosis cells (Annexin V + 7-AAD +) ratio; FlowJo V 10.0 software was used to reanalyze the data of each group.

[0118] The results are as follows Figure 4 As shown in the figure, flow cytometry showed the proportion of DC undergoing necroptosis after in vitro LPS+z-VAD stimulation and treatment with different concentrations of MSC-Exo for 24 hours. Regarding the inhibition of necroptosis, compared with the stimulation group (MSC-Exo dose of 0 μg / mL), the proportion of DC undergoing necroptosis (parents) was reduced after treatment with 0.4 μg / mL, 2 μg / mL, 10 μg / mL and 50 μg / mL of 0.1 ml MSC-Exo, among which the 2 μg / mL group (P < 0.05), 10 μg / mL group (P < 0.0001) and 50 μg / mL group (P < 0.0001) had a significant reduction in the proportion of DC undergoing necroptosis. In addition, compared with the 2μg / mL group, the proportion of DCs undergoing necroptosis in the 10μg / mL group (P<0.0001) and the 50μg / mL group (P<0.0001) was significantly reduced, but there was no significant difference between the 10μg / mL group and the 50μg / mL group. This result shows that MSC-Exo can effectively inhibit DCs from undergoing necroptosis under sepsis conditions, and the effects of the 10μg / mL and 50μg / mL dose groups are particularly significant.

[0119] 2SYTOX-Green staining

[0120] S1, prepare SYTOX-Green dead cell nucleic acid dye: dilute the SYTOX-Green dead cell nucleic acid dye stock solution with DMSO at a dilution ratio of 1:10000 to a final concentration of 50 nM;

[0121] S2, in vitro experimental part, 100 nM SYTOX-Green dye was added during the cell culture process, and the cell culture process was continued after addition;

[0122] S3, collect cells, count the cells and calculate the value of 2.0×10 6 The cells / tubes were collected in flow tubes and then placed in a centrifuge for centrifugation at 1500 rpm, room temperature, and 5 min. The cell pellets were then resuspended in 1 mL PBS and centrifuged again. The centrifugation conditions were the same and repeated 3 times.

[0123] S4, after washing the cells in the flow tube, discard the supernatant completely, resuspend the cell pellet with 60-70 μL PBS, and repeatedly blow and beat with a pipette to obtain a cell suspension;

[0124] S5, take 35 μL of cell suspension and drop it on a glass slide, cover it with a cover slip, spread it flat in a wet box, and send it for examination and filming;

[0125] S6, SYTOX-Green positive cells were observed and photographed using a fluorescence microscope, and Image J software was used to count and analyze the ratio of positive cells to the total number of cells in the field of view.

[0126] The results are as follows Figure 5 As shown in the figure, SYTOX-Green staining detected that compared with the stimulation group (MSC-Exo dose of 0 μg / mL), the proportion of DC necroptosis cells in the 10 μg / mL group (P < 0.0001) and 50 μg / mL group (P < 0.0001) treated with MSC-Exo after treatment was significantly reduced. In addition, the proportion of DC necroptosis in the 50 μg / mL group was higher than that in the 10 μg / mL group, but the difference between the two groups was not significant.

[0127] 3.ELISA evaluation

[0128] S1, sample processing method: in the in vitro experiment part, after LPS+z-VAD combined stimulation, the culture medium was collected, and then the culture medium was placed in a centrifuge for centrifugation at room temperature, with a centrifugal speed of 1500 rpm and a centrifugal time of 5 min, and then the supernatant was retained;

[0129] S2, cytokine levels in cell culture supernatant were detected by ELISA kit;

[0130] S3, take out the pre-coated and sealed 96-well plate, equilibrate to room temperature, add the standard and specimen universal diluent to the blank wells, add 100 μL of the diluted cell culture supernatant of each group to the remaining wells, seal the reaction wells with sealing tape, place in a 37°C constant temperature box, and incubate in the dark for 90 minutes;

[0131] S4, after the incubation is completed, the reaction solution in the ELISA plate is aspirated, and the biotinylated antibody working solution is prepared 20 minutes in advance. 100 μL of the biotinylated antibody diluted at 1:100 is added to the reaction well, and the reaction well is sealed with sealing tape, and the plate is placed in a 37°C incubator and incubated for 60 minutes in the dark;

[0132] S5, wash the plate with washing buffer, soak for 1 min each time, repeat 5 times;

[0133] S6, after washing, add 100 μL of diluted peroxidase-labeled avidin into the reaction wells at a dilution ratio of 1:100, seal the reaction wells with sealing tape, place in a 37°C incubator, and incubate in the dark for 30 min;

[0134] S7, wash the plate with washing buffer, soak for 90 seconds each time, repeat 5 times;

[0135] S8, add 90 μL of chromogenic substrate to the reaction well, place in a 37°C incubator, and incubate for 20 min in the dark;

[0136] S9. After the incubation is completed, add 100 μL of reaction stop solution to the reaction well and mix gently. According to the microplate reader operation method, detect the OD value of each well at a wavelength of 450 nm within 3 minutes after adding the reaction stop solution, and calculate the concentration of the measured cytokine.

[0137] The results are as follows Figure 6 As shown in the results, in terms of reducing the levels of necroptosis-related cytokines, the levels of HMGB1 (P < 0.0001), IL-1α (P < 0.01), IL-33 (P < 0.001), TNF-α (P < 0.0001) and LDH (P < 0.0001) were significantly reduced after treatment with 2 μg / mL MSC-Exo compared with the stimulation group. However, statistical analysis showed that the treatment effects of the 10 μg / mL and 50 μg / mL dose groups were better, and the statistical differences of the various indicators detected with the stimulation group were P < 0.0001. In addition, after treatment with 50 μg / mL MSC-Exo, the levels of LDH and TNF-α were lower than those of the 10 μg / mL dose group, and the differences were statistically significant (LDH: P < 0.01; TNF-α: P < 0.05), and there were no significant differences in the levels of HMGB1, IL-1α and IL-33.

[0138] Based on the above flow cytometry, SYTOX-Green staining and ELISA test results, this part of the experiment confirmed that MSC-Exo can effectively inhibit DC necroptosis under sepsis conditions, among which the 10μg / mL and 50μg / mL dose groups have particularly significant effects, while there is no significant difference in the therapeutic effect between the 10μg / mL dose group and the 50μg / mL dose group. Therefore, this part of the dose-effect relationship study determined that the MSC-Exo dose used in subsequent in vitro experiments was 10μg / mL.

[0139] Experiment 5: MSC-Exo inhibits DC necroptosis under sepsis conditions

[0140] 1 In vitro experiment: After determining that the effective dose and concentration of MSC-Exo in the in vitro experiment was 10 μg / mL, the present application detected the necroptosis of DC in the in vitro stimulation group and the treatment group at different time points (0h, 6h, 12h and 24h) by flow cytometry and SYTOX-Green staining in the in vitro experiment.

[0141] (1) Flow cytometry

[0142] S1, the cells of the corresponding groups were collected at 0h, 6h, 12h and 24h and counted.5 Place the cells / tube in a flow cytometry tube, add an appropriate amount of PBS to allow the cells to be evenly distributed in the flow cytometry tube, and then centrifuge at a speed of 1500 rpm for 5 min.

[0143] S2, discard the supernatant and resuspend the cell pellet with 1 mL Binding Buffer, centrifuge again, the speed of the centrifuge is 1500 rpm, the centrifugation time is 5 min, discard the supernatant and resuspend the cell pellet with 100 μL Binding Buffer;

[0144] S3, add 5 μL Annexin V and 2.5 μL 7-AAD dye to each flow tube, tap the tube wall to evenly disperse the cells after adding the reagents, and incubate at room temperature in the dark for 15 min;

[0145] S4, set up double-negative tubes (without Annexin V and 7-AAD), single-stained tubes (only Annexin V was added), and single-stained tubes (only 7-AAD was added) as controls;

[0146] S5. After incubation, add 200 μL Binding Buffer to each tube, flick the tube wall to mix, and then detect and analyze necrosis and apoptosis cells (Annexin V + 7-AAD + ) ratio; FlowJo V 10.0 software was used to reanalyze the data of each group.

[0147] The results are as follows Figure 7 As shown in the in vitro experiment, flow cytometry and SYTOX-Green staining showed that the proportion of DC undergoing necroptosis under in vitro sepsis conditions increased significantly, and increased in a time-dependent manner within 24 hours after stimulation. The treatment group was given MSC-Exo treatment after stimulation, and the proportion of DC undergoing necroptosis in the treatment group at each time point also increased, but its level was lower than that of the stimulation group at the same time point. Among them, the analysis of flow cytometry results showed that the differences between the groups at 12h (P < 0.05) and 24h (P < 0.05) after stimulation (treatment) were statistically significant;

[0148] (2) SYTOX-Green staining

[0149] S1, prepare SYTOX-Green dead cell nucleic acid dye: dilute the SYTOX-Green dead cell nucleic acid dye stock solution (5 mM) with DMSO at a dilution ratio of 1:10000 to a final concentration of 50 nM;

[0150] S2, in vitro experimental part, 100 nM SYTOX-Green dye was added during the cell culture process, and the cell culture process was continued after addition;

[0151] S3, the cells of the corresponding groups were collected at 0h, 6h, 12h and 24h, and the cells were counted and collected into flow tubes at 2.0×106 cells / tube, and then placed in a centrifuge for centrifugation at a speed of 1500rpm, room temperature, and a centrifugation time of 5min. Then, 1mL PBS was used to resuspend the cell pellet and centrifuge again. The centrifugation conditions were the same and repeated 3 times;

[0152] S4, after washing the cells in the flow tube, discard the supernatant completely, resuspend the cell pellet with 60-70 μL PBS, and repeatedly blow and beat with a pipette to obtain a cell suspension;

[0153] S5, take 35 μL of cell suspension and drop it on a glass slide, cover it with a cover slip, spread it flat in a wet box, and send it for examination and filming;

[0154] S6, SYTOX-Green positive cells were observed and photographed using a fluorescence microscope, and Image J software was used to count and analyze the ratio of positive cells to the total number of cells in the field of view.

[0155] The results are as follows Figure 8 As shown in the figure, the analysis of SYTOX-Green staining results showed that the differences among the groups at 6h (P<0.001), 12h (P<0.01) and 24h (P<0.001) after stimulation (treatment) were statistically significant. The experiment showed that MSC-Exo can effectively reduce the increased proportion of DC necroptosis caused by in vitro stimulation.

[0156] 2 In vivo experiments: A CLP mouse sepsis model was established and MSC-Exo (100 μg / mouse) was injected through the tail vein to carry out in vivo experiments. Flow cytometry was used to detect the necroptosis of splenic DCs in the sepsis group and the treatment group at different time points (0h, 6h, 12h and 24h).

[0157] The method of establishing a mouse sepsis model by the CLP method is as follows: the mice were fasted for 12 hours before surgery, but were not allowed to drink water. The mice were anesthetized by intraperitoneal injection of chloral hydrate (5%), and the anesthetic dose (10 mL / kg) was determined according to the weight of the mice. After anesthesia, the mice were fixed on the operating board in a supine position; the abdominal skin of the mice was disinfected with iodine tincture, and incisions were made layer by layer along the midline of the abdomen with a length of 1 cm; the cecum was quickly found after opening the abdomen, and sterile No. 4 thread was used to ligate it 1.0 cm away from the end of the cecum, and a 22G needle was used to penetrate the cecum once at the distal end of the ligation, and the ligated intestinal tube was squeezed to allow part of the intestinal contents to overflow, and the cecum was returned to the abdominal cavity in situ, sutured layer by layer, and the abdomen was closed; 1 mL of 0.9% saline was injected subcutaneously behind the neck of the mice, and the mice were transferred to a breeding cage, and insulation measures were taken. After waking up, the mice were free to eat and drink water.

[0158] The flow cytometry procedure is the same as that of the flow cytometry in the in vitro experiment in Example 5, and will not be described again here.

[0159] Test results such as Fig. 9 As shown in the figure, flow cytometry showed that the proportion of necroptosis of DC in the spleen of septic mice increased significantly, and increased in a time-dependent manner within 24 hours after stimulation. At each time point after MSC-Exo treatment, the proportion of necroptosis of DC in the spleen of the treated group mice also increased, but its level was lower than that of the stimulation group at the same time point, and the difference between the two groups was statistically significant at 24 hours (P < 0.01).

[0160] Experiment 6: MSC-Exo reduces the levels of DC necroptosis-related proteins under sepsis conditions

[0161] 1 In vitro experiment: After determining that the effective concentration of MSC-Exo in the in vitro experiment is 10 μg / mL, the present application detected the expression levels of necroptosis-related proteins in DCs of the in vitro stimulation group and the treatment group at different time points (0h, 6h, 12h and 24h) by Western Blot and laser confocal microscopy in the in vitro experiment.

[0162] (1) Western Blot method to detect protein levels

[0163] Step 1, SDS-PAGE gel preparation:

[0164] Before the experiment, clean the utensils used for preparing glue with distilled water, install the glass plates in order on the glue tank and clamp them, fill the glass plates with distilled water and test for leaks for 15-20 minutes;

[0165] Add separation gel buffer A and separation buffer B in a ratio of 1:1 into a centrifuge tube to prepare 9 ml of separation gel / block, then add 90 μl of 10% ammonium persulfate and mix thoroughly with a disposable pipette to obtain separation gel;

[0166] Meanwhile, in a centrifuge tube, mix the stacking gel buffer A and the stacking gel buffer B at a ratio of 1:1 to prepare 3 ml of stacking gel / block, and add 30 μl of 10% ammonium persulfate, and mix thoroughly with a disposable pipette to obtain stacking gel;

[0167] Add separation gel to the gap of the leak-tested glass plate to 4 / 5 of the height, then slowly add concentrated gel on top of the separation gel along the edge (without waiting for the separation gel to solidify), then quickly insert a 15 mm thick comb, make sure there are no obvious bubbles, and let it stand at room temperature for 23 minutes. After solidification, obtain SDS-PAGE gel;

[0168] Step 2, electrophoresis:

[0169] Place the SDS-PAGE gel in an electrophoresis tank, add the prepared 1× electrophoresis buffer (1× electrophoresis buffer is a mixture of 50 ml of 10× electrophoresis buffer and 450 ml of distilled water), remove the comb teeth and check the integrity of the gel; ensure that the electrophoresis buffer in the gel tank exceeds the upper edge of the gel, and continue to add electrophoresis buffer to the outside to half the height of the separation gel; use a pipette to add the protein sample to the well, and connect the electrophoresis tank to the power supply; use a constant voltage of 180V for electrophoresis for about 45 minutes; keep the electrophoresis tank in a low temperature environment during the electrophoresis process; after the electrophoresis is completed, remove the glass plates on both sides, take out the gel and soak it in the prepared 1× electrotransfer buffer (the 1× electrotransfer buffer is a mixture of 50 ml of 10× electrotransfer buffer, 350 ml of distilled water and 100 ml of anhydrous methanol added in sequence).

[0170] Step 3, electroporation:

[0171] Use the wet transfer method, prepare filter paper according to the size of the gel and soak it in 1× electrotransfer buffer to soak it. Take a PVDF membrane of appropriate size and soak it in anhydrous methanol for more than 5 minutes to charge the PVDF membrane. Place filter paper-SDS-PAGE gel-PVDF membrane-filter paper in order, remove bubbles and press tightly, and put it into the electrotransfer tank of 1× electrotransfer buffer. Transfer the membrane at a constant current of 200-300mA for 2-2.5h.

[0172] Adjust the current and transfer time according to the molecular weight of the target protein.

[0173] Step 4: Closure:

[0174] The PVDF membrane was blocked in a rapid blocking solution for 10 min, and then washed with the prepared 1×TBS-T (1×TBS-T is prepared by mixing 50 ml of 10×TBS-T with 450 ml of distilled water) for 3 times, each time for 15 min.

[0175] Step 5, incubate with antibodies:

[0176] Prepare the primary antibody with a dilution ratio of 1:1000, obtain the antibody diluent, place the PVDF membrane in the antibody diluent, and incubate overnight at 4°C; on the second day, wash the PVDF membrane three times with 1×TBS-T, incubate with anti-mouse or anti-rabbit HRP-coupled secondary antibody (secondary antibody dilution ratio of 1:5000) at room temperature for 60 minutes, and use 1×TBS-T for development after washing.

[0177] Step 6, protein blot development:

[0178] Prepare ECL developer and protect from light for subsequent use. Use ECL gel imaging system to detect protein expression, use β-actin expression as an internal reference, and use ImageJ software to analyze the gray value of the target band and perform statistical analysis.

[0179] The results are as follows Fig.10 As shown in the in vitro experiment, Western Blot showed that the phosphorylation levels of RIPK1, RIPK3 and MLKL in DC cells were significantly increased under in vitro sepsis conditions, and increased in a time-dependent manner within 24 hours after stimulation. The treatment group was treated with MSC-Exo after stimulation, and there was no significant increase in the phosphorylation levels of RIPK1, RIPK3 and MLKL in DC cells in the treatment group at each time point. Among them, the ratio of p-RIPK1 / t-RIPK1 in the treatment group was significantly lower than that in the in vitro stimulation group at 6h (P<0.05), 12h (P<0.01) and 24h (P<0.01); the ratio of p-RIPK3 / t-RIPK3 was lower than that in the sepsis group at 6h, 12h and 24h, but the difference between the two groups was statistically significant only at 24h (P<0.001); the ratio of p-MLKL / t-MLKL was significantly lower than that in the sepsis group at each time point, and the difference between the groups was statistically significant (6h: P<0.05; 12h: P<0.01; 24h: P<0.05).

[0180] (2) Laser confocal microscopy

[0181] Laser confocal microscopy imaging technology

[0182] S1, cells from each group were collected and counted, and the cells were counted at 2.0×10 6 Cells / tubes were collected in flow tubes;

[0183] S2, place the flow tube in a centrifuge for centrifugation at a speed of 1500 rpm, at room temperature, for 5 min, discard the supernatant completely, add 1 mL of PBS to resuspend the cell pellet, and centrifuge again. Repeat 3 times to wash the cells;

[0184] S3, use 30 μL Trixon and 9970 μL deionized water to prepare a 0.3% Trixon solution for cell membrane permeabilization, and vortex to mix;

[0185] S4, weigh 0.1 g of BSA and dissolve it in 10 mL of deionized water to obtain a 1% BSA solution;

[0186] S5, add 500 μL of 0.3% Trixon solution to each flow tube treated in step S2, tap the tube wall to mix the cells, and let stand at room temperature in the dark for 10 min;

[0187] S6, after the cell membrane is broken, the flow tube is placed in a centrifuge for centrifugation at a speed of 1500 rpm, at room temperature, for 5 min. Then, the cell pellet is resuspended in 1 mL PBS and centrifuged again. Repeat this 3 times for cell washing.

[0188] S7, add 200 μL of primary antibody solution to 1% BSA solution to obtain a mixed primary antibody solution;

[0189] S8, add 200 μL of mixed primary antibody solution to the flow tube treated in step S6, tap the tube wall to mix the cells, seal with sealing film, and incubate in a 4°C refrigerator overnight;

[0190] S9, after the primary antibody incubation is completed, remove the sealing film, put the flow tube into the centrifuge for centrifugation, the speed of the centrifuge is 1500rpm, the centrifugation temperature is room temperature, the centrifugation time is 5min, use 1mL PBS to resuspend the cell pellet and centrifuge again, repeat 3 times to wash the cells;

[0191] S10, adding 200 μL of secondary antibody solution to 1% BSA solution to obtain a diluted mixed secondary antibody solution;

[0192] S11, add 200 μL of mixed secondary antibody solution to the flow tube treated in step S9, tap the tube wall to mix the cells, seal it with sealing film, and stand it at room temperature away from light for 1 hour;

[0193] S12, after the secondary antibody incubation is completed, remove the sealing film, put the flow tube into the centrifuge for centrifugation, the speed of the centrifuge is 1500rpm, the centrifugation temperature is room temperature, the centrifugation time is 5min, use 1mL PBS to resuspend the cell pellet and centrifuge again, repeat 3 times to wash the cells, pay attention to avoid light during this process, and prepare the slide and cover slip at the same time;

[0194] S13, after washing the cells in the flow tube, discard the supernatant completely, use 60-70μL PBS to resuspend the cell pellet, repeatedly blow and mix evenly with a pipette, take 35μL of the cell suspension and drop it on the slide, then add 5μLDA I solution to the cell droplet, carefully cover with a coverslip, spread it flat in a wet box, and send it for inspection and filming.

[0195] The results are as follows Fig.11 and Fig.12 As shown, laser confocal microscopy showed that the fluorescence intensity of p-MLKL and p-RIPK3 in DC under in vitro sepsis conditions was significantly enhanced, and the proportion of positive cells was significantly increased, while the fluorescence intensity of p-MLKL and p-RIPK3 in DC in the treatment group at each time point was significantly weakened, and the proportion of positive cells was also less than that in the stimulation group.

[0196] 2 In vivo experiments: A CLP mouse sepsis model was established and in vivo experiments were carried out by tail vein injection of MSC-Exo (100 μg / mouse). Western Blot and laser confocal microscopy were used to detect the expression levels of necroptosis-related proteins in DCs in the sepsis group and during treatment at different time points (0h, 6h, 12h and 24h).

[0197] The in vivo Western Blot and laser confocal microscopy detection steps are the same as those in the in vitro experiments. The method of establishing a mouse sepsis model by the CLP method is the same as that of the CLP method in Experiment 5.

[0198] (1) The results are as follows Fig.13 As shown in the figure, MSC-Exo reduced the levels of DC necroptosis-related proteins under sepsis conditions. In the in vivo experiment, Western Blot showed that the phosphorylation levels of RIPK1, RIPK3 and MLKL in the spleen DC of septic mice were significantly increased, and increased in a time-dependent manner within 24 hours after stimulation. The treatment group was treated with MSC-Exo after the model was established, and there was no significant increase in the phosphorylation levels of RIPK1 and RIPK3 in the spleen DC of the treatment group mice at each time point. Among them, the ratio of p-RIPK1 / t-RIPK1 in the treatment group was significantly lower than that in the sepsis group at 12h (P<0.05) and 24h (P<0.01); the ratio of p-RIPK3 / t-RIPK3 was significantly lower than that in the sepsis group at 6h (P<0.05), 12h (P<0.05) and 24h (P<0.05), and the difference was statistically significant; the ratio of p-MLKL / t-MLKL was significantly lower than that in the sepsis group at each time point (6h: P<0.01; 12h: P<0.01; 24h: P<0.01).

[0199] (2) The results are as follows Fig.14 and Fig.15 As shown, laser confocal microscopy showed that the fluorescence intensity of p-MLKL and p-RIPK3 in the spleen DC of septic mice was significantly enhanced, and the proportion of positive cells was significantly increased, while the fluorescence intensity of p-MLKL and p-RIPK3 in the spleen DC of mice in the treatment group at each time point was significantly weakened, and the proportion of positive cells was also less than that in the sepsis group.

[0200] Experiment 7: MSC-Exo reduces DC necroptosis-associated molecular patterns (DAMPs) and cytokine levels under sepsis conditions

[0201] 1 In vitro experiment: After determining the effective dose concentration of MSC-Exo in the in vitro experiment, the present application first detected the levels of DC necroptosis-related DAMPs and cytokines in the in vitro stimulation group and the treatment group at different time points (0h, 6h, 12h and 24h) by ELISA in the in vitro experiment. The ELISA detection steps are the same as the ELISA in vitro detection steps in Experiment 4.

[0202] The results are as follows Fig.16 As shown, in the in vitro experiment, ELISA detection showed that the levels of HMGB1, IL-1α, IL-33, LDH and TNF-α in the supernatant of DC culture under in vitro sepsis conditions were significantly upregulated, and increased in a time-dependent manner within 24 hours after stimulation. The levels of necroptosis-related factors also increased within 24 hours after MSC-Exo treatment, but their levels were significantly lower than those in the stimulation group at the same time point, with statistically significant differences, including HMGB1 (6h: P < 0.05; 12h: P < 0.05; 24h: P < 0.01), IL-1α (6h: P < 0.05; 12h: P < 0.01; 24h: P < 0.01), IL-33 (6h: P < 0.01; 24h: P < 0.05), TNF-α (6h: P < 0.01; 12h: P < 0.05; 24h: P < 0.01) and LDH (6h: P < 0.01; 12h: P < 0.01; 24h: P < 0.01).

[0203] 2 In vivo experiments: A CLP mouse sepsis model was established and in vivo experiments were carried out by tail vein injection of MSC-Exo (100 μg / mouse). The levels of DC necroptosis-related DAMPs and cytokines in the serum of mice in the sepsis group and the treatment group at different time points (0h, 6h, 12h and 24h) were detected based on ELISA.

[0204] ELISA test processing method:

[0205] S1, in vivo experiment, the eyeballs of mice were removed at different times after CLP surgery to collect blood, and the blood was naturally coagulated at room temperature for 30 min, then centrifuged at a speed of 3000 rpm, at room temperature, for 20 min, and the serum was collected and retained;

[0206] S2, cytokine levels in serum were detected by ELISA kit;

[0207] S3, take out the pre-coated and sealed 96-well plate, equilibrate to room temperature, add standard and specimen universal diluent to the blank wells, add 100 μL of the diluted serum from each group to the remaining wells, seal the reaction wells with sealing tape, place in a 37°C constant temperature box, and incubate in the dark for 90 min;

[0208] S4, after the incubation is completed, the reaction solution in the ELISA plate is aspirated, and the biotinylated antibody working solution is prepared 20 minutes in advance. 100 μL of the biotinylated antibody diluted at 1:100 is added to the reaction well, and the reaction well is sealed with sealing tape, and the plate is placed in a 37°C incubator and incubated for 60 minutes in the dark;

[0209] S5, wash the plate with washing buffer, soak for 1 min each time, repeat 5 times;

[0210] S6, after washing, add 100 μL peroxidase-labeled avidin (1:100 dilution) to the reaction well, seal the reaction well with sealing tape, place in a 37°C incubator, and incubate in the dark for 30 min;

[0211] S7, wash the plate with washing buffer, soak for 90 seconds each time, repeat 5 times;

[0212] S8, add 90 μL of chromogenic substrate to the reaction well, place in a 37°C incubator, and incubate for 20 min in the dark;

[0213] S9. After the incubation is completed, add 100 μL of reaction stop solution to the reaction well and mix gently. According to the microplate reader operation method, detect the OD value of each well at a wavelength of 450 nm within 3 minutes after adding the reaction stop solution, and calculate the concentration of the measured cytokine.

[0214] The results are as follows Fig.17As shown, ELISA detection showed that the levels of HMGB1, IL-1α, IL-33, LDH and TNF-α in the supernatant of DC culture under in vitro sepsis conditions were significantly upregulated, and increased in a time-dependent manner within 24 hours after the model was established. The levels of necroptosis-related factors also increased within 24 hours after MSC-Exo treatment, but their levels were significantly lower than those in the sepsis group at the same time point, including HMGB1 (12h: P < 0.05; 24h: P < 0.05), IL-1α (6h: P < 0.05; 12h: P < 0.01; 24h: P < 0.01), IL-33 (12h: P < 0.05; 24h: P < 0.05), TNF-α (12h: P < 0.01; 24h: P < 0.01) and LDH (6h: P < 0.05; 12h: P < 0.01; 24h: P < 0.01).

[0215] The embodiments described above are only preferred implementation modes of the present invention and cannot be used to limit the protection scope of the present invention. Any non-substantial changes and modifications made by those skilled in the art on the basis of the invention shall fall within the protection scope of the present invention.

Claims

1. Application of mesenchymal stem cell exosomes in the preparation of a drug for treating sepsis-induced dendritic cell necroptosis.

2. Application of mesenchymal stem cell exosomes in the preparation of a drug for reducing the level of necroptosis-related proteins in splenic dendritic cells.

3. Application of mesenchymal stem cell exosomes in the preparation of drugs for reducing the levels of molecular patterns associated with dendritic cell necroptosis.

4. Application of mesenchymal stem cell exosomes in the preparation of drugs for reducing the levels of cytokines related to dendritic cell necroptosis.

Images