Application of adult stem cells containing neural spine source and composition thereof in treatment of pulmonary fibrosis

Abstract

The invention relates to the technical field of cell biology, in particular to application of adult stem cells containing neural spine sources and a composition of the adult stem cells in treatment of pulmonary fibrosis. The composition has high loading capacity, pharmacodynamic experiments show that the composition can reduce the content of TGF-beta1, increase the percentage of SaO2 and reduce pCO2, can efficiently enter cells to play a role, inhibit and reverse pulmonary fibrosis caused by lung diseases and improve the pulmonary respiratory function, has a good treatment effect, is high in development value and has good application prospects. The method is suitable for further research and development and productization.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to the use of adult stem cells derived from neural crest and their compositions in the treatment of pulmonary fibrosis. Background Art

[0002] While society is developing at a high speed, factors such as environmental pollutants, smoking, and pathogen infections are quietly damaging the health of the human lungs. The population suffering from lung diseases is expanding year by year. Idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, silicosis, chronic bronchitis, pneumonia, lung injury, etc. have gradually become the main threats to human health.

[0003] Pulmonary fibrosis (PF) is a lung disease characterized by chronic progressive fibrosis of lung tissue. Its characteristic is that the lung interstitium (i.e., the connective tissue supporting the alveoli) is gradually replaced by excessive fibrous tissue, resulting in the destruction of the lung structure and the loss of function. The disease usually presents symptoms such as shortness of breath, persistent cough, expectoration, and shortness of breath. And as the disease progresses, the symptoms gradually worsen, and the quality of life drops severely.

[0004] The pathogenesis of pulmonary fibrosis involves multiple factors, including genetics, immune abnormalities, environmental exposure, and cell-cell interactions, etc. The most common type is idiopathic pulmonary fibrosis (IPF), and other types include pulmonary fibrosis caused by drugs, infections, occupational exposure, connective tissue diseases, etc.

[0005] In recent years, with the progress of research, anti-fibrotic drugs have gradually been applied to clinical treatment. The main drugs include: Pirfenidone is an anti-fibrotic drug that can inhibit the proliferation, transformation, and collagen synthesis of fibroblasts. Research shows that pirfenidone can significantly slow down the progression of idiopathic pulmonary fibrosis. Nintedanib is a multi-target tyrosine kinase inhibitor that can slow down the process of pulmonary fibrosis by inhibiting the activation of multiple receptors. Research shows that nintedanib can effectively reduce the decline of lung function. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, the present invention provides the use of adult stem cells derived from neural crest and their compositions in the treatment of pulmonary fibrosis. The said composition can efficiently enter cells to play a role, inhibit and reverse pulmonary fibrosis caused by lung diseases, and improve lung respiratory function.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] A composition containing adult stem cells derived from neural crest, and the adult stem cells are derived from dental stem cells, including one or more of dental pulp stem cells derived from adult orthodontic teeth, dental pulp stem cells from exfoliated deciduous teeth, and stem cells from apical papilla, and more preferably dental pulp stem cells.

[0009] Further, the method for preparing the dental pulp stem cells is as follows: Healthy impacted and unerupted third molars of humans are extracted under anesthesia; after splitting the crown of the teeth, dental pulp tissue is taken, soaked in PBS, and cleaned; the dental follicle tissue is placed in a culture dish, minced, and placed in a solution containing type I collagenase and Dispase, and digested in a water bath until the tissue is completely digested; cells are collected by passing through a cell sieve, centrifuged, and the digestion solution is discarded. The tissue is resuspended in α-MEM medium, centrifuged, and the supernatant is discarded. Then the loose tissue is evenly spread on a T25 culture flask, and α-MEM medium containing FBS is added dropwise, and cultured. After the tissue blocks adhere, the medium is slowly added to the culture flask, and the medium is changed again after 3 days; when the cell density reaches 70%-80%, the medium is discarded, and digested with 0.25% trypsin in an incubator at 37°C for 1-2 minutes, and then digested and passaged with medium containing 10% FBS to obtain the cells.

[0010] Further, the preparation of exosomes from the dental pulp stem cells is as follows:

[0011] S1. The dental pulp stem cells are cultured and induced in DMEM supplemented with 3-8% FBS, 5-15 ng / mL chitosan, and 5-15 ng / mL nonadienol for 7 days; the cells are inoculated into a serum-containing medium at a cell density of 1-4×10 4 cells / mL, and after culturing for 2-4 days, the cell supernatant A is collected;

[0012] S2. The supernatant A is centrifuged at 400 rpm / min at 2-8°C for 5 minutes, and the supernatant B is collected. The supernatant B is centrifuged at 300 rpm / min for 10-20 minutes to obtain the supernatant C, and then washed with PBS buffer and centrifuged at 200 rpm / min for 20-40 minutes to obtain dental pulp stem cell exosomes.

[0013] Further, the preparation of the composition includes the following steps:

[0014] S1. L-cysteine and Sargentodoxa cuneata polysaccharide are added to an ethanol aqueous solution for standby

[0015] S2. The exosomes of dental pulp stem cells of the present invention are slowly added to S1 to obtain a mixed system, and then benzyl alcohol and lauroyl maltoside are added and stirred evenly to obtain a composition containing exosomes of dental pulp stem cells.

[0016] In a preferred embodiment of the present invention, calculated by weight ratio, the L-cysteine: Sargentodoxa cuneata polysaccharide: ethanol: water = 0.2 - 0.8: 0.07 - 0.12: 70 - 90: 15 - 25.

[0017] In a preferred embodiment of the present invention, calculated by weight ratio, the L-cysteine: Sargentodoxa cuneata polysaccharide: ethanol: water = 0.5: 0.1: 80: 20.

[0018] In a preferred embodiment of the present invention, calculated by mass-volume ratio, the dosage of benzyl alcohol is 0.1 - 0.3%, and the dosage of lauroyl maltoside is 0.03 - 0.08%.

[0019] In a preferred embodiment of the present invention, calculated by mass-volume ratio, the dosage of benzyl alcohol is 0.2%, and the dosage of lauroyl maltoside is 0.05%.

[0020] The role of the said composition in the treatment of lung diseases. Further, the said lung diseases include but are not limited to idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, silicosis, chronic bronchitis, pneumonia, lung injury and other lung diseases with pulmonary fibrosis as the final process.

[0021] In a preferred embodiment of the present invention, the preparation method of the said composition is as follows:

[0022] S1: Under anesthesia, healthy impacted and unerupted third molars of humans are extracted; after splitting the crowns of the teeth, dental pulp tissues are taken, soaked in PBS and cleaned; the dental follicle tissues are placed in a culture dish, cut into pieces, placed in a solution containing type I collagenase and Dispase, digested in a water bath until the tissues are completely digested; the cells are collected by passing through a cell sieve, centrifuged, and the digestion solution is discarded; the tissues are re-suspended in α-MEM medium, centrifuged, and the supernatant is discarded; then the loose tissues are evenly spread on a T25 culture flask, and α-MEM medium containing FBS is added dropwise, cultured, and when the tissue blocks adhere to the wall, the medium is slowly added to the culture flask, and the medium is changed again after 3 days; when the cell density reaches 70% - 80%, the medium is discarded, digested with 0.25% trypsin in a 37°C incubator for 1 - 2 min, and cultured by adding medium containing 10% FBS for passage 5 times to obtain;

[0023] S1. Cultivate and induce the dental pulp stem cells in step S1 for 7 days in DMEM supplemented with 5% FBS, 10 ng / ml chitosan and 10 ng / ml nonadienol; inoculate at a cell density of 2.5×10 4 cells / mL into a serum-containing medium, and after culturing for 3 days, collect the cell supernatant A;

[0024] S2. Centrifuge the supernatant A at 5°C and 400 rpm for 5 min, collect the supernatant B, then centrifuge the supernatant B at 300 rpm for 15 min to obtain the supernatant C. Then add PBS buffer for washing and centrifuge at 200 rpm for 30 min to obtain dental pulp stem cell exosomes.

[0025] S3. Add L-cysteine and rattan polysaccharide to the ethanol aqueous solution for standby; among them, calculated by weight ratio, the dosage of L-cysteine: rattan polysaccharide: ethanol: water is 0.5:0.1:80:20.

[0026] S4. Slowly add the dental pulp stem cell exosomes of S2 to S3 to obtain a mixed system, and add 0.2% benzyl alcohol and 0.05% lauroyl maltoside based on the mass of the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0027] In a preferred embodiment of the present invention, the composition is prepared into a drug, and the drug is administered by injection. The drug may contain clinically acceptable excipients.

[0028] Compared with the prior art, the present invention has achieved unexpected technical effects:

[0029] The present invention relates to a dental pulp stem cell exosome and its composition for treating lung diseases, which has a high loading capacity. Chitosan and nonadienol added during the culture process can form a stable environment suitable for the survival of dental pulp stem cells. The composition prepared from the finally obtained exosome reduces the content of TGF-β1, increases the percentage of SaO2 and reduces pCO2. The prepared composition improves lung function without causing adverse effects and side effects, and has good therapeutic effects. It has high development value and is suitable for further research and productization. Description of the Drawings

[0030] Figure 1 : The loading rate of alizarin from Rubia cordifolia L. by the dental pulp stem cell exosomes prepared in Examples 1-5 and Comparative Examples 1-3 at 0, 30, 60, 90, and 120 min.

[0031] Figure 2 : Effects of different examples on the proliferation of DFCs.

[0032] Figure 3 : Detection of the content of TGF-β1 in the lung tissue of rats in different examples.

[0033] Figure 4 : Detection results of alveolar lavage fluid ALB in different examples.

[0034] Figure 5: Changes in SaO2 (%) of each group of rats on the 21st day in different embodiments.

[0035] Figure 6 : Changes in pCO2 of each group of rats on the 21st day in different embodiments. Detailed implementation manners

[0036] In order to make the objectives and technical solutions of the present invention clearer, the following further describes the present invention in conjunction with embodiments. However, the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. Those skilled in the art should understand that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0037] I. Preparation of adult stem cells derived from neural crest used in the present invention

[0038] 1. Extraction of dental pulp stem cells

[0039] 1) After obtaining the informed consent of the patient, collect healthy impacted and unerupted third molars of humans aged 15 - 25 years old under anesthesia;

[0040] 2) After splitting the crown of the tooth, take the dental pulp tissue, soak it in PBS for 30 min, and wash it clean;

[0041] 3) Remove the hard tissue, place the dental follicle tissue in a culture dish, cut it into pieces, and set aside;

[0042] 4) Place the tissue in step 3) in a solution containing 10% type I collagenase and 12% Dispase, digest it in a 37°C water bath for 30 min, and shake it once every 10 min to completely digest the tissue;

[0043] 5) Pass the tissue in step 4) through a 70 μm cell sieve to collect cells, centrifuge at 1000 rpm for 5 min, discard the digestion solution, re-suspend the tissue in α-MEM medium (containing 10% fetal bovine serum), centrifuge at 1000 rpm for 5 min, discard the supernatant, and set aside the loose tissue;

[0044] 6) Evenly spread the loose tissue in a T25 culture flask, add a small amount of α-MEM medium with 15% FBS (5% CO2, 37°C), culture for 24 h, wait for the tissue block to adhere to the wall, slowly add 2 ml of medium to the culture flask, and change the medium again after 3 days;

[0045] 7) Observe the cell growth status under an inverted microscope every day. When the cell density reaches 70% - 80%, discard the medium, digest it with 0.25% trypsin in a 37°C incubator for 1 - 2 min, and add medium with 10% FBS for digestion and passage;

[0046] 8) Pipette the cells repeatedly to detach them from the bottom of the culture flask. Aspirate the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add medium to resuspend the cells, mix well, and then inoculate into a new culture flask for subculture at a ratio of 1:3.

[0047] 9) The cells for subculture are used for mesenchymal stem cell identification or subsequent exosome isolation. Passage 5 dental follicle stem cells are selected to extract exosomes.

[0048] 2. Identification of dental pulp stem cells

[0049] 1) Perform immunophenotypic detection on the dental pulp stem cells (i.e., odontogenic stem cells) obtained in "1": Label the cells with CD73, CD90, CD105, CD11b, CD19, CD34, CD45, HLA-DR antibodies labeled with FITC or PE and the corresponding isotype control antibodies, and detect with a flow cytometer.

[0050] 2) After osteogenic induction for 3 weeks, identify with alizarin red staining; after adipogenic induction for 2 weeks, identify with Oil Red O staining.

[0051] 3) Result analysis: The obtained dental pulp stem cells grow in a spindle shape, with a firm wall and spiral growth; highly express the mesenchymal stem cell-specific antigens CD73, CD90, and CD105, do not express the hematopoietic and immune cell surface markers CD11b, CD19, CD34, CD45, and do not express the surface marker HLA-DR related to transplantation immune rejection; after adipogenic induction, obvious lipid droplets can be seen in the cells stained with Oil Red O; after osteogenic induction, alizarin red staining is positive. It indicates that the isolated cells are dental pulp stem cells.

[0052] Example 1: A composition containing dental pulp stem cell exosomes is prepared as follows:

[0053] S1. Culture and induce dental pulp stem cells in DMEM supplemented with 5% FBS, 10 ng / ml chitosan, and 10 ng / ml nonadienol for 7 days; inoculate at a cell density of 2.5×10 4 cells / mL into the serum-containing medium, culture for 3 days, and collect the cell supernatant A.

[0054] S2. Centrifuge the supernatant A at 400 rpm / min at 5°C for 5 min, collect the supernatant B, centrifuge the supernatant B at 300 rpm / min for 15 min to obtain the supernatant C, then add PBS buffer for washing, and centrifuge at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes.

[0055] S3. Add L-cysteine and polysaccharide from Sargentodoxa cuneata to an ethanol aqueous solution for standby; (by weight ratio, L-cysteine: polysaccharide from Sargentodoxa cuneata: ethanol: water = 0.5:0.1:80:20)

[0056] S4. Slowly add the dental pulp stem cell exosomes from S3 to S2, and add 0.2% benzyl alcohol and 0.05% lauroyl maltoside based on the mass of the mixed system to the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0057] Example 2: A composition containing dental pulp stem cell exosomes is specifically prepared as follows:

[0058] S1. Culture and induce dental pulp stem cells in DMEM supplemented with 3% FBS, 5 ng / mL chitosan and 5 ng / mL nonadienol for 7 days; inoculate at a cell density of 1×10 4 cells / mL into a serum-containing medium, and after culturing for 2 - 4 days, collect cell supernatant A;

[0059] S2. Centrifuge supernatant A at 400 rpm / min at 2°C for 5 min, collect supernatant B, centrifuge supernatant B at 300 rpm / min for 10 min to obtain supernatant C, then add PBS buffer for washing, and centrifuge at 200 rpm / min for 20 min to obtain dental pulp stem cell exosomes;

[0060] S3. Add L-cysteine and polysaccharide from Sargentodoxa cuneata to an ethanol aqueous solution for standby; (by weight ratio, L-cysteine: polysaccharide from Sargentodoxa cuneata: ethanol: water = 0.2:0.07:70:15)

[0061] S4. Slowly add the dental pulp stem cell exosomes from S3 to S2, and add 0.1% benzyl alcohol and 0.03% lauroyl maltoside based on the mass of the mixed system to the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0062] Example 3: A composition containing dental pulp stem cell exosomes is specifically prepared as follows:

[0063] S1. Culture and induce dental pulp stem cells in DMEM supplemented with 8% FBS, 15 ng / mL chitosan and 15 ng / mL nonadienol for 7 days; inoculate at a cell density of 4×10 4 cells / mL into a serum-containing medium, and after culturing for 2 - 4 days, collect cell supernatant A;

[0064] S2. Centrifuge the supernatant A at 8 °C and 400 rpm for 5 min to collect the supernatant B. Then centrifuge the supernatant B at 300 rpm for 20 min to obtain the supernatant C. Next, add PBS buffer for washing and centrifuge at 200 rpm for 40 min to obtain dental pulp stem cell exosomes;

[0065] S3. Add L-cysteine and rattan polysaccharide to an ethanol aqueous solution for standby; (by weight ratio, L-cysteine: rattan polysaccharide: ethanol: water = 0.8:0.12:90:25)

[0066] S4. Slowly add the dental pulp stem cell exosomes from S3 to S2, and add 0.3% benzyl alcohol and 0.08% lauroyl maltoside based on the mass of the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0067] Example 4: A composition containing dental pulp stem cell exosomes is specifically prepared as follows:

[0068] S1. Culture and induce dental pulp stem cells in DMEM supplemented with 5% FBS, 10 ng / ml chitosan, and 10 ng / ml nonadienol for 7 days; inoculate at a cell density of 2.5×10 4 cells / mL into a serum-containing medium, and after culturing for 3 days, collect the cell supernatant A;

[0069] S2. Centrifuge the supernatant A at 5 °C and 400 rpm for 5 min to collect the supernatant B. Then centrifuge the supernatant B at 300 rpm for 15 min to obtain the supernatant C. Next, add PBS buffer for washing and centrifuge at 200 rpm for 30 min to obtain dental pulp stem cell exosomes;

[0070] S3. Add vitamin C and rattan polysaccharide to an ethanol aqueous solution for standby; (by weight ratio, L-cysteine: rattan polysaccharide: ethanol: water = 0.5:0.1:90:15)

[0071] S4. Slowly add the dental pulp stem cell exosomes from S3 to S2, and add 0.2% benzyl alcohol and 0.05% lauroyl maltoside based on the mass of the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0072] Example 5: A composition containing dental pulp stem cell exosomes is specifically prepared as follows:

[0073] S1. Culture induced dental pulp stem cells in DMEM supplemented with 3 - 8% FBS, 5 - 15 ng / mL chitosan, and 5 - 15 ng / mL nonadienol for 7 days; inoculate the cells at a cell density of 3×10 4 cells / mL into a serum-containing medium, culture for 2 - 4 days, and collect cell supernatant A;

[0074] S2. Centrifuge supernatant A at 400 rpm / min at 2 - 8°C for 5 min, collect supernatant B, then centrifuge supernatant B at 300 rpm / min for 20 min to obtain supernatant C, then add PBS buffer for washing and centrifuge at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes;

[0075] S3. Add sodium thiosulfate and rattan polysaccharide to an ethanol aqueous solution for standby; (by weight ratio, L-cysteine:rattan polysaccharide:ethanol:water = 0.5:0.12:80:20)

[0076] S4. Slowly add the dental pulp stem cell exosomes from S3 to S2, and add 0.3% benzyl alcohol and 0.08% lauroyl maltoside based on the mass of the mixed system, stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0077] Comparative Example 1: A composition containing dental pulp stem cell exosomes was prepared as follows:

[0078] S1. Culture induced dental pulp stem cells in DMEM supplemented with 5% FBS, 10 ng / mL chitosan, and 10 ng / mL nonadienol for 7 days; inoculate the cells at a cell density of 2.5×10 4 cells / mL into a serum-containing medium, culture for 3 days, and collect cell supernatant A;

[0079] S2. Centrifuge supernatant A at 5°C and 400 rpm / min for 5 min, collect supernatant B, then centrifuge supernatant B at 300 rpm / min for 15 min to obtain supernatant C, then add PBS buffer for washing and centrifuge at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes;

[0080] S3. Add L-cysteine and rattan polysaccharide to an ethanol aqueous solution for standby; (by weight ratio, L-cysteine:rattan polysaccharide:ethanol:water = 0.5:0.1:80:20)

[0081] S4. Slowly add the dental pulp stem cell exosomes from S3 to S2, stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0082] Comparative Example 2: A composition containing dental pulp stem cell exosomes was prepared as follows:

[0083] S1. Dental pulp stem cells were cultured in DMEM supplemented with 5% FBS, 10 ng / ml chitosan, and 10 ng / ml nonadienol for 7 days; they were inoculated into a serum-containing medium at a cell density of 2.5×10 4 cells / mL, and after 3 days of culture, the cell supernatant A was collected;

[0084] S2. The supernatant A was centrifuged at 400 rpm / min at 5°C for 5 min, and the supernatant B was collected. The supernatant B was centrifuged at 300 rpm / min for 15 min to obtain the supernatant C. Then, PBS buffer was added for washing, and it was centrifuged at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes;

[0085] S3. L-cysteine and rattan polysaccharide were added to an ethanol aqueous solution for standby; (calculated by weight ratio, L-cysteine: rattan polysaccharide: ethanol: water = 0.5:0.1:80:20)

[0086] S4. The dental pulp stem cell exosomes from S3 were slowly added to S2, and 0.2% benzyl alcohol and 0.05% sodium dodecyl sulfate based on the mass of the mixed system were added to the mixed system, and it was stirred evenly to obtain a composition containing dental pulp stem cell exosomes.

[0087] Comparative Example 3: A composition containing dental pulp stem cell exosomes was prepared as follows:

[0088] S1. Dental pulp stem cells were cultured in DMEM supplemented with 5% FBS, 10 ng / ml chitosan, and 10 ng / ml nonadienol for 7 days; they were inoculated into a serum-containing medium at a cell density of 2.5×10 4 cells / mL, and after 3 days of culture, the cell supernatant A was collected;

[0089] S2. The supernatant A was centrifuged at 400 rpm / min at 5°C for 5 min, and the supernatant B was collected. The supernatant B was centrifuged at 300 rpm / min for 15 min to obtain the supernatant C. Then, PBS buffer was added for washing, and it was centrifuged at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes;

[0090] S3. L-cysteine and rattan polysaccharide were added to an ethanol aqueous solution for standby; (calculated by weight ratio, L-cysteine: rattan polysaccharide: ethanol: water = 0.5:0.1:80:20)

[0091] S4. Slowly add the dental pulp stem cell exosomes of S3 to S2, and add 0.02% benzyl alcohol and 0.02% lauroyl maltoside based on the mass of the mixed system to the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0092] Comparative Example 4: A composition containing dental pulp stem cell exosomes was prepared as follows:

[0093] S1. Culture and induce dental pulp stem cells in DMEM supplemented with 5% FBS and 10 ng / ml chitosan for 7 days; inoculate at a cell density of 2.5×10 4 cells / mL into a serum-containing medium, and after culturing for 3 days, collect cell supernatant A;

[0094] S2. Centrifuge supernatant A at 400 rpm / min at 5°C for 5 min, collect supernatant B, centrifuge supernatant B at 300 rpm / min for 15 min to obtain supernatant C, then add PBS buffer for washing, and centrifuge at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes;

[0095] S3. Add L-cysteine and rattan polysaccharide to an ethanol aqueous solution for standby; (calculated by weight ratio, L-cysteine: rattan polysaccharide: ethanol: water = 0.5:0.1:80:20)

[0096] S4. Slowly add the dental pulp stem cell exosomes of S3 to S2, and add 0.2% benzyl alcohol and 0.05% lauroyl maltoside based on the mass of the mixed system to the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0097] Comparative Example 5: A composition containing dental pulp stem cell exosomes was prepared as follows:

[0098] S1. Culture and induce dental pulp stem cells in DMEM supplemented with 5% FBS, 10 ng / ml chitosan and 10 ng / ml ethanol for 7 days; inoculate at a cell density of 2.5×10 4 cells / mL into a serum-containing medium, and after culturing for 3 days, collect cell supernatant A;

[0099] S2. Centrifuge supernatant A at 400 rpm / min at 5°C for 5 min, collect supernatant B, centrifuge supernatant B at 300 rpm / min for 15 min to obtain supernatant C, then add PBS buffer for washing, and centrifuge at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes;

[0100] S3. Add L-cysteine and rattan polysaccharide into an ethanol aqueous solution for standby; (by weight ratio, L-cysteine: rattan polysaccharide: ethanol: water = 0.5:0.1:80:20)

[0101] S4. Slowly add the dental pulp stem cell exosomes of S3 into S2, and add 0.2% benzyl alcohol and 0.05% lauroyl maltoside based on the mass of the mixed system into the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0102] Comparative Example 6: A composition containing dental pulp stem cell exosomes was prepared as follows:

[0103] S1. Culture and induce dental pulp stem cells in DMEM supplemented with 5% FBS, 10 ng / ml chitosan, and 10 ng / ml nonadienol for 7 days; inoculate at a cell density of 2.5×10 4 cells / mL into a serum-containing medium, and after culturing for 3 days, collect cell supernatant A;

[0104] S2. Centrifuge supernatant A at 400 rpm / min at 5°C for 5 min, collect supernatant B, centrifuge supernatant B at 300 rpm / min for 15 min to obtain supernatant C, then add PBS buffer for washing, and centrifuge at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes;

[0105] S3. Add L-cysteine into an ethanol aqueous solution for standby; (by weight ratio, L-cysteine: ethanol: water = 0.5:80:20)

[0106] S4. Slowly add the dental pulp stem cell exosomes of S3 into S2, and add 0.2% benzyl alcohol and 0.05% lauroyl maltoside based on the mass of the mixed system into the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0107] Comparative Example 7: A composition containing dental pulp stem cell exosomes was prepared as follows:

[0108] S1. Culture and induce dental pulp stem cells in DMEM supplemented with 5% FBS, 10 ng / ml chitosan, and 10 ng / ml nonadienol for 7 days; inoculate at a cell density of 2.5×10 4 cells / mL into a serum-containing medium, and after culturing for 3 days, collect cell supernatant A;

[0109] S2. Centrifuge the supernatant A at 5°C and 400 rpm for 5 min to collect the supernatant B. Then centrifuge the supernatant B at 300 rpm for 15 min to obtain the supernatant C. Next, add PBS buffer for washing and centrifuge at 200 rpm for 30 min to obtain dental pulp stem cell exosomes.

[0110] S3. Add L-cysteine and polysaccharide from Dendrobium officinale to an ethanol aqueous solution for standby; (calculated by weight ratio, L-cysteine:polysaccharide from Sargentodoxa cuneata:ethanol:water = 0.5:0.1:80:20)

[0111] S4. Slowly add the dental pulp stem cell exosomes from S3 to S2, and add 0.2% benzyl alcohol and 0.05% lauroyl maltoside based on the mass of the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0112] Comparative Example 8: A composition containing dental pulp stem cell exosomes, which is specifically prepared as follows:

[0113] S1. Culture and induce dental pulp stem cells in DMEM supplemented with 5% FBS, 10 ng / ml chitosan and 10 ng / ml nonadienol for 7 days; inoculate at a cell density of 2.5×10 4 cells / mL into a serum-containing medium, and after culturing for 3 days, collect the cell supernatant A;

[0114] S2. Centrifuge the supernatant A at 5°C and 400 rpm for 5 min to collect the supernatant B. Then centrifuge the supernatant B at 300 rpm for 15 min to obtain the supernatant C. Next, add PBS buffer for washing and centrifuge at 200 rpm for 30 min to obtain dental pulp stem cell exosomes.

[0115] S3. Add L-cysteine and polysaccharide from Sargentodoxa cuneata to an ethanol aqueous solution for standby; (calculated by weight ratio, L-cysteine:polysaccharide from Sargentodoxa cuneata:ethanol:water = 0.5:0.1:80:20)

[0116] S4. Slowly add the dental pulp stem cell exosomes from S3 to S2, and add 0.2% benzyl alcohol and 0.05% alkyl sulfate to the mixed system, and stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0117] Comparative Example 9: A composition containing dental pulp stem cell exosomes, which is specifically prepared as follows:

[0118] S1. Induce dental pulp stem cells in DMEM supplemented with 5% FBS, 10 ng / ml chitosan, and 10 ng / ml nonadienol for 7 days; inoculate at a cell density of 2.5×10 4 cells / mL into the serum-containing medium, culture for 3 days, and collect cell supernatant A;

[0119] S2. Centrifuge supernatant A at 400 rpm / min at 5°C for 5 min, collect supernatant B, centrifuge supernatant B at 300 rpm / min for 15 min to obtain supernatant C, then add PBS buffer for washing and centrifuge at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes;

[0120] S3. Add L-cysteine and rattan polysaccharide to the ethanol aqueous solution for standby; (calculated by weight ratio, L-cysteine: rattan polysaccharide: ethanol: water = 0.5:0.1:80:20)

[0121] S4. Slowly add the dental pulp stem cell exosomes from S3 to S2, and add 0.2% benzyl alcohol and 0.05% hyaluronic acid based on the mass of the mixed system, stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0122] Comparative Example 10: A composition containing dental pulp stem cell exosomes is prepared as follows:

[0123] S1. Induce dental pulp stem cells in DMEM supplemented with 5% FBS, 10 ng / ml chitosan, and 10 ng / ml nonadienol for 7 days; inoculate at a cell density of 2.5×10 4 cells / mL into the serum-containing medium, culture for 3 days, and collect cell supernatant A;

[0124] S2. Centrifuge supernatant A at 400 rpm / min at 5°C for 5 min, collect supernatant B, centrifuge supernatant B at 300 rpm / min for 15 min to obtain supernatant C, then add PBS buffer for washing and centrifuge at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes;

[0125] S3. Add L-cysteine and rattan polysaccharide to the ethanol aqueous solution for standby; (calculated by weight ratio, L-cysteine: rattan polysaccharide: ethanol: water = 0.5:0.1:80:20)

[0126] S4. Slowly add the dental pulp stem cell exosomes from S3 to S2, and add 0.05% lauroyl maltoside to the mixed system, stir evenly to obtain a composition containing dental pulp stem cell exosomes.

[0127] II. Determination of the Loading Rate of Natural Antioxidant Rubimaillin by Dental Pulp Stem Cell Exosomes

[0128] The dental pulp stem cell exosome compositions obtained in Examples 1-5 and Comparative Examples 1-3 were resuspended with acetone respectively to disrupt the structure of the exosomes and denature the proteins for precipitation; centrifuged at 1000G for 15 min, the supernatant was taken, concentrated and dried on a rotary evaporator, and redissolved with methanol; the content of rubimaillin was detected by HPLC.

[0129] Chromatographic conditions and system suitability test: Using octadecylsilane chemically bonded silica gel as the filler; using methanol-acetonitrile-0.2% phosphoric acid solution (25:50:25) as the mobile phase; the detection wavelength was 250 nm. The number of theoretical plates calculated by the rubimaillin peak should be not less than 4000.

[0130] Calculation method: Loading rate = content of detected rubimaillin / content of initial rubimaillin. The results are as Figure 1 shown.

[0131] Figure 1 : As can be seen from the results, at 0, 30, 60, 90, and 120 min, the loading rates of the dental pulp stem cell exosomes prepared in Examples 1-5 for rubimaillin were significantly higher than those in Comparative Examples 1-3. Among them, the loading rate of Example 1 was as high as more than 50%. Compared with Comparative Example 1 (without surfactant), Comparative Example 2 (sodium dodecyl sulfate), and Comparative Example 3 (the dosage of surfactant was not within the scope of the present invention), the surfactants used in Examples 1-3 could effectively promote the loading of rubimaillin by exosomes.

[0132] III. Effects on the Proliferation of DFCs

[0133] 1) P5-generation DFCs were inoculated in 96-well plates at a density of 3000 cells / well, and 100 μL of DMEM in S1 prepared in Examples 1-3 and Comparative Examples 4-5 was added to each well;

[0134] 3) 10 μL of CCK8 solution was added to each well, incubated at 37 °C for 2 h, and the OD value was measured at a wavelength of 450 nm.

[0135] The results are as Figure 2 shown.

[0136] Figure 2 : As can be seen from the results, there were significant differences between Examples 1-3 and Comparative Examples 4-5 (P < 0.01), indicating that the preferred DMEM system of the present invention containing chitosan and nonadienol can form a stable environment suitable for the survival of dental pulp stem cells, which makes sufficient preparations for further extraction of their exosomes.

[0137] 4. Study on the effect of each group in the example on the rat fibrosis model

[0138] 4.1 Test drug: the composition of Example 1, Comparative Example 6 and Comparative Example 7.

[0139] Positive drug: Cyclic adenosine phosphate for injection, manufacturer: Guangdong China Resources Shunfeng Pharmaceutical Co., Ltd., drug standard code: 86900340000695, batch number: National Medicine Standard H20066680, specification: 20mg

[0140] 4.2 Animals

[0141] SD rats, SPF grade, Sibeifu (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2024-0001, use license number: SYXK (Beijing) 2024-0010SYXK (Tianjin) 2016-0009.

[0142] 2.3 Instruments

[0143] ECA522 electronic balance: Nanjing Bonita Scientific Instrument Co., Ltd.

[0144] ECC2201 electronic balance: Nanjing Bonita Scientific Instrument Co., Ltd.

[0145] RT-6100 microplate reader: product of Shenzhen Raydu Life Sciences Co., Ltd.

[0146] 4.3 Model preparation

[0147] After SD rats were anesthetized by intraperitoneal injection of 4% sodium pentobarbital (1.0 mL / kg), they were fixed on a mouse board in supine position, the neck hair was cut, the skin was disinfected with iodine, and a 1 cm long mid-cervical incision was made under aseptic operation. The trachea was separated and exposed layer by layer, and curved ophthalmic forceps were used to pass through the bottom of the trachea, and the trachea was slightly lifted, but it did not affect airway ventilation. Under direct vision, the needle of a 1 ml syringe was used to puncture between the two cartilage rings, and the head of the mouse board was raised to form an angle of 30 to 35 degrees with the table. Keep the needle consistent with the direction of the airway and in the center of the airway as much as possible. The bleomycin group was injected with 1.0 ml / kg (5 mg / kg) of bleomycin into the trachea at a time, and the control group was given an equal volume of normal saline. The needle was immediately pulled out, the mouse board was erected, the rat was kept in an upright position, and it was rotated back and forth for 1 to 2 minutes to make the drug solution reach the lungs on both sides as much as possible and evenly distributed. After surgery, the skin was sutured, and the rats were placed in the animal center animal room for feeding for one week, divided into groups according to body weight, and given drugs respectively. The indicators were observed after two weeks.

[0148] 4.4 Grouping and Dosage Regimen

[0149] Sham operation control group, model control group, positive control group, Example 1 group, Comparative Example 6 group, and Comparative Example 7 group. In both the sham operation and model control groups, normal saline (0.4 ml) was injected intravenously once. In the Example 1 group, Comparative Example 6 group, and Comparative Example 7 group, stem cells (3×10 6 cells / rat, 0.4 ml) were injected intravenously once, and adenosine cyclic phosphate for injection was administered intravenously at a dose of 0.126 mg / g body weight once a day for 14 consecutive days.

[0150] 4.3 Index detection

[0151] 4.3.1 Determination of the content of TGF-β1 in rat lung tissue by ELISA

[0152] TGF-β1 (transforming growth factor β1) is an important profibrotic factor that is widely involved in the fibrotic processes of various organs. It exacerbates the fibrotic response of tissues by activating fibroblasts and promoting the excessive synthesis of collagen and other extracellular matrix components. TGF-β1 is not only one of the key drivers of pulmonary fibrosis but also considered an early signal for the occurrence and progression of fibrosis, and its serum level is often closely related to the severity of pulmonary fibrosis. Therefore, TGF-β1 is widely used as a biomarker for evaluating the severity of pulmonary fibrosis. Based on this background, recent studies have shown that dental pulp stem cells (DPSCs) have significant antifibrotic effects and can inhibit the fibrotic process by regulating the TGF-β1 signaling pathway.

[0153] 4.3.2 Detection of ALB (albumin) in rat bronchoalveolar lavage fluid

[0154] Steps for bronchoalveolar lavage fluid: Fix the rat that has had blood collected. Insert one end of a butterfly needle into the rat's organ and connect the other end to a syringe. Flush the alveoli with 5 mL of normal saline, aspirate 3 mL of lavage fluid, centrifuge it, and retain the supernatant. The ALB content was determined by enzyme immunoassay.

[0155] 4.3.3 Arterial blood gas analysis

[0156] Check indicators such as the oxygenation status and carbon dioxide elimination in the blood.

[0157] 4.4 Result statistics

[0158] The statistical software GraphPad Prism 6 was used for processing. Measurement data were subjected to a normal test, and normally distributed data were expressed as mean ± standard deviation (X±s). For normal and homoscedastic data, one-way analysis of variance was used to compare the differences between groups, and the LSD method was used for comparison between two groups; for skewed data, the Kruskal wallis nonparametric test was used; P<0.05 indicated that the differences between groups were statistically significant.

[0159] 4.5 Result analysis

[0160] 4.5. Determination of the content of TGF-β1 by ELISA

[0161] Figure 3 : The results of ELISA determination showed that, compared with the sham operation group, the level of TGF-β1 in the model control group increased (P<0.01). Compared with the model control group, the positive control group, Example 1 and Comparative Examples 6-7 showed the effect of reducing TGF-β1, among which, there were significant differences between the positive control group, Example 1 and Comparative Examples 6-7 (P<0.05).

[0162] Figure 4 : It can be seen from the detection results of alveolar lavage fluid ALB that, compared with the sham operation group, the content of albumin ALB in the lavage fluid of the model group increased (P<0.01), and different degrees of improvement occurred in each treatment group. There were significant differences between Example 1, the positive control group and Comparative Examples 6-7; there was a significant difference between Example 1 and the positive control group (P<0.01). Among them, the change value of ALB = Example group - sham operation group.

[0163] 4.5.3 Results of arterial blood gas analysis

[0164] Figure 5 It shows that on the 21st day (14 days after treatment) of each group of rats, 0.3 ml of blood was drawn from the abdominal aorta and analyzed using a blood gas analyzer. Compared with the sham operation group, the SaO2 (percentage of hemoglobin saturated with oxygen) in the model group decreased significantly (P<0.01), and all groups of Example and the positive control group had improvement, among which the improvement in Example 1 group was the most obvious. Among them, the change value of SaO2 = sham operation group - Example group.

[0165] Figure 6 It shows that on the 21st day (14 days after treatment) of each group of rats, 0.3 ml of blood was drawn from the abdominal aorta and analyzed using a blood gas analyzer. Compared with the sham operation group, the pCO2 (partial pressure generated solely by dissolved carbon dioxide in plasma) in the model group increased. It indicates that DPSCs can improve the respiratory function of the lungs. Among them, the change value of pCO2 = Example group - sham operation group.

Claims

1. A method of producing adult stem cells derived from a neural crest, characterized in that: The adult stem cells are derived from dental stem cells, including one or more of dental pulp stem cells derived from adult orthodontic teeth, dental pulp stem cells from lost deciduous teeth, and apical dental papilla stem cells.

2. The adult stem cell according to claim 1, characterized in that The preparation method of the dental pulp stem cells is as follows: extracting a healthy human impacted and undeveloped third molar under anesthesia; taking dental pulp tissue, soaking it in PBS, and cleaning it; placing the dental sac tissue in a culture dish, cutting it into pieces, placing it in a type I collagenase and Dispase solution, and digesting it in a water bath to completely digest the tissue; sieving and collecting the cells, centrifuging, discarding the digestion solution, resuspending the tissue in an α-MEM culture medium, centrifuging, discarding the supernatant, and then evenly spreading the loose tissue in a T25 culture bottle, adding a-MEM culture medium containing FBS, culturing, and slowly adding culture medium to the culture bottle after the tissue block adheres to the wall, and changing the medium again after 3 days; when the cell density reaches 70%-80%, discarding the culture medium, digesting it with trypsin in a 37°C incubator for 1-2 minutes, adding a culture medium containing 10% FBS for digestion and subculture, and obtaining the obtained cells.

3. An exosome containing the dental pulp stem cells according to claim 1, characterized in that: The exosomes of dental pulp stem cells are prepared as follows: S1. Induce dental pulp stem cells by culturing in DMEM supplemented with 3-8% FBS, 5-15 ng / mL chitosan, and 5-15 ng / mL nonadienol for 7 days. 4 The cells were inoculated into serum-containing culture medium at a cell density of 10 cells / mL, and after culturing for 2-4 days, the cell supernatant A was collected; S2. Centrifuge supernatant A at 2-8°C and 400 rpm / min for 5 min, collect supernatant B, centrifuge supernatant B at 300 rpm / min for 10-20 min to obtain supernatant C, then add PBS buffer for washing, and centrifuge at 200 rpm / min for 20-40 min to obtain dental pulp stem cell exosomes.

4. A composition containing the dental pulp stem cell exosomes according to claim 1, characterized in that: The preparation of the composition comprises the following steps: S1, L-cysteine ​​and red safflower polysaccharide are added to the ethanol aqueous solution and set aside. S2. Slowly add the dental pulp stem cell exosomes of claim 3 into S1 to obtain a mixed system, then add benzyl alcohol and lauroyl maltoside, stir evenly, and obtain a composition containing dental pulp stem cell exosomes.

5. The adult stem cell according to claim 4, characterized in that Calculated by weight ratio, the L-cysteine:Caulis Sargentodoxae polysaccharide:ethanol:water=0.2-0.8:0.07-0.12:70-90:15-25.

6. The adult stem cell according to claim 4, characterized in that Calculated by weight ratio, the L-cysteine:Caulis Sargentodoxae polysaccharide:ethanol:water=0.5:0.1:80:

20.

7. The adult stem cell according to claim 4, characterized in that Calculated by mass volume ratio, the dosage of benzyl alcohol is 0.1-0.3%, and the dosage of lauroyl maltoside is 0.03-0.08%.

8. The adult stem cell according to claim 4, characterized in that Calculated by mass volume ratio, the amount of benzyl alcohol used is 0.2%, and the amount of lauroyl maltoside used is 0.05%.

9. A composition containing the dental pulp stem cell exosomes according to claim 1, characterized in that: The preparation method of the composition is as follows: S1: Extract the impacted and undeveloped third molar under anesthesia; After splitting the crown of the tooth, take the pulp tissue, soak it in PBS and clean it; Place the dental sac tissue in a culture dish, cut it into pieces, place it in a solution containing type I collagenase and Dispase, and digest it in a water bath to completely digest the tissue; Collect the cells through a cell sieve, centrifuge, discard the digestion solution, resuspend the tissue in α-MEM culture medium, centrifuge, discard the supernatant, and then evenly spread the loose tissue in a T25 culture bottle, add FBS a-MEM culture medium, culture, wait for the tissue block to adhere to the wall, slowly add culture medium to the culture bottle, and change the medium again after 3 days; When the cell density reaches 70%-80%, discard the culture medium, use 0.25% trypsin, digest it in a 37℃ incubator for 1-2 minutes, add 10% FBS culture medium to digest and subculture for 5 generations, and the result is obtained; S1, culture the dental pulp stem cells induced in step S1 in DMEM supplemented with 5% FBS, 10 ng / ml chitosan and 10 ng / ml nonadienol for 7 days; 4 The cells were inoculated into serum-containing medium at a cell density of 10 cells / mL and cultured for 3 days, and then the cell supernatant A was collected; S2, centrifuge supernatant A at 5°C and 400 rpm / min for 5 min, collect supernatant B, The supernatant B was centrifuged at 300 rpm / min for 15 min to obtain supernatant C, which was then washed with PBS buffer and centrifuged at 200 rpm / min for 30 min to obtain dental pulp stem cell exosomes; S3, L-cysteine ​​and red safflower polysaccharide are added to the ethanol aqueous solution for later use; wherein, calculated by weight ratio, the amount of L-cysteine: red safflower polysaccharide: ethanol: water is 0.5:0.1:80:20; S4, slowly adding the dental pulp stem cell exosomes of S2 into S3 to obtain a mixed system, adding 0.2% benzyl alcohol and 0.05% lauroyl maltoside by weight of the mixed system into the mixed system, stirring evenly to obtain a composition containing dental pulp stem cell exosomes.

10. The composition according to claim 9, characterized in that The composition is prepared into a medicine, and the medicine is administered by injection.

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