A method for preparing mesenchymal stem cell exosomes and its application in preparing aerosol preparations for treating pneumonia

Abstract

The present invention discloses a method for preparing mesenchymal stem cell exosomes and their application in preparing a nebulized preparation for treating pneumonia, and belongs to the field of biotechnology. The present invention uses centrifugation and suction filtration to clarify, concentrate and replace liquid, molecular exclusion chromatography, ion exchange chromatography and final concentration to separate exosomes in the supernatant. The exosomes prepared by this method have the characteristics of high purity, good uniformity, small amount of residual impurities, small loss of activity, high exosome concentration, etc. Animal experiments have found that the prepared exosomes can effectively alleviate and treat lung damage caused by pneumonia and bronchopneumonia through nebulization administration, and have the characteristics of convenient administration and high safety.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing mesenchymal stem cell exosomes and application thereof in preparing an aerosol preparation for treating pneumonia. Background Art

[0002] Mesenchymal stem cells are a type of adult stem cells with multidirectional differentiation potential, which can be obtained from various tissues such as bone marrow, peripheral blood, fat and skin. Exosomes are a double-layer vesicle structure with a diameter of 30-150nm. Stem cell exosomes have good immune regulation and tissue repair functions and have extremely high clinical value.

[0003] The most common method for preparing exosomes is to use gradient centrifugation or differential centrifugation. For very small amounts of scientific research, commercial kits are usually used to extract exosomes. Although the above methods can achieve the preparation of exosomes, they also have obvious disadvantages. For example: 1. Working principle of gradient centrifugation: Gradient centrifugation, also known as zonal centrifugation, can separate several or all components in a sample at the same time and has good resolution. Disadvantages: long centrifugation time, need to prepare inert gradient medium solution, strict operation, and need to use exogenous reagents as medium, which brings risks to later animal experiments.

[0004] Second, differential centrifugation is a method of using low-speed and high-speed centrifugation alternately to grade and separate substances of different masses with centrifugal forces of different strengths. This method is suitable for separating components with large differences in sedimentation coefficients in mixed samples. The advantage of differential centrifugation is that it has a large sample processing capacity and can be used for the initial separation of a large number of samples. Its disadvantage is that when separating complex samples and requiring a high separation purity, the number of centrifugations is large and the operation is complicated. It is impossible to completely separate and purify some components with similar sedimentation coefficients.

[0005] 3. The characteristics of the exosome extraction kit are high resolution, which can obtain relatively clean and high-purity exosome solutions. It is one of the preferred processes for scientific research and analysis of exosomes; but its disadvantages are high cost, low yield, and inability to be prepared on a large scale.

[0006] In order to overcome the above shortcomings, the inventor team used ultrafiltration concentration system plus composite chromatography to prepare exosomes in the early stage. Although this method solves the problems of high cost and uncontrollable quality, the ultrafiltration concentration system needs to use three steps of clarification filter column, concentration column and final concentration to achieve, which causes the exosomes to circulate repeatedly in the hollow fiber for a long time, causing the exosome particles to rupture, affecting the final recovery rate and integrity rate of the exosome particles. Although composite chromatography solves the problem of impurity removal, the collection flow-through volume of composite chromatography is large, which brings great pressure to the final concentration step and increases the experimental time. In the final concentration step, the number of exosome particles increases with the increase of unit concentration and the increase of the number of cycles, which greatly increases the risk of exosome particle rupture. Therefore, seeking a new exosome preparation process to solve the above-mentioned technical problems is of great significance for exosome scientific research and large-scale preparation. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing mesenchymal stem cell exosomes and their application in preparing an aerosol preparation for treating pneumonia, so as to solve the problems existing in the above-mentioned prior art. The exosome solution is purified by clarifying filtration, concentrating and replacing liquid, molecular exclusion chromatography, ion exchange chromatography and final concentration. The prepared exosomes have a higher number of particles per unit, a more complete number of particles, and a shorter concentration time. The obtained exosomes can significantly improve the pathological characteristics of pneumonia after aerosol administration, and can be used to prepare drugs for treating and repairing pneumonia and / or bronchopneumonia.

[0008] To achieve the above object, the present invention provides the following solutions: The present invention provides a method for preparing mesenchymal stem cell exosomes, comprising the following steps: Isolate and obtain exosome solution from umbilical cord mesenchymal stem cells; The exosome solution is centrifuged and filtered to collect the filtrate; The filtrate is concentrated and replaced with a hollow fiber column to obtain a preliminary ultrafiltration concentrate; Separating the preliminary ultrafiltration concentrate by size exclusion chromatography and ion exchange chromatography in sequence to obtain a high-purity exosome solution; The high-purity exosome solution is concentrated and replaced with a hollow fiber column to obtain mesenchymal stem cell exosomes.

[0009] Preferably, the step of isolating and obtaining the exosome solution from umbilical cord mesenchymal stem cells comprises: Umbilical cord mesenchymal stem cells are subcultured with stem cell culture medium, and after the cell fusion degree reaches 80-90%, the supernatant is collected and centrifuged to obtain the exosome solution; the conditions for the subculture are: constant temperature culture in an environment with a carbon dioxide content of 3%-8%, a temperature of 36°C-39°C, and a humidity of 85%-100%.

[0010] Preferably, the centrifugal conditions are: 1500g centrifugation for 10 min; the filtration is performed by using a filter membrane with a diameter of 100 mm and a pore size of 0.65 μm for clarification filtration, and the vacuum pressure is controlled within 5 MPa.

[0011] Preferably, the conditions for concentrating and replacing the filtrate with a hollow fiber column are: the molecular weight cutoff of the hollow fiber column is 300KD, the pressure is controlled at 0-14Psi, and the injection flow rate is 700mL / min.

[0012] Preferably, the filler used in the size exclusion chromatography is agarose gel 6FF, the flow rate is 15 mL / min, and the pressure is controlled at 0-0.3 MPa.

[0013] Preferably, the filler used in the ion exchange chromatography is Capto Q ImpRes, the flow rate is 10 mL / min, and the pressure is controlled at 0-0.3 MPa.

[0014] Preferably, the high-purity exosome solution is concentrated and replaced by a hollow fiber column under the following conditions: the molecular weight cutoff of the hollow fiber column is 100KD, the pressure is controlled at 0-14Psi, and the injection flow rate is 100mL / min.

[0015] Preferably, the high-purity exosome solution is concentrated and replaced by a hollow fiber column to obtain an exosome solution with a concentration of 7.4E+11 Particles / mL, and the exosome solution with a concentration of 1.5E+11 Particles / mL is obtained by dilution, which is the mesenchymal stem cell exosome.

[0016] The present invention also provides use of the mesenchymal stem cell exosomes prepared by the preparation method in preparing a drug for treating pneumonia and / or bronchopneumonia.

[0017] Preferably, the dosage form of the drug comprises an aerosolized preparation.

[0018] The present invention discloses the following technical effects: The present invention discloses a preparation process for purifying mesenchymal stem cell exosomes, specifically: clarification and filtration-concentration and liquid exchange-molecular exclusion chromatography-ion exchange chromatography-final concentration. In the preparation process, the clarification step uses a filtration technique, and each concentration step adopts a tangential flow process of a hollow fiber column. The clarification and filtration step can allow the exosome supernatant to pass through a 0.65 μm filter membrane, preventing some cell debris or some visible impurities produced by cell metabolism from filtering the filter membrane, thereby achieving sample clarification; concentration and liquid exchange and final concentration can intercept exosome particles larger than the pore size of the hollow fiber, causing them to reflux repeatedly, but the culture medium added during the exosome culture process can pass through the pore size, gradually reducing the sample volume and increasing the amount of exosomes contained in the unit volume. The present invention uses molecular exclusion chromatography plus ion exchange chromatography to process samples, which can effectively remove different impurities produced by cell metabolism and vesicle structures that are not within the scope of exosomes, thereby obtaining an exosome solution with a higher purity, and the number of exosome particles reaches 7.4E+11Particles / mL. Compared with the previous method of preparing exosomes by the inventor's team using an ultrafiltration concentration system plus composite chromatography, the number of exosome particles per unit is higher, the number of particles is more complete, and the concentration time is shorter. Animal experiments have shown that the exosome solution prepared by the present invention can significantly improve the pathological characteristics of pneumonia by atomization administration, indicating that the exosome atomized liquid prepared by the present invention can effectively alleviate and treat lung damage caused by pneumonia and bronchopneumonia, and has the characteristics of convenient administration and high safety.

[0019] The exosome atomized liquid prepared by the present invention improves the purity of the exosomes, is also conducive to industrial production, and reduces production costs, providing a new method for large-scale industrial production of exosomes; in addition, it provides a new source of drugs for the treatment of pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 is the size exclusion chromatogram; Figure 2 is the ion exchange chromatogram; Figure 3 This is a schematic diagram of the number of unit particles in exosome solution detected by electron microscopy; Figure 4 The effects of different concentrations of exosomes on the treatment cycle of pneumonia in mice; Figure 5Results of hematoxylin-eosin staining of lung tissue sections after administration in different groups; A: control group; B: model group; C: treatment group. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Example 1 A method for preparing mesenchymal stem cell exosomes Umbilical cord mesenchymal stem cells were subcultured in stem cell serum-free medium (gibco, catalog number C12571500CP 10X500ml, English name MEM Alphabasic (1X)) and subcultured to P1-P10 generations. The culture conditions were 3%-8% carbon dioxide, temperature of 36℃-39℃, and humidity of 85%-100%. After 48 hours, the cell adhesion was observed under a microscope, and the adhesion was good. The culture medium was gently aspirated and the cells were gently rinsed once with serum-free medium to remove tissue residues and cell debris. 10mL of fresh serum-free medium was added and the cells were cultured in a constant temperature incubator. The carbon dioxide concentration in the incubator was controlled at 5%, the temperature was 37℃, and the humidity was 90%; incubated, and serum-free medium was added for culture. After the culture medium was replaced with fresh culture medium, the cells were digested with trypsin. After the cultured cell confluence reached 80-90%, the supernatant was collected to obtain the exosome supernatant, with a total volume of 4.2L of supernatant.

[0028] The process of purifying exosomes includes the following steps: (1) Clarification and filtration The harvested cell supernatant was centrifuged at 1500 g for 10 min at 4°C to remove preliminary impurities, and then clarified and filtered using a filter membrane with a diameter of 100 mm and a pore size of 0.65 μm: the vacuum pressure was controlled within 5 MPa, and the filtrate was collected.

[0029] (2) Concentration and fluid replacement Use a 300KD hollow fiber column to concentrate and replace the liquid: insert the silicone tubes at the inlet and reflux ends into the clarified filtrate obtained in step (1), and insert the permeate end into the waste liquid bucket; set the injection flow rate to 700mL / min, start concentration, control all pressures to 0-14Psi during the concentration process, after concentrating to 200mL, add 200mL of replacement liquid, and concentrate and replace the liquid 3 times; finally, push out all the solution in the hollow fiber column to obtain the ultrafiltration concentrate.

[0030] (3) Size Exclusion Chromatography Use size exclusion chromatography (filler: Sepharose 6FF) to remove most of the impurities in the cell supernatant: Balance: The flow rate is 15 mL / min, the pressure is controlled at 0-0.3 MPa, and the column is balanced with PBS until the baseline is stable.

[0031] Sample loading: The ultrafiltration concentrate obtained in step (2) was loaded at a flow rate of 15 mL / min. When the UV signal appeared, peak 1 was collected, and peak 2 was the impurity.

[0032] Balance: Flow rate is 15mL / min, pressure is controlled at 0-0.3MPa, PBS is used to balance to the UV baseline again, and collection is stopped. Figure 1 shown.

[0033] (4) Use Capto Q ImpRes for ion exchange to remove trace impurities, reduce the volume collected by size exclusion chromatography, and achieve concentration: Balance: The flow rate is 10 mL / min, the pressure is controlled at 0-0.3 MPa, and the column is balanced with PBS until the baseline is stable.

[0034] Sample loading: Peak 1 flow-through obtained in step (3) was loaded at a flow rate of 10 mL / min until all the flow-through was loaded.

[0035] Re-balance: The flow rate is 10 mL / min, the pressure is controlled at 0-0.3 MPa, and the column is re-balanced with PBS until the baseline is stable.

[0036] Elution: Use 1M NaCl pH 7.5 in PBS as the eluent. When the UV peak starts to rise, start collecting until the entire elution peak is completely collected. Figure 2 shown.

[0037] (5) Final concentration Use a 100KD hollow fiber column for concentration: insert the silicone tubes at the inlet and reflux ends of the 100KD hollow fiber column into the ion exchange eluate obtained in step (4), and insert the permeate end into the waste liquid bucket; set the injection flow rate to 100mL / min, start concentration, and control all pressures to 0-14Psi during the concentration process. When concentrated to 20mL, add 10 times the volume of physiological sodium chloride injection and continue to concentrate to 20mL. After repeating the concentration and liquid replacement 3 times, all the solution in the hollow fiber column is pushed out to obtain the final exosome solution with physiological sodium chloride injection as the buffer, which is the mesenchymal stem cell exosomes.

[0038] (6) Sterile filtration and packaging The exosome solution obtained in step (5) was filtered through a 0.2 μm sterile disposable filter membrane and packaged at 1 mL / tube to obtain the final exosome solution to be stored. A total of 32 tubes were taken, and 3 of them were tested. The concentration of exosomes was tested using an NTA detector. The results were as follows: Figure 3 shown.

[0039] The detection result of step (5) is 7.4E+11Particles / mL. The exosomes of this concentration are diluted to 1.5E+11Particles / mL with physiological sodium chloride injection. The exosome solution of this concentration is called exosome aerosol solution.

[0040] Comparative Example 1 The difference from Example 1 is that the purification process of the collected exosome supernatant is different, specifically: (1) Clarification and filtration Use a 0.45 μm hollow fiber column for clarification filtration: the inlet end, permeate end, and reflux end of the 0.45 μm hollow fiber column are connected to the instrument and fixed respectively; set the flow rate to 1500 mL / min, control the pressure at the feed end below 14 Psi, and collect the filtrate at the permeate end.

[0041] (2) Concentration and fluid replacement Use a 300KD hollow fiber column to concentrate and replace the liquid: insert the silicone tubes at the inlet and reflux ends into the clarified filtrate obtained in step (1), and insert the permeate end into the waste liquid bucket; set the flow rate to 420mL / min and start concentration. During the concentration process, control all pressures to 0-14Psi. After concentrating to 200mL, add 200mL of replacement liquid, and concentrate and replace the liquid three times; finally, push out all the solution in the hollow fiber column to obtain the ultrafiltration concentrate.

[0042] (3) Composite chromatography Using Core700 for composite chromatography: Balance: 25mL / min flow rate, balance the column with PBS until the baseline is stable.

[0043] Sample loading: The ultrafiltration concentrate obtained in step (2) was loaded at a flow rate of 25 mL / min. When the UV signal appeared, the flow-through was collected.

[0044] Balance: 25 mL / min flow rate, use PBS to balance to the UV baseline again, and stop collecting flow-through.

[0045] The liquid collected from the flow-through is the composite chromatography liquid.

[0046] (4) Final concentration Use a 100KD hollow fiber column for concentration: insert the silicone tubes at the inlet and reflux ends of the 100KD hollow fiber column into the composite chromatographic solution obtained in step (3), and insert the permeate end into the waste liquid bucket; set the flow rate to 100mL / min, start concentration, and control all pressures to 0-14Psi during the concentration process. When concentrated to 20mL, add 10 times the volume of physiological sodium chloride injection and continue to concentrate to 20mL. After repeating the concentration and liquid replacement 3 times, all the solution in the hollow fiber column is pushed out to obtain the final exosome solution with physiological sodium chloride injection as the buffer.

[0047] The purification efficiency, exosome quality and recovery rate of Example 1 and the comparative example were compared, and the results are shown in Tables 1 and 2 below.

[0048] Table 1 Purification efficiency comparison results

[0049] Table 2 Statistical results of exosome plasmids and recovery rates

[0050] As can be seen from Table 1 and Table 2, the method for purifying and preparing exosomes of the present invention takes less time and has higher purification efficiency than Comparative Example 1, and the recovery rate and the percentage of intact exosome particles recovered are higher, and the number of exosome particles per unit volume is also greater. After analysis, the specific reasons are: Compared with the three-step ultrafiltration concentration plus composite chromatography method of comparative example 1, the present invention reduces the disadvantage of repeatedly circulating the exosome particles in the clarification step. At the same time, the molecular exclusion chromatography plus ion exchange chromatography is used for purification. Compared with composite chromatography, the collection volume will increase, and the ion exchange plays a role in concentration, so that the volume of exosomes eluted by ion exchange is more than 10 times smaller than the flow-through volume collected by composite chromatography. After a final concentration step, high-concentration exosomes can be produced, and the number of exosome particles per unit volume is higher, the number of particles is more complete, and the concentration time is shorter.

[0051] Animal experiments 1. Animal grouping Normal mice, aged 3-5 weeks and weighing 18-25 g, were selected. After 7 days of adaptive feeding, the mice were randomly divided into control group, infection group and treatment group, with 5 mice in each group. Another group of 10 mice was selected as the aerosol concentration exploration group. This group was the first group of experiments, and the other three groups were then tested.

[0052] 2. Animal modeling The mice in the exploratory group were intraperitoneally injected with 100 μL of cyclophosphamide to cause low immune function in the mice. On this basis, the prepared Staphylococcus aureus solution was slowly dripped into one side of the mouse's nose. After the dripping, the mice were immediately placed on a fixed platform for about 1 minute and fed for one week. The mice in the control group were treated with sodium chloride solution for injection.

[0053] A mouse from the exploratory group was selected, and the lung tissue was collected and fixed for one week. The lung tissue was then dehydrated with ethanol, transparentized with xylene, embedded in paraffin, and routinely sectioned for hematoxylin-eosin staining and observation. It was found that the mice in the model group and the control group were successfully infected with pneumonia, and the control group was normal.

[0054] The remaining 9 mice were numbered 01-09 and administered with exosomes at different concentrations (see Table 3). The drugs were administered by nebulization using an animal nebulizer for 30 minutes each time, twice a day. The treatment status was determined based on the respiratory rate and status of the mice.

[0055] Table 3 Method of administration

[0056] The data showed that the treatment cycle of mice No. 01 and No. 02 was significantly longer, and they could only reach the respiratory rate and status of mice No. 03-09 on the seventh and fifth days respectively. When the concentration reached 1.5E+11 Particles / mL or higher, the treatment cycle did not shorten with the increase of the concentration. Therefore, 1.5E+11 Particles / mL was determined to be the optimal concentration. Figure 4 shown.

[0057] 3. Animal medication In the same way, mice in the model group and treatment group were intraperitoneally injected with 100 μL of cyclophosphamide to cause low immune function in mice. On this basis, the prepared bacterial solution was slowly dripped into one side of the mouse's nose. After the dripping, the mice were immediately placed on a fixed platform for about 1 minute and raised for one week. The mice in the control group were treated with sodium chloride solution for injection.

[0058] Then one mouse each was taken from the model group, treatment group and control group. The lung tissue was collected and fixed for one week, and then dehydrated with ethanol, transparentized with xylene, embedded in paraffin, and routinely sectioned for hematoxylin-eosin staining and observation. It was found that the mice in the model group and the control group were successfully infected with pneumonia, while the control group was normal.

[0059] The drug was administered by nebulization using an animal nebulizer for 30 minutes each time, twice a day, for 2 weeks. The control group and the infection group were administered with saline solution, and the treatment group was administered with exosome nebulization solution. 4. Evaluation of lung tissue sections The three groups of mice were routinely killed, and the lung tissues were collected and fixed for one week, then dehydrated with ethanol, transparentized with xylene, embedded in paraffin, and routinely sectioned for hematoxylin-eosin staining and observation.

[0060] 5. Experimental results The results of section staining showed that the control group was normal lung tissue (see Figure 5 In the middle A, obvious inflammatory tissue infection was found in the infection group (see Figure 5 In the middle B, the group that received the exosome aerosol solution showed a significant reduction in inflammatory infiltration in the lung tissue and a significant improvement in goblet cell hyperplasia in the airways (see Figure 5 Middle C).

[0061] The above results indicate that the mesenchymal stem cell exosome aerosol solution prepared in the example can be administered by inhalation with a nebulizer device, which can greatly improve the therapeutic effect.

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing mesenchymal stem cell exosomes, characterized in that: The following steps are involved: Isolate and obtain exosome solution from umbilical cord mesenchymal stem cells; The exosome solution is centrifuged and filtered to collect the filtrate; The filtrate is concentrated and replaced with a hollow fiber column to obtain a preliminary ultrafiltration concentrate; Separating the preliminary ultrafiltration concentrate by size exclusion chromatography and ion exchange chromatography in sequence to obtain a high-purity exosome solution; The high-purity exosome solution is concentrated and replaced with a hollow fiber column to obtain mesenchymal stem cell exosomes.

2. The preparation method according to claim 1, characterized in that The steps of isolating and obtaining exosome solution from umbilical cord mesenchymal stem cells include: Umbilical cord mesenchymal stem cells are subcultured with stem cell culture medium, and after the cell fusion degree reaches 80-90%, the supernatant is collected and centrifuged to obtain the exosome solution; the conditions for the subculture are: constant temperature culture in an environment with a carbon dioxide content of 3%-8%, a temperature of 36°C-39°C, and a humidity of 85%-100%.

3. The preparation method according to claim 1, characterized in that: The centrifugal conditions are: 1500g centrifugation for 10 minutes; the filtration is performed by using a filter membrane with a diameter of 100mm and a pore size of 0.65μm for clarification filtration, and the vacuum pressure is controlled within 5Mpa.

4. The preparation method according to claim 1, characterized in that: The conditions for concentrating and replacing the filtrate with a hollow fiber column are as follows: the molecular weight cutoff of the hollow fiber column is 300KD, the pressure is controlled at 0-14Psi, and the injection flow rate is 700mL / min.

5. The preparation method according to claim 1, characterized in that: The filler used in the molecular exclusion chromatography is agarose gel 6FF, the flow rate is 15 mL / min, and the pressure is controlled at 0-0.3 MPa.

6. The preparation method according to claim 1, characterized in that: The filler used in the ion exchange chromatography is CaptoQ ImpRes, the flow rate is 10 mL / min, and the pressure is controlled at 0-0.3 MPa.

7. The preparation method according to claim 1, characterized in that: The conditions for concentrating and replacing the high-purity exosome solution through the hollow fiber column are as follows: the molecular weight cutoff of the hollow fiber column is 100KD, the pressure is controlled at 0-14Psi, and the injection flow rate is 100mL / min.

8. The preparation method according to claim 1, characterized in that: The high-purity exosome solution is concentrated and replaced by a hollow fiber column to obtain an exosome solution with a concentration of 7.4E+11 Particles / mL, and is diluted to obtain an exosome solution with a concentration of 1.5E+11 Particles / mL, which is the mesenchymal stem cell exosome.

9. Use of mesenchymal stem cell exosomes prepared by the preparation method according to any one of claims 1 to 8 in the preparation of a medicament for treating pneumonia and / or bronchopneumonia.

10. The use according to claim 9, characterized in that The dosage form of the drug includes an aerosol preparation.

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