Exosomes for treating lung injury and preparation method and application thereof

Abstract

The present invention discloses an exosome for treating lung injury, a preparation method and application thereof, and relates to the field of biotechnology. The exosome for treating lung injury is derived from aquatic animal cells; the aquatic animal is a freshwater fish; the freshwater fish include: crucian carp, grass carp, black carp, snakehead carp and catfish; the exosome for treating lung injury prepared by the present invention has multiple characteristics such as small size, evasion of phagocytosis, easy penetration of the respiratory tract endothelium layer, etc., can be applied to lung injury, can be used to prepare drugs for lung injury, regulate the lung cell repair mechanism, shorten the process of lung injury repair, and improve the lung injury repair effect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular to an exosome for treating lung injury, and a preparation method and application thereof. Background Art

[0002] Exosomes refer to small membrane vesicles (30-150nm) containing complex RNA and proteins. Nowadays, they specifically refer to discoid vesicles with a diameter of 40-100nm. In 1983, exosomes were first discovered in sheep reticulocytes, and Johnstone named them "exosomes" in 1987. A variety of cells can secrete exosomes under normal and pathological conditions. They are mainly derived from multivesicular bodies formed by the invagination of intracellular lysosomal microparticles, and are released into the extracellular matrix after the multivesicular outer membrane fuses with the cell membrane.

[0003] Exosomes can be secreted by all cultured cell types and are naturally present in body fluids, including blood, saliva, urine, cerebrospinal fluid, and breast milk. Exosomes are considered to be specifically secreted membrane vesicles that participate in intercellular communication and can be used for a variety of medical purposes.

[0004] The lungs are one of the most important organs in the human body. Therefore, damage to the lungs can induce a series of lung diseases, including pulmonary fibrosis, bronchitis, bullae, lung abscess, pneumonia, pneumothorax, tuberculosis, lung nodules, emphysema, lung tumors, chronic obstructive pulmonary disease, pulmonary embolism, etc.

[0005] Therefore, the repair of lung damage is directly related to whether the lung organ functions normally and has a great correlation with human health.

[0006] Based on this, an exosome for treating lung injury is provided to solve the existing technical problems. Summary of the invention

[0007] The purpose of the present invention is to provide an exosome for treating lung injury and a preparation method thereof, so as to make up for the deficiencies in the prior art.

[0008] The technical solution adopted by the present invention is as follows:

[0009] An exosome for treating lung injury, wherein the exosome for treating lung injury is derived from aquatic animal cells;

[0010] The aquatic animal is a freshwater fish;

[0011] The freshwater fish include: crucian carp, grass carp, black carp, snakehead carp and catfish;

[0012] The exosomes for treating lung injury contain proteins, miRNAs, and mRNAs;

[0013] The proteins include specific proteins, and the specific proteins are: CD9, CD63, CD82, EpCAM;

[0014] The exosomes used to treat lung injury have a diameter of 80-85 nm.

[0015] As a further technical solution, the aquatic animal cells are aquatic animal embryonic cells.

[0016] As a further technical solution, the aquatic animal embryonic cell is any one of the embryonic cells of crucian carp, grass carp, black carp, snakehead carp and catfish.

[0017] As a further technical solution, the aquatic animal embryonic cells are obtained by separation and culture:

[0018] Specific methods include:

[0019] Firstly, embryonic cells were isolated from gastrula-stage aquatic animals by trypsin digestion.

[0020] Inoculating the separated embryonic cells into a culture medium, culturing at 27.5° C. and 5% carbon dioxide concentration for 7-8 days, replacing the culture medium every 3 days, and obtaining an aquatic animal embryonic cell culture medium after the culture is completed;

[0021] The mass ratio of the separated embryos to the culture medium is 1:10-12.

[0022] As a further technical solution, the method for isolating embryonic cells is:

[0023] Take the fertilized eggs of aquatic animals fertilized by natural mating, wash them with sterilized pure water, then take 30-35 embryos out of them and put them into a culture dish, add 5 times the weight of trypsin treatment solution to treat for 2 minutes, then add an equal volume of DMEM culture solution to terminate digestion for 1 minute, and high-speed centrifuge for 3-4 minutes, discard the supernatant to obtain separated embryos, wherein the speed of high-speed centrifugation is 1000r / min;

[0024] The method for preparing the trypsin treatment solution is as follows:

[0025] 0.25 g trypsin and 0.05 g ethylenediaminetetraacetic acid were added to 95 ml PBS buffer, stirred magnetically until completely dissolved, and the volume was fixed to 100 ml;

[0026] Then filter through a 0.22μm filter membrane to obtain;

[0027] The DMEM culture medium preparation method is:

[0028] Take 13.4g high-glucose DMEM powder, 3.7g sodium bicarbonate, and newborn calf serum, add them to 900ml distilled water, stir with a magnetic stirrer until completely dissolved, then make up to 1000ml, adjust the pH to 7.1, and filter through a 0.22μm filter membrane to obtain;

[0029] The mass fraction of newborn calf serum in the DMEM culture medium is 11.2%.

[0030] As a further technical solution, the culture medium preparation method is:

[0031] Dissolve 60 g of M199 medium powder in 600 ml of deionized water, then add 16 g of sodium bicarbonate, stir and mix evenly, and then make up to 1000 mL, then add sterilized newborn calf serum, sterilized aquatic animal serum, sodium pyruvate, integrin α5β1, fibroblast growth factor, penicillin and streptomycin, stir and mix evenly, adjust the pH to 7.3, filter through a 0.22 μm filter membrane for sterilization, and store at 4°C for use;

[0032] As a further technical solution:

[0033] The mass fraction of the newborn calf serum in the culture medium is 8-9%;

[0034] The mass fraction of the aquatic animal's own serum is 0.6-0.8%;

[0035] The sodium pyruvate concentration is 0.7-0.8 mmol / L;

[0036] The concentration of integrin α5β1 is 0.5-0.7 mmol / L;

[0037] The fibroblast growth factor concentration is 3-3.5 ng / mL;

[0038] The penicillin concentration is 75UI / mL;

[0039] The streptomycin concentration is 75 UI / mL.

[0040] A method for preparing exosomes for treating lung injury, comprising:

[0041] 1) Centrifuging the aquatic animal embryo cell culture fluid once and taking the supernatant once;

[0042] 2) Centrifuging the primary supernatant for a second time and taking the secondary supernatant;

[0043] The secondary supernatant was centrifuged three times, and the supernatant was ultracentrifuged at 150,000 g for 1 hour using PBS buffer. The precipitate was collected and resuspended with PBS buffer to obtain a crude product, which was purified by anion exchange column chromatography to obtain the desired exosomes.

[0044] The anion exchanger used for anion exchange column chromatography was: CaptoMMC;

[0045] The anion exchange column chromatography was performed in the presence of 0.85 M NaCl. The column condition of the anion exchange column chromatography was selected such that the pH of the column eluent was 7.

[0046] As a further technical solution, the centrifugation condition in step 1) is 3000 g for 15-20 min;

[0047] Step 2) centrifugation conditions are 30000g for 30-35min;

[0048] Step 3) The centrifugation condition is 100000g for 2-2.5h.

[0049] The exosomes prepared by the method of the present invention are used to treat lung injury.

[0050] Beneficial effects:

[0051] The exosomes prepared by the present invention for treating lung injury have multiple characteristics such as small size, evasion of phagocytosis, and easy penetration of the respiratory tract endothelial layer. They can be used to prepare drugs for lung injury, regulate the lung cell repair mechanism, shorten the process of lung injury repair, and improve the lung injury repair effect.

[0052] The present invention introduces integrin α5β1 and sodium pyruvate into the culture medium, and the two cooperate to induce and promote the generation of exosomes, thereby increasing the extraction concentration of exosomes to a certain extent.

[0053] After a large number of experimental studies, the present invention adopts exosomes obtained from embryonic cells of aquatic animals. Through their introduction, they have a certain specificity, which is mainly manifested in the inhibition of the expression of serine / threonine kinase 39. After its expression is inhibited, it will promote autophagy of alveolar macrophages, reduce the level of inflammatory factors and inhibit lung inflammation and oxidative stress, thereby alleviating lung damage and enhancing lung function.

[0054] The exosomes prepared by the present invention can be absorbed by the lungs, which can effectively improve the delivery efficiency of exosome drug delivery and exert a positive anti-inflammatory effect, thereby accelerating the repair of lung damage.

[0055] Since the aquatic animal embryo exosomes prepared by the present invention have unique biological characteristics such as low toxicity and low immunogenicity, and can enhance immunity, reduce lung inflammation and repair lung tissue, they have significant therapeutic and alleviating effects on the symptoms of lung damage tissue.

[0056] The exosomes prepared by the present invention are applied to lung injuries, which will activate apoptosis genes to a certain extent and promote smooth muscle cell apoptosis and excretion from the body, thereby promoting cell lung proliferation, thereby promoting the repair of lung tissue, achieving a balance between lung cell proliferation and apoptosis, ensuring the health of vascular wall cells, ensuring the normal delivery of nutrients in the blood, and improving the repair efficiency of lung cell tissue.

[0057] The exosomes obtained by the present invention can be atomized or intravenously infused to regulate the microenvironment of damaged lung tissue, initiate self-repair programs, and promote the transformation of macrophages to an anti-inflammatory phenotype by restoring mitochondrial integrity, thereby playing an anti-inflammatory role and repairing lung epithelial tissue, and can achieve the effect of promoting angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a schematic diagram of the structure of exosomes used to treat lung injury;

[0059] Figure 2 This is the electron microscopic image of exosomes in Example 1;

[0060] Figure 3 It is a bar graph of the malondialdehyde content in lung cells in each experimental group;

[0061] Figure 4 The figure is a bar graph showing the catalase activity of lung cells in each experimental group. DETAILED DESCRIPTION

[0062] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] Example 1: Obtaining exosomes from black carp embryonic cells:

[0064] A. Black carp embryo cell culture

[0065] Isolation of embryonic cells:

[0066] The fertilized eggs of the black carp fertilized by natural mating were taken, washed with sterilized pure water, and then 30 embryos were taken into a culture dish, and 5 times the weight of the trypsin treatment solution was added for 2 minutes, and then an equal volume of DMEM culture solution was added to terminate the digestion for 1 minute, and the supernatant was discarded after high-speed centrifugation for 3 minutes to obtain separated embryos, wherein the speed of the high-speed centrifugation was 5000r / min;

[0067] The preparation method of trypsin treatment solution is as follows:

[0068] 0.25 g trypsin and 0.05 g ethylenediaminetetraacetic acid were added to 95 ml PBS buffer, stirred magnetically until completely dissolved, and the volume was fixed to 100 ml;

[0069] Then filter through a 0.22μm filter membrane to obtain;

[0070] The preparation method of DMEM culture medium is as follows:

[0071] Take 13.4g high-glucose DMEM dry powder, 3.7g sodium bicarbonate, and newborn calf serum, where the mass fraction of newborn calf serum in the DMEM culture medium is 11.2%; add them to 900ml distilled water, stir with a magnetic stirrer until completely dissolved, then make the volume 1000ml, adjust the pH to 7.1, and filter through a 0.22μm filter membrane to obtain;

[0072] The separated embryo cells are inoculated into a culture medium, wherein the mixed mass ratio of the separated embryos to the culture medium is 1:10; the culture is carried out at 27.5° C. and 5% carbon dioxide concentration for 7 days, and the culture medium is replaced every 3 days. After the culture is completed, a black carp embryo cell culture medium is obtained; the culture medium preparation method is as follows:

[0073] Dissolve 60g of M199 culture medium powder in 600ml of deionized water, then add 16g of sodium bicarbonate, stir and mix evenly, and then adjust the volume to 1000mL, and then add sterilized newborn calf serum and sterilized aquatic animal serum. The mass fraction of newborn calf serum in the culture medium is 8%; the mass fraction of aquatic animal serum is 0.6%; sodium pyruvate, integrin α5β1, fibroblast growth factor, penicillin and streptomycin, the concentration of sodium pyruvate is 0.7mmo l / L; the concentration of integrin α5β1 is 0.5mmo l / L; the concentration of fibroblast growth factor is 3ng / mL; the concentration of penicillin is 75UI / mL; the concentration of streptomycin is 75UI / mL. After stirring and mixing evenly, adjust the pH value to 7.3, filter and sterilize through a 0.22μm filter membrane, and store at 4℃ for use;

[0074] B. Centrifuge the aquatic animal embryonic cell culture medium once at 3000 g for 15-20 min and take the supernatant once;

[0075] The primary supernatant was centrifuged for a second time at 30000 g for 30 min, and the secondary supernatant was collected;

[0076] The secondary supernatant was centrifuged three times at 100,000 g for 2.5 h, and the supernatant was ultracentrifuged at 150,000 g for 1 h using PBS buffer. The precipitate was collected and resuspended with PBS buffer to obtain a crude product, which was purified by anion exchange column chromatography to obtain the desired exosomes.

[0077] The anion exchanger used for anion exchange column chromatography was: CaptoMMC;

[0078] The anion exchange column chromatography was performed in the presence of 0.85 M NaCl. The column condition of the anion exchange column chromatography was selected such that the pH of the column eluent was 7.

[0079] The isolated exosomes are vesicle-shaped with a diameter of about 80 nm.

[0080] Example 2 Obtaining exosomes from crucian carp embryonic cells:

[0081] A. Crucian carp embryo cell culture

[0082] Isolation of embryonic cells:

[0083] The fertilized eggs of the black carp fertilized by natural mating were taken, washed with sterilized pure water, and then 30 embryos were taken into a culture dish, and 5 times the weight of the trypsin treatment solution was added for 2 minutes, and then an equal volume of DMEM culture solution was added to terminate the digestion for 1 minute, and the supernatant was discarded after high-speed centrifugation for 3 minutes to obtain separated embryos, wherein the speed of the high-speed centrifugation was 5000r / min;

[0084] The preparation method of trypsin treatment solution is as follows:

[0085] 0.25 g trypsin and 0.05 g ethylenediaminetetraacetic acid were added to 95 ml PBS buffer, stirred magnetically until completely dissolved, and the volume was fixed to 100 ml;

[0086] Then filter through a 0.22μm filter membrane to obtain;

[0087] The preparation method of DMEM culture medium is as follows:

[0088] Take 13.4g high-glucose DMEM dry powder, 3.7g sodium bicarbonate, and newborn calf serum, where the mass fraction of newborn calf serum in the DMEM culture medium is 11.2%; add them to 900ml distilled water, stir with a magnetic stirrer until completely dissolved, then make the volume 1000ml, adjust the pH to 7.1, and filter through a 0.22μm filter membrane to obtain;

[0089] The separated embryo cells are inoculated into a culture medium, wherein the mixed mass ratio of the separated embryos to the culture medium is 1:10; the culture is carried out at 27.5° C. and 5% carbon dioxide concentration for 7 days, and the culture medium is replaced every 3 days. After the culture is completed, a black carp embryo cell culture medium is obtained; the culture medium preparation method is as follows:

[0090] Dissolve 60g of M199 culture medium powder in 600ml of deionized water, then add 16g of sodium bicarbonate, stir and mix evenly, and then adjust the volume to 1000mL, and then add sterilized newborn calf serum and sterilized aquatic animal serum. The mass fraction of newborn calf serum in the culture medium is 8%; the mass fraction of aquatic animal serum is 0.6%; sodium pyruvate, integrin α5β1, fibroblast growth factor, penicillin and streptomycin, the concentration of sodium pyruvate is 0.7mmo l / L; the concentration of integrin α5β1 is 0.5mmo l / L; the concentration of fibroblast growth factor is 3ng / mL; the concentration of penicillin is 75U I / mL; the concentration of streptomycin is 75U I / mL. After stirring and mixing evenly, adjust the pH value to 7.3, filter and sterilize through a 0.22μm filter membrane, and store at 4℃ for use;

[0091] B. Centrifuge the aquatic animal embryonic cell culture medium once at 3000 g for 15-20 min and take the supernatant once;

[0092] The supernatant was centrifuged for a second time at 30000 g for 30 min, and the secondary supernatant was collected;

[0093] The secondary supernatant was centrifuged three times at 100,000 g for 2.5 h, and the supernatant was ultracentrifuged at 150,000 g for 1 h using PBS buffer. The precipitate was collected and resuspended with PBS buffer to obtain a crude product, which was purified by anion exchange column chromatography to obtain the desired exosomes.

[0094] The anion exchanger used for anion exchange column chromatography was: CaptoMMC;

[0095] The anion exchange column chromatography was performed in the presence of 0.85 M NaCl. The column condition of the anion exchange column chromatography was selected such that the pH of the column eluent was 7.

[0096] The isolated exosomes are vesicle-shaped with a diameter of about 80 nm.

[0097] Example 3 Obtaining exosomes from catfish embryonic cells:

[0098] A. Catfish embryonic cell culture

[0099] Isolation of embryonic cells:

[0100] The fertilized eggs of the black carp fertilized by natural mating were taken, washed with sterilized pure water, and then 30 embryos were taken into a culture dish, and 5 times the weight of the trypsin treatment solution was added for 2 minutes, and then an equal volume of DMEM culture solution was added to terminate the digestion for 1 minute, and the supernatant was discarded after high-speed centrifugation for 3 minutes to obtain separated embryos, wherein the speed of the high-speed centrifugation was 5000r / min;

[0101] The preparation method of trypsin treatment solution is as follows:

[0102] 0.25 g trypsin and 0.05 g ethylenediaminetetraacetic acid were added to 95 ml PBS buffer, stirred magnetically until completely dissolved, and the volume was fixed to 100 ml;

[0103] Then filter through a 0.22μm filter membrane to obtain;

[0104] The preparation method of DMEM culture medium is as follows:

[0105] Take 13.4g high-glucose DMEM dry powder, 3.7g sodium bicarbonate, and newborn calf serum, where the mass fraction of newborn calf serum in the DMEM culture medium is 11.2%; add them to 900ml distilled water, stir with a magnetic stirrer until completely dissolved, then make the volume 1000ml, adjust the pH to 7.1, and filter through a 0.22μm filter membrane to obtain;

[0106] The separated embryo cells are inoculated into a culture medium, wherein the mixed mass ratio of the separated embryos to the culture medium is 1:10; the culture is carried out at 27.5° C. and 5% carbon dioxide concentration for 7 days, and the culture medium is replaced every 3 days. After the culture is completed, a black carp embryo cell culture medium is obtained; the culture medium preparation method is as follows:

[0107] Dissolve 60g of M199 culture medium powder in 600ml of deionized water, then add 16g of sodium bicarbonate, stir and mix evenly, and then adjust the volume to 1000mL, and then add sterilized newborn calf serum and sterilized aquatic animal serum. The mass fraction of newborn calf serum in the culture medium is 8%; the mass fraction of aquatic animal serum is 0.6%; sodium pyruvate, integrin α5β1, fibroblast growth factor, penicillin and streptomycin, the concentration of sodium pyruvate is 0.7mmo l / L; the concentration of integrin α5β1 is 0.5mmo l / L; the concentration of fibroblast growth factor is 3ng / mL; the concentration of penicillin is 75U I / mL; the concentration of streptomycin is 75U I / mL. After stirring and mixing evenly, adjust the pH value to 7.3, filter and sterilize through a 0.22μm filter membrane, and store at 4℃ for use;

[0108] B. Centrifuge the aquatic animal embryonic cell culture medium once at 3000 g for 15-20 min and take the supernatant once;

[0109] The primary supernatant was centrifuged for a second time at 30000 g for 30 min, and the secondary supernatant was collected;

[0110] The secondary supernatant was centrifuged three times at 100,000 g for 2.5 h, and the supernatant was ultracentrifuged at 150,000 g for 1 h using PBS buffer. The precipitate was collected and resuspended with PBS buffer to obtain a crude product, which was purified by anion exchange column chromatography to obtain the desired exosomes.

[0111] The anion exchanger used for anion exchange column chromatography was: CaptoMMC;

[0112] The anion exchange column chromatography was performed in the presence of 0.85 M NaCl, and the column condition of the anion exchange column chromatography was selected such that the pH of the column eluent was 7.

[0113] The isolated exosomes are vesicle-shaped with a diameter of about 80 nm.

[0114] Example 4 Obtaining exosomes from snakehead fish embryonic cells:

[0115] A. Snakehead carp embryo cell culture

[0116] Isolation of embryonic cells:

[0117] The fertilized eggs of the black carp fertilized by natural mating were taken, washed with sterilized pure water, and then 30 embryos were taken into a culture dish, and 5 times the weight of the trypsin treatment solution was added for 2 minutes, and then an equal volume of DMEM culture solution was added to terminate the digestion for 1 minute, and the supernatant was discarded after high-speed centrifugation for 3 minutes to obtain separated embryos, wherein the speed of the high-speed centrifugation was 5000r / min;

[0118] The preparation method of trypsin treatment solution is as follows:

[0119] 0.25 g trypsin and 0.05 g ethylenediaminetetraacetic acid were added to 95 ml PBS buffer, stirred magnetically until completely dissolved, and the volume was fixed to 100 ml;

[0120] Then filter through a 0.22μm filter membrane to obtain;

[0121] The preparation method of DMEM culture medium is as follows:

[0122] Take 13.4g high-glucose DMEM dry powder, 3.7g sodium bicarbonate, and newborn calf serum, where the mass fraction of newborn calf serum in the DMEM culture medium is 11.2%; add them to 900ml distilled water, stir with a magnetic stirrer until completely dissolved, then make the volume 1000ml, adjust the pH to 7.1, and filter through a 0.22μm filter membrane to obtain;

[0123] The separated embryo cells are inoculated into a culture medium, wherein the mixed mass ratio of the separated embryos to the culture medium is 1:10; the culture is carried out at 27.5° C. and 5% carbon dioxide concentration for 7 days, and the culture medium is replaced every 3 days. After the culture is completed, a black carp embryo cell culture medium is obtained; the culture medium preparation method is as follows:

[0124] Dissolve 60g of M199 culture medium powder in 600ml of deionized water, then add 16g of sodium bicarbonate, stir and mix evenly, and then adjust the volume to 1000mL, and then add sterilized newborn calf serum and sterilized aquatic animal serum. The mass fraction of newborn calf serum in the culture medium is 8%; the mass fraction of aquatic animal serum is 0.6%; sodium pyruvate, integrin α5β1, fibroblast growth factor, penicillin and streptomycin, the concentration of sodium pyruvate is 0.7mmo l / L; the concentration of integrin α5β1 is 0.5mmo l / L; the concentration of fibroblast growth factor is 3ng / mL; the concentration of penicillin is 75U I / mL; the concentration of streptomycin is 75U I / mL. After stirring and mixing evenly, adjust the pH value to 7.3, filter and sterilize through a 0.22μm filter membrane, and store at 4℃ for use;

[0125] B. Centrifuge the aquatic animal embryonic cell culture medium once at 3000 g for 15-20 min and take the supernatant once;

[0126] The primary supernatant was centrifuged for a second time at 30000 g for 30 min, and the secondary supernatant was collected;

[0127] The secondary supernatant was centrifuged three times at 100,000 g for 2.5 h, and the supernatant was ultracentrifuged at 150,000 g for 1 h using PBS buffer. The precipitate was collected and resuspended with PBS buffer to obtain a crude product, which was purified by anion exchange column chromatography to obtain the desired exosomes.

[0128] The anion exchanger used for anion exchange column chromatography was: CaptoMMC;

[0129] The anion exchange column chromatography was performed in the presence of 0.85 M NaCl. The column condition of the anion exchange column chromatography was selected such that the pH of the column eluent was 7.

[0130] The isolated exosomes are vesicle-shaped with a diameter of about 80 nm.

[0131] Example 5 Obtaining exosomes from grass carp embryonic cells:

[0132] A. Grass carp embryo cell culture

[0133] Isolation of embryonic cells:

[0134] The fertilized eggs of the black carp fertilized by natural mating were taken, washed with sterilized pure water, and then 30 embryos were taken into a culture dish, and 5 times the weight of the trypsin treatment solution was added for 2 minutes, and then an equal volume of DMEM culture solution was added to terminate the digestion for 1 minute, and the supernatant was discarded after high-speed centrifugation for 3 minutes to obtain separated embryos, wherein the speed of the high-speed centrifugation was 5000r / min;

[0135] The preparation method of trypsin treatment solution is as follows:

[0136] 0.25 g trypsin and 0.05 g ethylenediaminetetraacetic acid were added to 95 ml PBS buffer, stirred magnetically until completely dissolved, and the volume was fixed to 100 ml;

[0137] Then filter through a 0.22μm filter membrane to obtain;

[0138] The preparation method of DMEM culture medium is as follows:

[0139] Take 13.4g high-glucose DMEM dry powder, 3.7g sodium bicarbonate, and newborn calf serum, where the mass fraction of newborn calf serum in the DMEM culture medium is 11.2%; add them to 900ml distilled water, stir with a magnetic stirrer until completely dissolved, then make the volume 1000ml, adjust the pH to 7.1, and filter through a 0.22μm filter membrane to obtain;

[0140] The separated embryo cells are inoculated into a culture medium, wherein the mixed mass ratio of the separated embryos to the culture medium is 1:10; the culture is carried out at 27.5° C. and 5% carbon dioxide concentration for 7 days, and the culture medium is replaced every 3 days. After the culture is completed, a black carp embryo cell culture medium is obtained; the culture medium preparation method is as follows:

[0141] Dissolve 60g of M199 culture medium powder in 600ml of deionized water, then add 16g of sodium bicarbonate, stir and mix evenly, and then adjust the volume to 1000mL, and then add sterilized newborn calf serum and sterilized aquatic animal serum. The mass fraction of newborn calf serum in the culture medium is 8%; the mass fraction of aquatic animal serum is 0.6%; sodium pyruvate, integrin α5β1, fibroblast growth factor, penicillin and streptomycin, the concentration of sodium pyruvate is 0.7mmo l / L; the concentration of integrin α5β1 is 0.5mmo l / L; the concentration of fibroblast growth factor is 3ng / mL; the concentration of penicillin is 75U I / mL; the concentration of streptomycin is 75U I / mL. After stirring and mixing evenly, adjust the pH value to 7.3, filter and sterilize through a 0.22μm filter membrane, and store at 4℃ for use;

[0142] B. Centrifuge the aquatic animal embryonic cell culture medium once at 3000 g for 15-20 min and take the supernatant once;

[0143] The supernatant was centrifuged for a second time at 30000 g for 30 min, and the secondary supernatant was collected;

[0144] The secondary supernatant was centrifuged three times at 100,000 g for 2.5 h, and the supernatant was ultracentrifuged at 150,000 g for 1 h using PBS buffer. The precipitate was collected and resuspended with PBS buffer to obtain a crude product, which was purified by anion exchange column chromatography to obtain the desired exosomes.

[0145] The anion exchanger used for anion exchange column chromatography was: CaptoMMC;

[0146] The anion exchange column chromatography was performed in the presence of 0.85 M NaCl, and the column condition of the anion exchange column chromatography was selected such that the pH of the column eluent was 7.

[0147] The isolated exosomes are vesicle-shaped with a diameter of about 80 nm.

[0148] Comparative Example 1:

[0149] Based on Example 1, the aquatic animal embryonic cells are replaced by pig embryonic cells, and the rest of the technical solutions remain unchanged.

[0150] Comparative Example 2:

[0151] Based on Example 1, integrin α5β1 and fibroblast growth factor were not added to the culture medium, and the rest of the technical scheme remained unchanged.

[0152] test:

[0153] The experiment selected 80 10-week-old SPF adult SD rats, half male and half female, with an average body weight of (255±20) g. The rats were raised in an SPF environment with good ventilation, temperature (24±2)℃, and humidity of 50%. There were no restrictions on the diet of adult SD rats. After getting familiar with the environment for 1 week, they were divided into 8 groups, each with 10 rats, of which 7 groups were used for modeling and 1 group was set as a normal control group:

[0154] Except for the normal group, the other groups were injected with 5.5 mg / kg lipopolysaccharide through the tail vein to establish the model, with an injection dose of 0.52 ml. The normal group was given an equal volume of normal saline;

[0155] The exosomes of the embodiment and the control case carried the drug component: the exosomes of the embodiment and the control case were dissolved in PBS buffer to prepare an injection sample with an exosome concentration of 1.5ug / μL;

[0156] After modeling, 1 ml of the sample of the embodiment and the sample of the comparative example were subcutaneously injected into the experimental group rats every day for 14 consecutive days, and the normal group and the model group were injected with an equal amount of physiological saline. After the injection, the rats were free to eat without restriction.

[0157] Detection of respiratory function in each group: The animal pulmonary function tester was used to measure tidal volume, respiratory rate and minute ventilation on the second day after administration;

[0158] Table 1

[0159]

[0160]

[0161] As can be seen from Table 1, the exosomes prepared by the present invention can have a significant repair effect on lung damage.

[0162] The malondialdehyde (MDA) content in the lung cells of each group of experimental rats was determined using a malondialdehyde (MDA) detection kit, as shown in Table 2:

[0163] Table 2

[0164] Malondialdehyde nmol / mgprot Example 1 0.46 Example 2 0.47 Example 3 0.42 Example 4 0.44 Comparative Example 1 0.69 Comparative Example 2 0.51 Normal group 0.31 Model Group 0.88

[0165] As can be seen from Table 2, the exosomes of the present invention can significantly reduce the content of malondialdehyde.

[0166] The SOD activity in the lung cells of each group of experimental rats was measured using a superoxide dismutase (SOD) detection kit, as shown in Table 3:

[0167] Table 3

[0168]

[0169] As can be seen from Table 3, the exosomes prepared by the present invention can increase the activity of superoxide dismutase.

[0170] The CAT activity in the lung cells of each group of experimental rats was determined using a catalase (CAT) detection kit, as shown in Table 4:

[0171] Table 4

[0172]

[0173]

[0174] As can be seen from Table 4, the exosomes prepared by the present invention can increase the activity of catalase.

[0175] The above description is only a preferred embodiment of the present invention, but the present invention is not limited to the scope of implementation shown. Any changes made in accordance with the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which still do not exceed the spirit covered by the specification, should be within the protection scope of the present invention.

Claims

1. A use of exosomes in preparing a drug for treating lung injury, characterized in that: The exosomes are derived from aquatic animal cells; The aquatic animal cells are aquatic animal embryonic cells; the aquatic animal embryonic cells are any one of the embryonic cells of crucian carp, grass carp, black carp, black carp and catfish.

2. The use of exosomes according to claim 1 in preparing a drug for treating lung injury, characterized in that: The aquatic animal embryonic cells are obtained by isolating embryonic cells and culturing embryonic cells: Specific methods include: Firstly, embryonic cells were isolated from gastrula-stage aquatic animals by trypsin digestion. The separated embryonic cells are inoculated into a culture medium, and cultured at 27.5° C. and 5% carbon dioxide concentration for 7-8 days, and the culture medium is replaced every 3 days. After the culture is completed, an aquatic animal embryonic cell culture medium is obtained; The mass ratio of the separated embryos to the culture medium is 1:10-12.

3. The use of exosomes according to claim 2 in preparing a drug for treating lung injury, characterized in that: The method for separating embryonic cells is: Take the fertilized eggs of aquatic animals fertilized by natural mating, wash them with sterilized pure water, then take 30-35 embryos out of them and put them into a culture dish, add 5 times the weight of trypsin treatment solution to treat for 2 minutes, then add an equal volume of DMEM culture solution to terminate digestion for 1 minute, and high-speed centrifuge for 3-4 minutes, discard the supernatant to obtain separated embryos, wherein the speed of high-speed centrifugation is 1000r / min; The preparation method of the trypsin treatment solution is as follows: 0.25 g trypsin and 0.05 g ethylenediaminetetraacetic acid were added to 95 ml PBS buffer, stirred magnetically until completely dissolved, and the volume was adjusted to 100 ml; Then filter through a 0.22μm filter membrane to obtain; The DMEM culture medium preparation method is: Take 13.4g high-glucose DMEM powder, 3.7g sodium bicarbonate, and newborn calf serum, add them to 900ml distilled water, stir with a magnetic stirrer until completely dissolved, then make up to 1000ml, adjust the pH to 7.1, and filter through a 0.22μm filter membrane to obtain; The mass fraction of newborn calf serum in the DMEM culture medium is 11.2%.

4. The use of exosomes according to claim 2 in preparing a drug for treating lung injury, characterized in that: The method for preparing the culture medium for culturing embryonic cells is as follows: Dissolve 60 g of M199 medium powder in 600 ml of deionized water, then add 16 g of sodium bicarbonate, stir and mix evenly, and then make up to 1000 mL. Then add sterilized newborn calf serum, sterilized aquatic animal serum, sodium pyruvate, integrin α5β1, fibroblast growth factor, penicillin and streptomycin, stir and mix evenly, adjust the pH to 7.3, filter through a 0.22 μm filter membrane for sterilization, and store at 4°C for use.

5. The use of exosomes according to claim 4 in preparing a drug for treating lung injury, characterized in that: The mass fraction of the newborn calf serum in the culture medium is 8-9%; The mass fraction of the aquatic animal's own serum is 0.6-0.8%; The sodium pyruvate concentration is 0.7-0.8 mmol / L; The concentration of integrin α5β1 is 0.5-0.7 mmol / L; The fibroblast growth factor concentration is 3-3.5 ng / mL; The penicillin concentration is 75UI / mL; The streptomycin concentration is 75 UI / mL.

6. The use of exosomes in the preparation of a drug for treating lung injury according to claim 1, characterized in that: The exosome preparation method comprises: 1) Centrifuging the aquatic animal embryo cell culture fluid once and taking the supernatant once; 2) Centrifuging the primary supernatant for a second time and taking the secondary supernatant; 3) taking the secondary supernatant and centrifuging it three times, taking the supernatant three times, ultracentrifuging the supernatant three times at 150,000 g for 1 hour using PBS buffer, collecting the precipitate and resuspending the precipitate with PBS buffer solution to obtain a crude product, and purifying it by anion exchange column chromatography to obtain the desired exosomes; The anion exchanger used for anion exchange column chromatography was: CaptoMMC; The anion exchange column chromatography was performed in the presence of 0.85 M NaCl, and the column condition of the anion exchange column chromatography was selected to elute when the pH of the column eluent was 7; Step 1) The centrifugation condition is 3000g for 15-20min; Step 2) centrifugation conditions are 30000g for 30-35min; Step 3) The centrifugation condition is 100000g for 2-2.5h.

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