CN112043686A - Application of umbilical cord mesenchymal stem cell exosome atomization liquid in preparation of product for treating asthma - Google Patents
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Abstract
The invention discloses application of an umbilical cord mesenchymal stem cell exosome atomized liquid in preparation of a product for treating asthma, wherein the atomized liquid is the application of a mesenchymal stem cell exosome atomized liquid consisting of a mesenchymal stem cell exosome and physiological saline in any one of the following steps of (1) preparing a product for reducing inflammation of a lung trachea and a lung trachea side; (2) preparing a product for treating asthma; (3) preparing a product for preventing asthma; (4) preparing the product for relieving asthma. The preparation method of the umbilical cord mesenchymal stem cell exosome atomized liquid for treating asthma can obtain high-purity and high-density exosomes with biological activity in a short time; in the preparation process, the centrifugal temperature is 0-20 ℃, so that the biological activity of exosome is not influenced in the extraction process.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of an umbilical cord mesenchymal stem cell exosome atomization liquid in preparation of a product for treating asthma.
Background
Mesenchymal stem cells (Mesenchymal stem cells) are a class of adult stem cells with multipotentiality, originate from early-developing mesoderm, mainly exist in connective tissues and organ interstitium, and can be obtained from various tissues such as bone marrow, peripheral blood, fat, skin and the like. In recent years, it has been found that stem cells can secrete exosomes containing a variety of bioactive substances into a culture medium during culture. The exosome is a double-layer vesicle structure with the diameter of 30-150nm and is used as a signal transmission substance between cells in an organism, and the exosome contains various bioactive substances such as microRNA, signal protein, active peptide, cytokine and the like. And a large number of researches show that the stem cell exosome has good functions of immunoregulation, tissue repair and the like, and has extremely high clinical value in the aspects of treating various diseases such as burn, chronic obstructive pulmonary disease, asthma, aging resistance and the like.
Asthma is a common clinical respiratory disease characterized by chronic airway inflammation, airway hyperresponsiveness and airway remodeling, and is characterized by a variety of respiratory symptoms, including wheezing, shortness of breath, chest tightness, cough, and often by expiratory airflow limitation during pulmonary function examinations. Asthma is a chronic inflammatory disease of the airways involving a variety of cells including eosinophils, mast cells, T lymphocytes, neutrophils, smooth muscle cells, airway epithelial cells, etc., and cellular components. The clinical manifestations of the disease are recurrent wheezing, chest distress or cough, which often attacks or aggravates at night and in the morning, and most patients can relieve themselves or relieve after treatment, accompanied by variable restriction of wheezing and airway hyperreactivity, and a series of airway structural changes can be caused along with the prolongation of the course of the disease, namely airway remodeling. According to GINA (global guidelines for asthma control), treatment of bronchial asthma mainly involves inhalation of hormones in combination with inhalation of beta receptor agonists. However, many asthmatics, despite optimal treatment, fail to manage asthma and severely affect the quality of life of the patient. In recent years, a large number of basic experiments show that in an asthma model, the mesenchymal stem cell exosome can play a therapeutic role in the aspects of resisting inflammation, promoting tissue cell repair, regulating immune response and the like, and improve asthma-related symptoms.
At present, the exosome preparation for treating asthma generally has the defects of low exosome extraction efficiency, damage to an exosome self-structure in the extraction process, unsatisfactory administration mode and the like.
Disclosure of Invention
Therefore, the invention provides the application of the umbilical cord mesenchymal stem cell exosome atomization liquid in preparing a product for treating asthma.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a preparation method of mesenchymal stem cell exosome atomized liquid, which comprises the following steps: subculturing umbilical cord mesenchymal stem cells in a stem cell culture medium, collecting supernatant after the fusion degree of the cultured cells reaches 80-90%, separating to obtain an exosome solution, filtering the exosome solution by a sterile filter membrane of 0.22 mu m, centrifuging the collected filtrate for 30-90min at the rotating speed of 50000-;
and dissolving the exosome in physiological saline to obtain the mesenchymal stem cell exosome atomization liquid.
In one embodiment of the invention, the subculture mesenchymal stem cells are transferred to P1-P10 generations under the conditions of 3% -8% carbon dioxide, 36-39 ℃ and 85% -100% humidity and constant temperature culture.
In one embodiment of the invention, the umbilical cord mesenchymal stem cells are obtained by removing arteriovenous blood vessels from an umbilical cord, shearing into tissue homogenate blocks, filtering by using a tissue homogenate block filter screen, incubating the tissue homogenate blocks, adding a serum-free culture medium for culture, replacing a fresh culture medium for culture, and digesting by using pancreatin.
In one embodiment of the invention, the volume of the tissue homogenate is 0.5 to 3mm3。
In one embodiment of the present invention, the filter screen is 200 mesh.
In one embodiment of the invention, the ambient temperature of the centrifugation is 0-20 ℃.
The invention also provides the mesenchymal stem cell exosome atomization liquid prepared by the method.
In one embodiment of the invention, the mesenchymal stem cells are human primary umbilical cord mesenchymal stem cells.
The application of the mesenchymal stem cell exosome atomized liquid prepared by any one of the methods in any one of the following steps of (1) preparing a product for reducing pulmonary trachea and pulmonary paratracheal inflammation; (2) preparing a product for treating asthma; (3) preparing a product for preventing asthma; (4) the product for relieving asthma is also covered by the invention.
In the invention, the exosome is a double-layer vesicle structure with the diameter of 30-150nm secreted by cells, and contains various bioactive substances, such as microRNA, signal protein, cytokine, active polypeptide and the like.
The invention has the following advantages:
the preparation method of the umbilical cord mesenchymal stem cell exosome atomized liquid for treating asthma can obtain high-purity and high-density exosomes with biological activity in a short time; in the preparation process, the centrifugal temperature is 0-20 ℃, so that the biological activity of exosomes is not influenced in the extraction process; according to the invention, the extraction rate and purity of exosome extraction are improved to a certain extent by improving the isolated culture method of umbilical cord mesenchymal stem cells; the content of the exosome is that 0.1-10ml of exosome atomization liquid can be prepared from the exosome contained in every 100ml of umbilical cord mesenchymal stem cell supernatant, and the exosome has good biological activity under the concentration; the aerosol inhalation administration scheme is not limited to the use with an ultrasonic nebulizer, a compression nebulizer, a mesh nebulizer, or other atomization device, and improves the therapeutic effect.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is a microscope observation image of a P3 generation umbilical cord mesenchymal stem cell of an embodiment of the invention;
fig. 2 is a diagram of the results of flow assay of mesenchymal stem cells according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of the exosome diameter distribution range of an embodiment of the present invention;
FIG. 4 is a morphology under a scanning electron microscope of exosomes prepared according to the example of the present invention;
FIG. 5 is a HE-stained lung histomorphogram of a mouse according to an embodiment of the present invention, wherein A is a control group, B is an asthma model group, and C is a human umbilical cord mesenchymal stem cell exosome nebulization liquid treatment group.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the invention, the serum-free stem cell culture medium has the following formula: 100-300mg/L of anhydrous calcium chloride, 0.03-0.2mg/L of ferric nitrate nonahydrate, 500mg/L of potassium chloride 200, 50-150mg/L of anhydrous magnesium sulfate, 5000-7200mg/L of sodium chloride, 75-165mg/L, L of anhydrous sodium dihydrogen phosphate, 65-105mg/L, L of arginine hydrochloride, 42-86mg/L, L of cystine hydrochloride, 320-760mg/L of glycine, 14-32mg/L, L of histidine hydrochloride, 30-54mg/L, L of isoleucine, 75-165mg/L, L of leucine, 80-125mg/L, L of lysine hydrochloride 120, 165mg/L, L-methionine, 12-48mg/L of methionine, 30-80mg/L, L mg/25-62 mg/L, L-72-125 mg/L, L-tryptophan 8-22.5mg/L, L-tyrosine sodium salt 75-162mg/L, L-valine 80-160mg/L, D-calcium pantothenate 0.5-6.5mg/L, choline chloride 2.5-8mg/L, folic acid 2.5-6.5mg/L, inositol 4.2-8.6mg/L, nicotinamide 3-7.5mg/L, riboflavin 0.2-1.2mg/L, thiamine hydrochloride 1.6-5.8mg/L, pyridoxine hydrochloride 2.4-6mg/L, glucose 2000-8500mg/L, sodium pyruvate 80-135mg/L, 0.01-0.03mg/L of sodium selenite, 10-45mg/L of insulin and 2-80mg/L of total cytokine.
Example 1 preparation of mesenchymal Stem cell exosome atomization solution
The preparation method of the human umbilical cord mesenchymal stem cell exosome atomized liquid provided by the embodiment comprises the following steps:
(1) aseptically collecting umbilical cords, selecting healthy, full-term and caesarean born primiparies, and after the parturients and family members agree, taking the whole umbilical cords and quickly placing the whole umbilical cords in sterile PBS (phosphate buffer solution) containing 1% of penicillin sodium and 1% of streptomycin sulfate in an operating room under an aseptic environment;
(2) repeatedly flushing the umbilical cord with sterile PBS solution in a super clean bench, flushing to remove residual blood in umbilical vein and artery, longitudinally dissecting the umbilical cord, and removing arteriovenous vessels in the umbilical cord;
(3) fixing one segment of umbilical cord with hemostatic forceps, cutting into 1cm length segments with surgical scissors, and cutting into 1mm length segments with tissue scissors3Tissue mass of size. Filtering the obtained tissue block by using a 200-mesh filter screen, removing smaller tissue remains and cell fragments, and selecting an umbilical cord tissue block with the size meeting the requirement;
(4) uniformly spreading the obtained tissue blocks in a culture dish of 10cm, and culturing in a constant-temperature incubator for 1 hour, wherein the constant-temperature incubator has a carbon dioxide concentration of 3%, a temperature of 36 ℃ and a humidity of 100%;
(5) after the tissue blocks are cultured until the adherence is relatively firm, gently adding a proper amount of serum-free culture medium special for stem cells, and placing the mixture in a constant-temperature incubator for static culture, wherein the concentration of carbon dioxide in the incubator is 3%, the temperature is 36 ℃, and the humidity is 100%;
(6) after 24 hours, the cells were observed by microscope for good adherence. Gently sucking the culture medium, gently rinsing the culture medium once by using a serum-free culture medium to remove tissue residues and cell fragments, adding 10ml of fresh serum-free culture medium, and performing static culture in a constant-temperature culture box, wherein the concentration of carbon dioxide in the culture box is 3%, the temperature is 36 ℃, and the humidity is 100%;
(7) on the 5 th day, when the adherent cells in the culture dish are fully spread by about 60 percent, digesting for 3 minutes by using 1ml of 0.25 percent recombinant trypsin, observing cytoplasm retraction under a microscope, and beginning to fall off and float, at the moment, adding 10ml of serum-free culture medium to stop digestion, slightly blowing by using a 10ml pipette to prepare single cell suspension, centrifuging for 10 minutes at 1000rpm, removing supernatant, and re-suspending by using 10ml of fresh serum-free culture medium to obtain the cells, namely the primary umbilical cord mesenchymal stem cells;
(8) subculturing the primary umbilical cord mesenchymal stem cell suspension obtained in the step (7) according to the ratio of 1:2, marking the obtained cell as a first generation mesenchymal stem cell (P1), and as shown in figure 2, obtaining a mesenchymal stem cell flow detection result diagram;
(9) repeating the steps (7) and (8) when the cultured mesenchymal stem cells are fully grown by about 60-90%, and continuing to passage to P3 generation, wherein the passage is shown in figure 1 and is a microscope observation picture of umbilical cord mesenchymal stem cells of P3 generation;
(10) after the fusion degree of the cultured mesenchymal stem cells P3 generation reaches 80-90%, sucking out the mesenchymal stem cells from the culture medium, replacing the serum-free culture medium for culturing for 24h, collecting the supernatant, and removing cell fragments to obtain a solution containing exosomes;
(11) taking 100ml of the solution containing the exosomes obtained in the step (10), filtering the solution through a sterile filter membrane of 0.22 mu m to obtain a filtrate, then placing the filtrate at 120000g of rotation speed for centrifugation for 60 minutes, and discarding the supernatant, wherein the obtained precipitate is the exosomes, and is shown in fig. 3, which is a schematic diagram of the diameter distribution range of the exosomes, and is shown in fig. 4, which is a morphological diagram of the exosomes under a scanning electron microscope.
(12) And (4) resuspending the exosome obtained in the step (11) by using 2ml of sterile normal saline to obtain the human umbilical cord mesenchymal stem cell exosome atomization liquid.
Example 2 preparation of mesenchymal Stem cell exosome atomization solution
The preparation method of the human umbilical cord mesenchymal stem cell exosome atomized liquid provided by the embodiment comprises the following steps:
(1) aseptically collecting umbilical cords, selecting healthy, full-term and caesarean born primiparies, and after the parturients and family members agree, taking the whole umbilical cords and quickly placing the whole umbilical cords in sterile PBS (phosphate buffer solution) containing 1% of penicillin sodium and 1% of streptomycin sulfate in an operating room under an aseptic environment;
(2) in the clean bench, the umbilical cord was repeatedly flushed with sterile PBS to remove residual blood from the umbilical vein and artery. Longitudinally cutting open the umbilical cord, and removing arteriovenous vessels in the umbilical cord;
(3) fixing one segment of umbilical cord with hemostatic forceps, cutting into 1cm length segments with surgical scissors, and cutting into 1mm length segments with tissue scissors3Tissue mass of size. Filtering the obtained tissue block by using a 200-mesh filter screen, removing smaller tissue remains and cell fragments, and selecting an umbilical cord tissue block with the size meeting the requirement;
(4) uniformly spreading the obtained tissue blocks in a culture dish of 10cm, and culturing in a constant-temperature incubator for 2 hours, wherein the concentration of carbon dioxide in the incubator is 5%, the temperature is 37 ℃, and the humidity is 90%;
(5) after the tissue block adheres to the wall firmly, gently adding a proper amount of serum-free culture medium special for stem cells, and placing the mixture in a constant-temperature incubator for standing culture, wherein the concentration of carbon dioxide in the incubator is controlled to be 5%, the temperature is controlled to be 37 ℃, and the humidity is controlled to be 90%;
(6) after 48 hours, the cells were observed by microscope for good adherence. The medium was gently aspirated and gently rinsed once with serum-free medium to remove tissue debris and cell debris. Adding 10ml of fresh serum-free culture medium, and performing static culture in a constant-temperature incubator, wherein the concentration of carbon dioxide is controlled to be 5%, the temperature is controlled to be 37 ℃, and the humidity is controlled to be 90%;
(7) on the 7 th day, when the adherent cells in the culture dish are approximately 80% full, 1ml of 0.25% recombinant trypsin is used for digesting for 3 minutes, the cytoplasm is observed to retract under a microscope, the cells begin to fall off and float, at the moment, 10ml of serum-free culture medium is added to stop digestion, a 10ml pipettor is used for blowing gently to form single cell suspension, the single cell suspension is centrifuged at 1000rpm for 10 minutes, the supernatant is discarded, and the suspension is resuspended by 10ml of fresh serum-free culture medium, so that the obtained cells are the primary umbilical cord mesenchymal stem cells;
(8) subculturing the primary umbilical cord mesenchymal stem cell suspension obtained in the step (7) according to the proportion of 1:4, and marking the obtained cells as first-generation mesenchymal stem cells (P1);
(9) repeating the steps (7) and (8) when the cultured mesenchymal stem cells are fully grown by about 60-90%, and continuously carrying out passage until P5 generation;
(10) after the fusion degree of the cultured mesenchymal stem cells P5 generation reaches 80-90%, sucking out the mesenchymal stem cells from the culture medium, replacing the serum-free culture medium for culturing for 24h, collecting the supernatant, and removing cell fragments to obtain a solution containing exosomes;
(11) and (3) taking 100ml of the solution containing the exosome prepared in the step (10), filtering the solution through a sterile filter membrane with the diameter of 0.22 mu m, taking the filtrate, then placing the filtrate at the rotating speed of 100000g for centrifuging for 90 minutes, and removing the supernatant to obtain the precipitate, namely the exosome.
(12) And (4) resuspending the exosome obtained in the step (11) by using 8ml of sterile normal saline to obtain the human umbilical cord mesenchymal stem cell exosome atomization liquid.
Example 3 preparation of mesenchymal Stem cell exosome atomization solution
The preparation method of the mesenchymal stem cell exosome atomized liquid provided by the embodiment comprises the following steps:
(1) aseptically collecting umbilical cords, selecting healthy, full-term and caesarean born primiparies, and after the parturients and family members agree, taking the whole umbilical cords and quickly placing the whole umbilical cords in sterile PBS (phosphate buffer solution) containing 1% of penicillin sodium and 1% of streptomycin sulfate in an operating room under an aseptic environment;
(2) repeatedly flushing the umbilical cord with sterile PBS solution in a super clean bench, flushing to remove residual blood in umbilical vein and artery, longitudinally dissecting the umbilical cord, and removing arteriovenous vessels in the umbilical cord;
(3) fixing one segment of umbilical cord with hemostatic forceps, cutting into 1cm length segments with surgical scissors, and cutting into 1mm length segments with tissue scissors3Tissue mass of size. Filtering the obtained tissue block by using a 200-mesh filter screen, removing smaller tissue remains and cell fragments, and selecting an umbilical cord tissue block with the size meeting the requirement;
(4) uniformly spreading the obtained tissue blocks in a culture dish of 10cm, and culturing in a constant-temperature incubator for 1 hour, wherein the concentration of carbon dioxide in the constant-temperature incubator is 8%, the temperature is 38 ℃, and the humidity is 85%;
(5) after the tissue blocks are cultured until the adherence is relatively firm, gently adding a proper amount of serum-free culture medium special for stem cells, and placing the mixture in a constant-temperature incubator for static culture, wherein the concentration of carbon dioxide in the incubator is 3%, the temperature is 36 ℃, and the humidity is 100%;
(6) after 24 hours, the cells were observed by microscope for good adherence. Gently sucking the culture medium, gently rinsing the culture medium once by using a serum-free culture medium to remove tissue residues and cell fragments, adding 10ml of fresh serum-free culture medium, and performing static culture in a constant-temperature culture box, wherein the concentration of carbon dioxide in the culture box is 8%, the temperature is 38 ℃, and the humidity is 85%;
(7) on the 5 th day, when the adherent cells in the culture dish are fully spread by about 60 percent, digesting for 3 minutes by using 1ml of 0.25 percent recombinant trypsin, observing cytoplasm retraction under a microscope, and beginning to fall off and float, at the moment, adding 10ml of serum-free culture medium to stop digestion, slightly blowing by using a 10ml pipette to prepare single cell suspension, centrifuging for 10 minutes at 1000rpm, removing supernatant, and re-suspending by using 10ml of fresh serum-free culture medium to obtain the cells, namely the primary umbilical cord mesenchymal stem cells;
(8) subculturing the primary umbilical cord mesenchymal stem cell suspension obtained in the step (7) according to the proportion of 1:3, and marking the obtained cells as first-generation mesenchymal stem cells (P1);
(9) repeating the steps (7) and (8) when the cultured mesenchymal stem cells are fully grown by about 60-90%, and continuously carrying out passage until P8 generation;
(10) after the fusion degree of the cultured mesenchymal stem cells P8 generation reaches 80-90%, sucking out the mesenchymal stem cells from the culture medium, replacing the serum-free culture medium for culturing for 24h, collecting the supernatant, and removing cell fragments to obtain a solution containing exosomes;
(11) taking 100ml of the solution containing the exosome obtained in the step (10), filtering the solution through a sterile filter membrane with the diameter of 0.22 mu m to obtain a filtrate, then placing the filtrate at 190000g of rotation speed for centrifuging for 40 minutes, and removing the supernatant to obtain a precipitate, namely the exosome.
(12) And (4) resuspending the exosome obtained in the step (11) by using 2ml of sterile normal saline to obtain the umbilical cord mesenchymal stem cell exosome atomization liquid.
In embodiments 1 to 3 of the present invention, in the preparation process of the mesenchymal stem cell exosome atomization liquid, each 100ml of exosome prepared from umbilical cord mesenchymal stem cell supernatant can be used to prepare 0.1 to 10ml of the mesenchymal stem cell exosome atomization liquid of the present invention.
In embodiments 1 to 3 of the present invention, the administration of the mesenchymal stem cell exosome aerosol by aerosol inhalation is not limited to the use with an ultrasonic nebulizer, a compression nebulizer, a mesh nebulizer or other atomization device, and the therapeutic effect can be greatly improved.
Experimental example treatment of mesenchymal stem cell exosome atomized liquid on asthma mouse model
The experimental process of treating the asthma small animal model by the human umbilical cord mesenchymal stem cell exosome atomized liquid is as follows:
first, animal grouping
Selecting Balb/c mice, female, 4-6 weeks old, after the mice are bred for 7 days in an adaptive way, grouping the mice by adopting a random digital table method, and dividing the mice into a normal group, a model group and a treatment group, wherein each group comprises 6 mice.
Preparation of mouse COPD model
The mice in the model group and the treatment group are sensitized by intraperitoneal injection for 3 times by 200 mu l of ovalbumin plus aluminum hydroxide solvent (containing 40 mu g of ovalbumin and 2mg of aluminum hydroxide, and the volume is adjusted to 200 mu l by PBS), and the treatment time is 1, 7 and 14 days respectively. On days 22-26, nebulization with 5% by volume egg white albumin solution was continued for 30 minutes with 5 consecutive days of challenge.
Control mice were treated with PBS instead of ovalbumin.
Administration to animals
The administration is carried out by using an animal aerosol administration box, wherein each aerosol administration is carried out for 10 minutes, 2 times a day and the duration is 8 weeks. Wherein, the normal group and the model group are administrated with normal saline, and the treatment group is human umbilical cord mesenchymal stem cell exosome atomized liquid.
Pathological morphological evaluation of lung tissue
Rats were sacrificed routinely, lung tissue was collected and fixed for one week, ethanol dehydrated, xylene cleared, paraffin embedded, routinely sectioned for hematoxylin-eosin staining and observation.
Fifth, experimental results
As shown in fig. 5, compared with the control group, the human umbilical cord mesenchymal stem cell exosome atomized liquid treatment group of the present invention has the advantages that the mouse model group has obvious inflammatory cell infiltration beside the trachea, the airway epithelium goblet cell proliferation is obvious, the lung endotracheal inflammation infiltration is obviously reduced, and the goblet cell proliferation in the airway is obviously improved after the mesenchymal stem cell exosome atomized treatment of the present invention.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. A preparation method of mesenchymal stem cell exosome atomized liquid is characterized by comprising the following steps: subculturing umbilical cord mesenchymal stem cells in a stem cell culture medium, collecting supernatant after the fusion degree of the cultured cells reaches 80-90%, separating to obtain an exosome solution, filtering the exosome solution by a sterile filter membrane of 0.22 mu m, centrifuging the collected filtrate for 30-90min at the rotating speed of 50000-;
and dissolving the exosome in physiological saline to obtain the mesenchymal stem cell exosome atomization liquid.
2. The method for preparing an exosome nebulizing liquid of mesenchymal stem cells according to claim 1,
the subculture mesenchymal stem cells are transferred to P1-P10 generations, and the cells are cultured under the conditions of 3% -8% of carbon dioxide, the temperature of 36-39 ℃ and the humidity of 85% -100% at constant temperature.
3. The method for preparing an exosome nebulizing liquid of mesenchymal stem cells according to claim 1,
the umbilical cord mesenchymal stem cells are obtained by removing artery and vein blood vessels from an umbilical cord, shearing into tissue homogenate blocks, filtering by using a tissue homogenate block filter screen, incubating the tissue homogenate blocks, adding a serum-free culture medium for culture, replacing a fresh culture medium for culture, and digesting by using pancreatin.
4. The method for preparing an exosome nebulizing liquid of mesenchymal stem cells according to claim 3,
the volume of the tissue homogenate block is 0.5-3mm3。
5. The method for preparing an exosome nebulizing liquid of mesenchymal stem cells according to claim 3,
the filter screen adopts 200 meshes.
6. The method for preparing an exosome nebulizing liquid of mesenchymal stem cells according to claim 1,
the environment temperature of the centrifugation is 0-20 ℃.
7. The mesenchymal stem cell exosome atomized liquid prepared by the method of any one of claims 1-6.
8. The mesenchymal stem cell exosome atomization liquid of claim 1, wherein,
the mesenchymal stem cell is a human primary umbilical cord mesenchymal stem cell.
9. Use of a mesenchymal stem cell exosome nebulised solution prepared by the method of any one of claims 1 to 6 in any one of,
(1) preparing a product for reducing inflammation of lung trachea and pulmonary trachea bypass;
(2) preparing a product for treating asthma;
(3) preparing a product for preventing asthma;
(4) preparing the product for relieving asthma.
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