Use of cytokines to promote secretion of exosomes by dental pulp stem cells

Abstract

This invention provides the application of cytokines in promoting exosome secretion by dental pulp stem cells. This invention uses cytokines as stimulants. Dental pulp stem cells express five receptors selected in this invention on their surface. All five cytokines can stimulate the proliferation, differentiation, or migration of dental pulp stem cells. Cell proliferation, differentiation, and migration are all processes of cell activation and vigorous cell metabolism, and cells in an activated state can secrete large amounts of exosomes. Therefore, after stimulation with the five cytokines, dental pulp stem cells are in an activated state, thereby significantly increasing the amount of exosomes secreted by dental pulp stem cells. The resulting exosomes can be used for anti-aging or other medical applications and have broad market value.

Description

[0001] This application is a divisional application of the invention patent application filed on December 20, 2019, with application number 2019113277049, entitled "Application of Cytokines in Promoting Exosome Secretion by Dental Pulp Stem Cells". Technical Field

[0002] This invention relates to the field of biotechnology, and in particular to the application of a cytokine in promoting the secretion of exosomes by dental pulp stem cells. Background Technology

[0003] Mesenchymal stem cells (MSCs) are a group of adult stem cells with osteogenic, chondrogenic, and adipogenic functions. MSCs can exert functions such as immune regulation, angiogenesis promotion, and hematopoietic support through paracrine signaling. MSCs are abundant, found in various tissues including the placenta, umbilical cord, bone marrow, dental pulp, and adipose tissue. Therefore, MSCs have been approved as drugs in several countries for the treatment of graft-versus-host disease, ulcerative colitis, and the recovery period from myocardial infarction. However, regardless of whether they are injected intravenously, intramuscularly, or intramyocardially, exogenously transplanted MSCs mostly die within 72 hours. Regarding how dead MSCs exert their aforementioned functions, previous studies have shown that cultured MSCs can release large amounts of exosomes under hypoxic and serum withdrawal conditions. These exosomes can promote endothelial cell proliferation and the formation of capillary networks in endothelial cells, accelerating the recovery of blood flow in ischemic models. In fact, MSC-derived exosomes have entered clinical trials for the treatment of diabetes and renal insufficiency. Therefore, MSC-derived exosomes have potential practical value as a material for "cell-free" cell therapy. To improve treatment efficacy and explore ways to obtain more functional exosomes, it is essential to conduct clinical trials of MSC exosomes.

[0004] However, there has been no specific research on the promoting factors of stem cell exosome secretion in the existing technology.

[0005] Chinese patent application publication number CN 106492194 A discloses a stem cell exosome preparation, its preparation method and application. The cytokine used is only basic fibroblast growth factor. The preparation is added to the exosomes as a supplement. The patent uses dental pulp stem cells + exosomes + basic fibroblast growth factor (10ng / ml) as a mixed preparation to treat gastritis. It does not conduct specific research on the promoting factors of stem cell exosome secretion, nor does it involve stem cell culture that can be used for skin. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and to provide the application of cytokines in the preparation of stimulants, which promote the secretion of exosomes by dental pulp stem cells.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides the application of cytokines in the preparation of stimulants, which promote the secretion of exosomes by dental pulp stem cells. These exosomes can be specifically used in the preparation of anti-skin aging drugs and other applications.

[0008] Optionally, the cytokine is selected from at least one of epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), human platelet-derived growth factor BB (PDGF-BB), stem cell growth factor (SCF), and interferon-gamma (IFN-γ).

[0009] Preferably, the cytokine is a combination of epidermal growth factor (EGF) and human platelet-derived growth factor BB (PDGF-BB).

[0010] Preferably, the mass ratio of the epidermal growth factor to the human platelet-derived growth factor BB is 1:1.

[0011] The present invention also provides a method for preparing exosomes, comprising: adding cytokines to a culture medium to obtain a culture medium containing cytokines, adding dental pulp stem cells to the culture medium for culture, and obtaining exosomes secreted by dental pulp stem cells.

[0012] Optionally, the cytokine is selected from at least one of epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), human platelet-derived growth factor BB (PDGF-BB), stem cell growth factor (SCF), and interferon-gamma (IFN-γ).

[0013] Preferably, the cytokine is a combination of epidermal growth factor (EGF) and human platelet-derived growth factor BB (PDGF-BB).

[0014] Preferably, the mass ratio of the epidermal growth factor to the human platelet-derived growth factor BB is 1:1.

[0015] Optionally, the concentration of the epidermal growth factor in the culture medium is 1-20 ng / mL, preferably 10-20 ng / mL, including but not limited to 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, etc.

[0016] Optionally, the concentration of the insulin-like growth factor 1 in the culture medium is 1-100 ng / mL, including but not limited to 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, and 100 ng / mL.

[0017] Optionally, the concentration of human platelet-derived growth factor BB in the culture medium is 1-50 ng / mL, including but not limited to 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, and 50 ng / mL.

[0018] Optionally, the concentration of the stem cell growth factor in the culture medium is 1-50 ng / mL, including but not limited to 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, and 50 ng / mL.

[0019] Optionally, the concentration of the γ-interferon in the culture medium is 1-50 ng / mL, including but not limited to 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, and 50 ng / mL.

[0020] Optionally, the culture medium includes a culture medium for promoting the growth of dental pulp stem cells, specifically a serum-free MSC culture medium, which can be purchased commercially.

[0021] Optionally, the culture medium also contains physiological saline for culturing exosomes.

[0022] Optionally, the dental pulp stem cells are added to the culture medium and cultured for 72 hours.

[0023] Optionally, after the culture is completed, the supernatant is collected, centrifuged, and the exosomes are obtained.

[0024] This invention provides exosomes prepared by the above method.

[0025] The present invention also provides a formulation containing the above-mentioned exosomes, the formulation being used to promote fibroblast proliferation and / or promote angiogenesis, thereby playing an anti-skin aging role.

[0026] The present invention has the following beneficial effects: This invention uses cytokines as stimulants. Dental pulp stem cells express receptors for five selected cytokines on their surface. All five cytokines can stimulate the proliferation, differentiation, and migration of dental pulp stem cells. These processes are all characterized by cell activation and vigorous cell metabolism, during which cells can secrete large amounts of exosomes. Therefore, stimulation with these five cytokines activates dental pulp stem cells, significantly increasing their exosome secretion. The resulting exosomes can be used for anti-aging or other medical applications, possessing broad market value. Attached Figure Description

[0027] Figure 1 The image shown is a 50× morphological photograph of human dental pulp stem cells, which is an embodiment of the present invention.

[0028] Figure 2 and Figure 3 The figure shown is a flow cytometry analysis result of human dental pulp stem cells according to an embodiment of the present invention.

[0029] Figure 4 The image shown is a morphological observation (transmission electron microscope) of exosomes according to an embodiment of the present invention.

[0030] Figure 5 The image shown is a Western blot detection result of exosomes according to an embodiment of the present invention.

[0031] Figure 6 The figure shows the effect of exosomes secreted by dental pulp stem cells stimulated by different cytokines on fibroblast proliferation in an embodiment of the present invention.

[0032] Figure 7 The figure shows the effect of exosomes secreted by dental pulp stem cells stimulated by different cytokines on the formation of capillary-like structures by vascular endothelial cells, according to an embodiment of the present invention. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] This invention selects dental pulp stem cells, which has the following advantages: 1) Abundant sources.

[0036] The teeth that fall out naturally in children aged 6-12 and the wisdom teeth that adults need to have extracted both contain abundant dental pulp stem cells.

[0037] 2) Few side effects.

[0038] Dental pulp stem cells are a type of mesenchymal stem cell. They possess the general characteristics of mesenchymal stem cells, including low immunogenicity, no need for strict pairing, no strong rejection reaction, and immunomodulatory function. Allogeneic transplantation will not cause rejection.

[0039] 3) No ethical controversy.

[0040] Since dental pulp stem cells are taken from children's naturally shed teeth and adults' wisdom teeth, they are considered waste materials and do not harm life, so there is no ethical controversy.

[0041] Dental pulp stem cells can secrete exosomes via paracrine signaling. Exosomes are small vesicles, 30-100 nm in diameter, secreted by various cells. Their outer membranes are mainly composed of lipids and proteins, expressing specific markers CD63, CD9, and CD81, as well as some surface markers of their source cells. Exosomes carry various functional proteins, lipids, DNA, RNA, and microRNAs (miRNAs), mediating cell-to-cell communication. Exosomes deliver the bioactive substances of their source stem cells, performing stem cell-like functions while avoiding the potential risks of direct stem cell transplantation. Exosomes can cross the skin, mucous membranes, and blood-brain barrier to enter tissue cells, avoiding removal by filters. Exosomes can be artificially modified as drug and drug-targeted delivery carriers, facilitating storage, transportation, and industrial production.

[0042] Exosomes are substances proven to penetrate the stratum corneum of the skin. We hypothesize that dental pulp stem cell exosomes contain a large number of anti-aging factors and can penetrate the dermis to exert anti-aging effects. Exosomes carry the bioactive substances of dental pulp stem cells, performing stem cell-like functions while avoiding the potential risks of direct stem cell transplantation. Simultaneously, exosomes can cross the skin, mucous membranes, and blood-brain barrier to enter tissue cells, avoiding removal by filtration organs. Exosomes can also be artificially modified for use as drug and targeted drug delivery carriers. Furthermore, exosomes are easier to store, transport, and industrially produce than stem cells.

[0043] In view of this, the present invention is proposed. The present invention relates to a method for increasing the number of exosomes secreted by dental pulp stem cells (MSCs). This method involves adding specific cytokines, preferably EGF+PDGF-BB, during the culture of MSCs, thereby increasing the number of exosomes released by MSCs and simultaneously obtaining more anti-skin aging cytokines, exhibiting stronger ability to promote fibroblast proliferation and angiogenesis. This invention provides a powerful tool for the clinical application of dental pulp stem cells.

[0044] This invention provides a method for culturing dental pulp stem cell exosomes, which can yield a larger quantity of exosomes.

[0045] Example 1 1. Main equipment Table 1

[0046] 2. Main reagents Table 2

[0047] 3. Experimental steps: 3.1 Isolation and Culture of Dental Pulp Stem Cells 3.1.1 Tooth surface cleaning Soak the collected healthy, intact teeth in 75% vol% alcohol for 1 minute, then rinse 2-3 times with sterile saline. Use scissors to scrape away tissue debris from the tooth surface (especially at the root), and rinse repeatedly.

[0048] 3.1.2 Pulp Extraction Place the treated tooth on the flat side of a bone hammer and strike it with another bone hammer to expose the pulp cavity. Mix 2 mL each of the prepared 0.2% collagenase and 0.4% dispersant enzyme. Place the exposed tooth and pulp cavity fragments into the mixture and shake on a shaker at low speed (100 r / min) at 37°C for 1 h.

[0049] 3.1.3 Cell Culture The digested cell clusters were resuspended by pipetting to form a single-cell suspension. The suspension was passed through a cell strainer, and the cell culture was transferred to another centrifuge tube and centrifuged at 1500 r / min for 5 min. The cells were then resuspended in MSC medium, seeded, and 5% (v / v) of additive was added. The cells were incubated at 37℃ in a 5% CO2 incubator. When the cell confluence reached 80%, the cells were passaged using 0.25% trypsin, and cells from passages P3-P5 with good growth were used for experiments.

[0050] 3.1.4 When the cell confluence reaches 80%-90%, remove the culture supernatant, replace it with an equal volume of physiological saline, add AF group stimulating factor and continue culturing for 72 hours, then collect the supernatant.

[0051] The addition of each group of stimulating factors is as follows: A: No addition; B: Add 10 ng / mL EGF; C: Add 100 ng / mL IGF-1; D: Add 50 ng / mL PDGF-BB; E: Add 50 ng / mL SCF; F: Add 50 ng / mL IFN-γ.

[0052] The concentrations of the above-mentioned stimulating factors refer to the final concentrations of each stimulating factor in the culture medium.

[0053] 3.1.5 Exosome collection: Exosomes were collected by density gradient centrifugation, following these steps: 1) Centrifuge at 300g for 10 minutes to remove cells.

[0054] 2) Centrifuge at 2000g for 10 minutes to remove cell debris.

[0055] 3) Centrifuge at 10000g for 30 minutes to remove small cell debris.

[0056] 4) Centrifuge a 100KD ultrafiltration tube at 40000g for 20 minutes and collect exosomes.

[0057] 4. Testing 4.1 Identification of dental pulp stem cells 4.1.1 Obtain morphological photographs (50×) of dental pulp stem cells, see... Figure 1 .

[0058] 4.1.2 Flow cytometry was used to analyze surface markers of dental pulp stem cells.

[0059] Eight antigens of dental pulp mesenchymal stem cells were detected by flow cytometry: CD73, CD90, CD105, HLA-DR, CD45, CD19, CD34, and CD11b. The flow cytometry results are shown below. Figure 2 and Figure 3 .

[0060] 4.2 Exosome Identification 4.2.1 Observation of exosome morphology and size under transmission electron microscopy: Take 10 μL of isolated and purified exosomes, dilute them with PBS solution at 4℃ at a volume ratio of 1:1, and drop them onto a 2 mm copper grid. After standing at room temperature for 1 min, gently absorb the excess liquid with filter paper pulp. Negatively stain with 3% (w / v) sodium phosphotungstenate solution (pH 6.8) at room temperature for 5 minutes. Gently wash half of the stain with double-distilled water and air dry at room temperature. Observe and photograph the exosomes under a transmission electron microscope, and measure their diameter. The transmission electron microscopy results are shown in the figure. Figure 4 .

[0061] 4.2.2 Western blot detection of characteristic protein expression on exosome membrane surface The expression of exosome characteristic proteins was detected using an exosome CD63 & TSG101 protein detection kit. Dental pulp stem cells and exosome samples were collected, and proteins were extracted by lysing the samples using cell lysis buffer. After 10% SDS-PAGE gel electrophoresis according to standard methods, the samples were transferred to PVDF membranes and blocked with 5% skim milk blocking buffer for 1 hour. Then, CD63 and TSG101 primary antibodies were added and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST for 10 minutes each time, and then reacted with HRP-labeled secondary antibody at room temperature for 50 minutes. After washing three times with TBST for 10 minutes each time, chemiluminescent substrate was added for color development and observation.

[0062] 4.3 Determine exosome protein concentration. Calculate the number of cells and their protein expression levels. "Cells" refers to the total number of cells obtained, and "protein" refers to the total content of exosome proteins. This determines which cytokine can stimulate the maximum secretion of exosomes without affecting cell proliferation.

[0063] A: No irritant added (as a negative control); B: Exosomes produced by EGF stimulation; C: Exosomes produced by IGF-1 stimulation; D: Exosomes produced by PDGF-BB stimulation; E: Exosomes produced by SCF stimulation; F: Exosomes produced by IFN-γ stimulation.

[0064] 4.4 The contents of VEGF, IL-6, TGF and bFGF in exosomes were determined by ELISA.

[0065] A: No irritant added (as a negative control); B: Exosomes produced by EGF stimulation; C: Exosomes produced by IGF-1 stimulation; D: Exosomes produced by PDGF-BB stimulation; E: Exosomes produced by SCF stimulation; F: Exosomes produced by IFN-γ stimulation.

[0066] 4.5 Functional identification of exosomes secreted by dental pulp stem cells and experiments on their promotion of fibroblast (HF-1) proliferation.

[0067] HF-1 cells (purchased from Shanghai Jining Industrial Co., Ltd.) were cultured in α-MEM containing 10% FBS (v / v). After digestion and collection, cell counts were performed, and the cells were resuspended in α-MEM containing 10% FBS. Cells were seeded in 96-well plates for MTT assays at 1000 cells / well. Cell groups were as follows: A: 10 μg / mL of supernatant without added irritant (as a negative control); B: Exosomes produced by EGF stimulation at 10 μg / mL; C: Exosomes produced by IGF-1 stimulation at a concentration of 10 μg / mL; D: 10 μg / mL of exosomes produced by PDGF-BB stimulation; E: 10 μg / mL of exosomes produced by SCF stimulation; F: 10 μg / mL of exosomes produced by IFN-γ stimulation; G: 10 μg / mL of exosomes produced by stimulation with EGF+PDGF-BB.

[0068] To determine which cytokines stimulate exosomes to better stimulate cell proliferation.

[0069] The effect of different exosome groups on the proliferation of HF-1 cells was observed using the MTT assay. HF-1 cells were seeded in 96-well plates at a density of 1000 cells / well. Exosomes treated with the AG group (10 μg / mL) were added to the culture system, with three replicates per group. After 72 hours of culture, MTT was added, and the cells were cultured for another 4 hours. Then, dimethyl sulfoxide (DMSO) was added, and the optical density was measured at 490 nm.

[0070] 4.6 Experiment on the promotion of capillary-like structure formation by dental pulp stem cell exosomes The Matrigel assay is a classic method for observing endothelial cell function, especially its ability to form capillary networks, and the results represent angiogenesis activity in vivo. Endothelial cells are seeded in Matrigel-coated culture plates, and the culture system is supplemented with exosomes at a concentration of 10 μg / mL from the untreated, EGF, IGF-1, PDGF-BB, SCF, IFN-γ, and EGF+PDGF-BB stimulation groups, respectively. After 24 hours, the number of reticular structures in each high-power field is counted.

[0071] 5. Results 5.1 Isolation, expansion and identification of human dental pulp stem cells.

[0072] 5.1.1 Figure 1 This image shows the cellular morphology of human dental pulp stem cells.

[0073] 5.1.2 Figure 2 and Figure 3 The image shows the flow cytometry analysis results of human dental pulp stem cells. According to the "Minimum Criteria for Identification of Mesenchymal Stem Cells" issued by the International Society for Cell Therapy (ISCT) in 2006, the expression of the three surface antigens CD73, CD90, and CD105 was no less than 95%; the expression of CD34, CD45, CD11b, CD19, and HLA-DR was no more than 2.0%. The control group was an isotype control of the eight antigens corresponding to dental pulp mesenchymal stem cells. The flow cytometry results of the control group are shown below. Figure 2 The sample group shows the expression of eight antigens. The flow cytometry results for the sample group are as follows: Figure 3 As shown, the expression of CD73, CD90, and CD105 in the sample group was higher than 95.0%; the expression of negative markers (HLA-DR, CD45, CD19, CD34, and CD11b) in the sample group was lower than 2.0%, which is consistent with the surface antigen characteristics of stem cells. This indicates that the dental pulp stem cells have not differentiated and still maintain the characteristics of stem cells.

[0074] 5.2 A large number of exosomes were collected from dental pulp stem cells and the exosomes were identified.

[0075] 5.2.1 Observe the size and morphology of exosomes using transmission electron microscopy. Figure 4 The image shown is a transmission electron microscope image of exosomes. The exosomes range in size from 30 to 120 nm, with most being 30-60 nm bilayer structures that are hollow cystic, consistent with the morphological characteristics of exosomes.

[0076] 5.2.2 Western blot was used to detect the expression of CD63 and TSG101, characteristic proteins on the surface of exosomes. Figure 5 The results of Western blot analysis of exosomes are shown. The exosome-specific markers CD63 and TSG101 are clearly visible, while the dental pulp stem cell group does not express CD63 and TSG101 antibodies.

[0077] 5.3 Protein content in exosomes was determined by ELISA. The statistical results of exosomal protein expression levels in each group are shown in Table 3. When a single stimulant was used, the two groups with the highest protein content were groups B and D, namely the EGF and PDGF-BB groups. Therefore, we tried to combine the two stimulants, EGF and PDGF-BB, as group G. The results showed that the exosomal protein content was significantly increased, reaching 2507±733 μg / 10. 8 Each cell.

[0078] Table 3 5.4 The levels of VEGF, IL-6, TGF, and bFGF were measured using the ELISA method to determine the optimal stimulating factors as EGF and PDGF-BB.

[0080] Table 4

[0081] The statistical results of VEGF, IL-6, TGF, and bFGF levels in each group are shown in Table 4. When a single stimulant was used, the levels of VEGF, IL-6, TGF, and bFGF were significantly higher in groups B and D, indicating a positive correlation between EGF, PDGF-BB, and exosome protein levels. Meanwhile, we tested the levels of VEGF, IL-6, TGF, and bFGF in group G, and the results were significantly higher than those in group AF, still showing a positive correlation with exosome protein levels.

[0082] 5.5 Verify that dental pulp stem cell exosomes stimulated by various stimulating factors EGF, IGF-1, PDGF-BB, SCF, IFN-γ, and EGF+PDGF-BB have better stimulating effect on fibroblast (HF-1) proliferation.

[0083] Figure 6 The diagram shows the effect of exosomes secreted by dental pulp stem cells (HF-1) stimulated by different cytokines on fibroblast proliferation. HF-1 HF-1 cells were stimulated with various stimulating factors: Untreated, EGF, IGF-1, PDGF-BB, SCF, IFN-γ, and EGF+PDGF-BB. MTT assay was used to observe their proliferation. The ordinate is OD490nm. Untreated represents the experimental group without stimulating exosomes. The results showed that the OD values ​​of the factor-treated groups were significantly higher than those of the Untreated group (P<0.05), and the mixed factor group (EGF+PDGF-BB) was slightly higher than the single factor treatment. These results indicate that exosomes released by dental pulp stem cells treated with EGF, IGF-1, PDGF-BB, SCF, IFN-γ, and EGF+PDGF-BB significantly promoted the in vitro proliferation of HF-1 fibroblasts. Exosomes released by dental pulp stem cells after combined stimulation with EGF+PDGF-BB exhibited a stronger ability to promote HF-1 proliferation.

[0084] 5.6 Effects of exosomes secreted by dental pulp stem cells stimulated by different cytokines on the formation of capillary-like structures by vascular endothelial cells.

[0085] Figure 7The results, showing the effects of exosomes secreted by dental pulp stem cells stimulated by different cytokines on the formation of capillary-like structures by vascular endothelial cells, indicated that the number of reticular structures per field of view in endothelial cells was 2.3±1.2, 9.4±0.89, 7±1.42, 8.3±1.33, 7±0.54, 8±1.15, and 10±1.06 in the untreated group, respectively. Endothelial cells in the untreated group showed limited lumen-forming ability. The lumen-forming ability of endothelial cells in the cell growth factor-treated groups was significantly higher than that in the untreated group, and the mixed factor (EGF+PDGF-BB) treatment group was higher than that in the single factor treatment group. These results suggest that exosomes released by dental pulp stem cells after combined stimulation with EGF+PDGF-BB have a stronger pro-angiogenic capacity.

[0086] In summary, this invention produces high-concentration exosomes (with a basic fibroblast growth factor content of 2000 pg / mL), which can be directly applied to the skin. This invention uses cytokines as stimulants to significantly increase the secretion of exosomes from stem cells. The resulting exosomes can be used for anti-aging or other medical applications, possessing broad market value.

[0087] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of a cytokine for the preparation of a stimulating agent, characterized in that, The stimulant is used to promote the secretion of exosomes by dental pulp stem cells; wherein the cytokine is insulin-like growth factor 1.

2. A method for preparing exosomes, characterized in that, The method includes the following steps: adding cytokines to a culture medium to prepare a culture medium containing the cytokines; adding dental pulp stem cells to the culture medium for culture to obtain exosomes secreted by the dental pulp stem cells; wherein the cytokines are insulin-like growth factor 1, and the concentration of insulin-like growth factor 1 in the culture medium is 100 ng / mL.

3. The preparation method according to claim 2, characterized in that, The culture medium includes a medium for promoting the growth of dental pulp stem cells and physiological saline.

4. The preparation method according to claim 2, characterized in that, After the culture was completed, the supernatant was collected, centrifuged, and the exosomes were obtained.

5. Exosomes prepared by the method according to any one of claims 2 to 4.

6. A formulation containing the exosomes of claim 5, characterized in that, The formulation is used to promote fibroblast proliferation and / or promote angiogenesis.

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