CN116515748A - Application of cytokine in promoting secretion of dental pulp stem cells into exosome - Google Patents
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Abstract
The invention provides the use of cytokines in promoting secretion of pulp stem cells into exosomes. The invention takes cytokines as a stimulator, the surfaces of dental pulp stem cells express 5 factor receptors selected by the invention, the 5 cytokines can stimulate proliferation, differentiation or migration of the dental pulp stem cells, the processes of cell proliferation, differentiation, migration and the like are all processes of cell activation and vigorous cell metabolism, and the cells can secrete a large amount of exosomes in an activated state. Therefore, after 5 cytokines are stimulated, the dental pulp stem cells are in an activated state, so that the secretion of exosomes of the dental pulp stem cells is remarkably improved, and the obtained exosomes can be used for anti-aging or other medical purposes and have a wide market value.
Description
The present application is a divisional application of an invention patent application of which the application date is 2019, 12, 20 and application number is 2019113277049, and the invention name is "application of cytokines in promoting secretion of dental pulp stem cells into exosomes".
Technical Field
The invention relates to the field of biotechnology, in particular to application of a cytokine in promoting secretion of dental pulp stem cells into exosomes.
Background
Mesenchymal Stem Cells (MSCs) are a group of adult stem cells with osteogenic, chondrogenic and adipogenic functions. MSCs can exert functions of regulating immunity, promoting angiogenesis, supporting hematopoiesis, and the like through paracrine forms. MSC is abundant in source and exists in various tissues such as placenta, umbilical cord, bone marrow, dental pulp, fat, etc. Therefore, MSC has been approved as a drug by various countries for the treatment of graft versus host disease, ulcerative colitis, myocardial infarction recovery phase, and the like. However, exogenously transplanted MSCs die within 72 hours, whether by intravenous injection, intramuscular injection, or intramyocardial injection. Then, the previous study on how to exert the above effect on dead MSCs shows that MSCs cultured in vitro can release a large amount of exosomes under conditions of hypoxia and serum withdrawal, and the exosomes can promote endothelial cell proliferation in vitro, promote endothelial cells to form capillary network structure, and accelerate recovery of blood flow of an ischemia model. In fact, exosomes derived from MSC have entered the clinical trial stage for the treatment of diabetes and renal insufficiency. Therefore, the exosomes derived from MSC have potential practical value as materials for "cell-free" cell therapy. To improve the therapeutic effect, it is necessary to search for ways to obtain exosomes with stronger functions, and to develop clinical trial studies of MSC exosomes.
No special research has been done in the prior art on the promotion of stem cell exosome secretion.
The Chinese patent with application publication number of CN 106492194A discloses a stem cell exosome preparation, a preparation method and application thereof, wherein the adopted cytokine is only basic fibroblast growth factor, the prepared preparation is used as supplement of exosomes and is additionally added into exosomes, the dental pulp stem cell + exosomes + basic fibroblast growth factor (10 ng/ml) is used as a mixed preparation for treating gastritis, no special research is carried out on the promoting factors secreted by the stem cell exosomes, and no stem cell culture capable of being used for skin is involved.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art and provide the application of cytokines in the preparation of the stimulant, wherein the stimulant promotes the secretion of exosomes by dental pulp stem cells.
The above object of the present invention is achieved by the following technical scheme:
the present invention provides the use of a cytokine in the preparation of a stimulator for promoting secretion of exosomes by dental pulp stem cells. The exosome can be particularly used for preparing anti-skin aging medicines and the like.
Optionally, the cytokine is selected from at least one of Epidermal Growth Factor (EGF), insulin-like growth factor No. 1 (IGF-1), human platelet-derived growth factor BB (PDGF-BB), stem cell growth factor (SCF), and gamma-interferon (IFN-gamma).
Preferably, the cytokine is a combination of Epidermal Growth Factor (EGF), human platelet-derived growth factor BB (PDGF-BB).
Preferably, the mass ratio of the epidermal growth factor to the human platelet-derived growth factor BB is 1:1.
the invention also provides a preparation method of the exosome, which comprises the following steps: and adding the cell factor into the culture medium to prepare a culture medium containing the cell factor, and adding the dental pulp stem cells into the culture medium for culture to obtain exosomes secreted by the dental pulp stem cells.
Optionally, the cytokine is selected from at least one of Epidermal Growth Factor (EGF), insulin-like growth factor No. 1 (IGF-1), human platelet-derived growth factor BB (PDGF-BB), stem cell growth factor (SCF), and gamma-interferon (IFN-gamma).
Preferably, the cytokine is a combination of Epidermal Growth Factor (EGF), human platelet-derived growth factor BB (PDGF-BB).
Preferably, the mass ratio of the epidermal growth factor to the human platelet-derived growth factor BB is 1:1.
alternatively, the concentration of the epidermal growth factor in the medium is 1-20ng/mL, preferably 10-20ng/mL, including but not limited to 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, 10ng/mL, 11ng/mL, 12ng/mL, 13ng/mL, 14ng/mL, 15ng/mL, 16ng/mL, 17ng/mL, 18ng/mL, 19ng/mL, 20ng/mL, etc.
Alternatively, the insulin-like growth factor number 1 is present in the medium at a concentration of 1-100ng/mL, including but not limited to 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL.
Alternatively, the human platelet-derived growth factor BB is present in the medium at a concentration of 1-50ng/mL, including but not limited to 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL.
Alternatively, the concentration of the stem cell growth factor in the medium is 1-50ng/mL, including but not limited to 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL.
Alternatively, the concentration of the gamma interferon in the medium is 1-50ng/mL, including but not limited to 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL.
Alternatively, the medium includes a medium for promoting growth of dental pulp stem cells, and the medium for promoting growth of dental pulp stem cells may be, in particular, MSC serum-free medium, which is commercially available.
Optionally, the medium further comprises physiological saline for culturing exosomes.
Alternatively, the dental pulp stem cells are added to the medium for a period of 72 hours.
Optionally, after the culture is completed, collecting supernatant, and centrifuging to obtain the exosomes.
The invention provides the exosome prepared by the method.
The invention also provides a preparation containing the exosome, which is used for promoting fibroblast proliferation and/or promoting angiogenesis so as to play a role in resisting skin aging.
The invention has the following beneficial effects:
the invention takes cytokines as the stimulant, the surfaces of dental pulp stem cells all express 5 factor receptors selected by the invention, the 5 cytokines can stimulate proliferation, differentiation, migration and other processes of dental pulp stem cells, the processes of cell proliferation, differentiation, migration and the like are all processes of cell activation and vigorous cell metabolism, and the cells can secrete a large amount of exosomes in an activated state. Therefore, after 5 cytokines are stimulated, the dental pulp stem cells are in an activated state, so that the secretion of exosomes of the dental pulp stem cells is remarkably improved, and the obtained exosomes can be used for anti-aging or other medical purposes and have a wide market value.
Drawings
FIG. 1 shows a photograph (50X) of the cell morphology of human dental pulp stem cells of the example of the present invention.
Fig. 2 and 3 are graphs showing the results of flow cytometry analysis of human dental pulp stem cells according to an embodiment of the present invention.
Fig. 4 shows a morphological observation (transmission electron microscopy) of an exosome according to an embodiment of the invention.
FIG. 5 shows a Western blot detection result diagram of exosomes according to an embodiment of the invention.
FIG. 6 is a graph showing the effect of different cytokines on fibroblast proliferation of exosomes secreted by dental pulp stem cells according to the examples of the present invention.
FIG. 7 is a graph showing the effect of different cytokines on vascular endothelial cells to form capillary-like structures.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The invention selects dental pulp stem cells, and has the following advantages:
1) The source is rich.
Teeth naturally shed by children aged 6-12 are rich in dental pulp stem cells in wisdom teeth that adults need to pluck.
2) Has small side effects.
Dental pulp stem cells are mesenchymal stem cells, have the universality of the mesenchymal stem cells, have low immunogenicity, can be used without strict pairing, cannot cause strong rejection reaction, have an immunoregulatory function, and cannot cause rejection reaction after allograft transplantation.
3) There is no ethical dispute.
The dental pulp stem cells are taken from the teeth naturally fallen by children and the wisdom teeth of adults, belong to waste articles, do not damage life, and have no ethical disputes.
Dental pulp stem cells are capable of secreting exosomes by paracrine means. Exosomes are small vesicles 30-100nm in diameter secreted by a variety of cells, the outer membrane consisting mainly of lipids and proteins, expressing the specific markers CD63, CD9 and CD81, while expressing some of the surface markers of the cells from which it is derived. Exosomes carry a variety of functional proteins, lipids, DNA, RNA, micrornas (mirnas), etc., that mediate communication between cells. The exosomes carry bioactive substances of stem cells from which the exosomes are derived, exert similar functions of the stem cells, and can avoid potential risks of direct transplantation of the stem cells; the exosomes can enter tissue cells through skin, mucous membrane and blood brain barrier, and can be prevented from being cleared by a filter tube; the exosomes can be artificially modified, and can be used as a drug and a drug targeting delivery carrier, thereby being beneficial to storage, transportation and industrialized production.
Exosomes are substances that have been shown to penetrate the stratum corneum of the skin, and we speculate that pulp stem cell exosomes contain a large amount of anti-aging factors and can penetrate into the dermis layer of the skin to exert an anti-aging effect. The exosomes carry bioactive substances of dental pulp stem cells, exert similar functions of the stem cells, and avoid potential risks of direct transplantation of the stem cells; meanwhile, the exosomes can enter tissue cells through skin, mucous membrane and blood brain barrier, and can be prevented from being cleared by a filtering organ; the exosomes can also be artificially modified to be used as drugs and drug targeting delivery carriers; and compared with stem cells, the exosomes are more beneficial to storage, transportation and industrial production.
In view of this, the present invention has been made. The present invention relates to a method for increasing the number of exosomes secreted by dental pulp stem cells, which increases the number of exosomes released by MSCs while obtaining more cytokines against skin aging, having a greater capacity to promote fibroblast proliferation and angiogenesis, by adding specific cytokines, preferably egf+pdgf-BB, when culturing dental pulp stem cells. The invention provides a powerful tool for applying the dental pulp stem cells to clinic.
The present invention provides a method for culturing dental pulp stem cell exosomes, which can obtain more exosomes.
Example 1
1. Main equipment
TABLE 1
2. Main reagent
TABLE 2
| Reagent name | Reagent manufacturer |
| MSC culture medium and additive | Beijing friend kang science and technology Co Ltd |
| ɑ-MEM | Beijing friend kang science and technology Co Ltd |
| FBS (fetal bovine serum) | Gibco |
| Exosome CD63&TSG101 protein detection kit | SHANGHAI UMIBIO SCIENCE AND TECHNOLOGY Co.,Ltd. |
| MSC stem cell flow analysis reagent | BD |
| Epidermal Growth Factor (EGF) | BEIJING T&L BIOTECHNOLOGY Co.,Ltd. |
| Insulin-like growth factor (IGF-1) | BEIJING T&L BIOTECHNOLOGY Co.,Ltd. |
| Platelet derived growth factor (PDGF-BB) | BEIJING T&L BIOTECHNOLOGY Co.,Ltd. |
| Stem Cell Factor (SCF) | BEIJING T&L BIOTECHNOLOGY Co.,Ltd. |
| Recombinant human interferon-gamma (IFN-gamma) | BEIJING T&L BIOTECHNOLOGY Co.,Ltd. |
| PBS | Gibco |
| Disperse enzyme | Gibco |
| Collagenase enzyme | Gibco |
| Elisa kit of VEGF, TGF-beta, IL-6 and bFGF | Beijing Crystal America technologies Co.Ltd |
| BCA protein concentration determination kit | Beijing Crystal America technologies Co.Ltd |
| 0.25% trypsin | Gibco |
3. The experimental steps are as follows:
3.1 dental pulp Stem cell isolated culture
3.1.1 tooth surface cleaning
The collected healthy and intact teeth are soaked in 75 volume percent alcohol for 1min, and then rinsed with sterile physiological saline for 2 to 3 times. The scissors scratch, remove tissue scraps (especially at the tooth root) on the tooth surface, and repeatedly rinse.
3.1.2 dental pulp removal
The treated teeth were placed on the flat side of the hammer and struck with another hammer to expose the pulp cavity. 2mL of each of the prepared collagenase and dispase in a volume ratio of 0.2% and 0.4% was mixed. The teeth and fragments exposing the pulp cavity were placed in the mixed solution and the shaker was shaken at a low speed (100 r/min) at 37℃for 1h.
3.1.3 cell culture
The digested cell pellet is re-blown to form a single cell suspension. Sieving with a cell sieve, transferring cell sap to another centrifuge tube, and centrifuging at 1500r/min for 5min. Suspending MSC culture medium, inoculating, adding volume ratio of5% of additive. 37 ℃ 5% CO 2 Culturing in an incubator, and taking P3-P5 generation cells with good growth state for experiment after the cell fusion degree reaches 80% and carrying out digestion and passage by 0.25% trypsin.
3.1.4 when the cell fusion degree reaches 80% -90%, removing the culture supernatant, replacing with equal volume of physiological saline, adding A-F group stimulating factors, continuously culturing for 72 hours, and collecting the supernatant.
The addition of each group of stimulus factors was as follows:
a: the method is not added;
b: adding 10ng/mL EGF;
c: adding 100ng/mL IGF-1;
d: adding 50ng/mL PDGF-BB;
e: adding 50ng/mL SCF;
f: 50ng/mL IFN-. Gamma.was added.
The concentration of each of the above-mentioned stimulating factors means the final concentration of each stimulating factor in the medium.
3.1.5 exosome collection, density gradient centrifugation collection exosome, specifically carried out according to the following steps in turn:
1) Cells were removed by centrifugation at 300g for 10 min.
2) Centrifugation at 2000g for 10min removed cell debris.
3) Centrifugation at 10000g for 30min, and small cell debris is removed.
4) The exosomes were collected by centrifugation at 40000g for 20min in a 100KD ultrafiltration tube.
4. Detection of
4.1 identification of dental pulp Stem cells
4.1.1 dental pulp stem cell morphology photographs (50×) were taken, see fig. 1.
4.1.2 analysis of surface markers of dental pulp stem cells using flow-through.
Flow assay of 8 antigens from dental pulp mesenchymal stem cells: CD73, CD90, CD105, HLA-DR, CD45, CD19, CD34 and CD11b, and the results of the flow assays are shown in FIGS. 2 and 3.
4.2 identification of exosomes
4.2.1 viewing exosome morphology and size under transmission electron microscopy: taking 10 microliters of separated and purified exosomes, wherein the volume ratio=1: 1 adding 4 ℃ PBS solution for dilution, then dripping the diluted solution onto a 2mm sample-carrying copper net, standing at room temperature for 1min, then lightly sucking the solution by using filter paper pulp, carrying out negative dyeing for 5min at room temperature by using 3% (W/V) sodium phosphotungstate solution (PH 6.8), lightly washing half the solution by using double distilled water, airing at room temperature, observing and photographing by using a transmission electron microscope, measuring the diameter of the solution, and obtaining a transmission electron microscope result shown in figure 4.
4.2.2Western-blot detection of exosome Membrane surface characteristic protein expression
Exosome CD63& TSG101 protein detection kit is adopted to detect exosome characteristic protein expression. Dental pulp stem cells and exosome samples are collected, protein is extracted by lysing the samples by using cell lysate, 10% SDS-PAGE gel electrophoresis is carried out according to a standard method, the samples are transferred to a PVDF membrane, and after the samples are blocked for 1h by using a skim milk powder blocking solution with the volume fraction of 5%, CD63 and TSG101 primary antibodies are respectively added for incubation at 4 ℃ for overnight. The membranes were washed 3 times per day for 10min with TBST, reacted with HRP-labeled secondary antibodies for 50min at room temperature, washed 3 times per day for 10min, and then developed with chemiluminescent substrate.
4.3 determination of exosome protein concentration. The number of cells, which is the total number of cells finally obtained, and the protein expression amount thereof, which is the total amount of the obtained exosome protein, were calculated. Thus, it was determined which cytokines stimulated the greatest secretion of exosomes without affecting the proliferation of the cells.
A: no stimulant (as negative control);
b: adding EGF to stimulate the produced exosomes;
c: adding IGF-1 to stimulate the produced exosomes;
d: adding PDGF-BB to stimulate the generated exosomes;
e: adding SCF to stimulate exosomes;
f: exosomes produced by IFN-gamma stimulation are added.
4.4ELISA method for determining VEGF, IL-6, TGF and bFGF content in exosomes.
A: no stimulant (as negative control);
b: adding EGF to stimulate the produced exosomes;
c: adding IGF-1 to stimulate the produced exosomes;
d: adding PDGF-BB to stimulate the generated exosomes;
e: adding SCF to stimulate exosomes;
f: exosomes produced by IFN-gamma stimulation are added.
4.5 functional identification of exosomes secreted by dental pulp stem cells, facilitating fibroblast (HF-1) proliferation experiments.
HF-1 cells (available from Shanghai Ji Ning Utility Co., ltd.) were cultured with alpha-MEM containing 10% FBS in a volume ratio, and the cells were counted after digestion and collection and suspended in alpha-MEM containing 10% FBS. The cells were inoculated in 96-well plates for MTT assay, 1000 cells/well. The grouping is as follows:
a: 10. Mu.g/mL of the supernatant without the addition of the stimulant (as a negative control);
b: adding 10 mug/mL of exosomes produced by EGF stimulation;
c: adding 10 mug/mL of exosomes produced by IGF-1 stimulation;
d: adding 10 mug/mL of exosomes produced by PDGF-BB stimulation;
e: adding 10 mug/mL of exosomes produced by SCF stimulation;
f: adding 10 mug/mL of exosomes produced by IFN-gamma stimulation;
g: the resulting exosomes were stimulated with EGF+PDGF-BB at 10 μg/mL.
It was determined which cytokine-stimulated exosomes had better function in stimulating cell proliferation.
The effect of different groups of exosomes on proliferation of HF-1 cells was observed by MTT assay, HF-1 cells were seeded in 96 well plates according to 1000 cells/well, 10 μg/mL of exosomes treated in groups A-G were added to the culture system, 3 multiplex wells per group, respectively. After 72 hours of incubation, MTT was added and incubation continued for 4 hours. After that, dimethyl sulfoxide (DMSO) was added, and the optical density at 490nm was measured.
4.6 experiments of pulp Stem cell exosomes promoting capillary-like Structure formation
Matrigel experiments are classical methods for observing endothelial cell function, especially capillary network formation, the results of which represent in vivo neovascularization activity. Endothelial cells were inoculated into Matrigel-plated plates, and the culture system was added with 10. Mu.g/mL of exosomes treated with Untreated, EGF, IGF-1, PDGF-BB, SCF, IFN-gamma, EGF+PDGF-BB stimulation groups, respectively, and the number of reticulocytes in each high-power field was counted after 24 hours.
5. Results
5.1 isolation, expansion and identification of human dental pulp stem cells.
5.1.1 figure 1 shows a photograph of the cell morphology of human dental pulp stem cells.
5.1.2 FIGS. 2 and 3 show graphs of results of flow cytometry analysis of human dental pulp stem cells, according to the minimum standard for mesenchymal stem cell identification issued by International cell therapy Association (ISCT) in 2006, the expression of three surface antigens CD73, CD90, CD105 is not less than 95%; CD34, CD45, CD11b, CD19, HLA-DR is expressed by not more than 2.0%. The control group is a isotype control corresponding to 8 antigens of dental pulp mesenchymal stem cells, and the flow result of the control group is shown in fig. 2. The sample group is the expression condition of 8 antigens, the flow result of the sample group is shown in figure 3, and the expression of CD73, CD90 and CD105 of the sample group is higher than 95.0%; the negative markers (HLA-DR, CD45, CD19, CD34, CD11 b) of the sample group expressed less than 2.0% and were consistent with the surface antigen characteristics of stem cells, indicating that dental pulp stem cells did not differentiate and still maintained the characteristics of stem cells.
5.2A large number of exosomes were collected from pulp stem cells and identified.
5.2.1 the exosome size and morphology were observed using transmission electron microscopy, and fig. 4 shows a transmission electron microscopy image of the exosome. The exosomes are 30-120nm in size, most of the exosomes are 30-60nm in double-layer structure, are hollow cystic structure, and accord with morphological characteristics of exosomes.
5.2.2 detection of the expression of the exosome membrane surface characteristic proteins CD63 and TSG101 by using Western-blot, and the result of the exosome Western blot detection is shown in FIG. 5, the exosome specific markers CD63 and TSG101 are clearly visible, and the pulp stem cell group does not express the CD63 and TSG101 antibodies.
5.3Elisa method for measuring protein content in exosomes
Exosome eggs of each groupAs shown in Table 3, when a certain stimulant was used alone, the two groups with the highest protein content were group B and group D, namely EGF and PDGF-BB, and therefore, we tried to combine EGF and PDGF-BB, and found that the exosome protein content was significantly increased as high as 2507.+ -.733. Mu.g/10 as group G 8 Individual cells.
TABLE 3 Table 3
| Group of irritants | Exosome stimulators | Exosome protein content |
| A | Non-stimulated group | 222±115μg/10 8 |
| B | EGF | 830±265μg/10 8 |
| C | IGF-1 | 556±124μg/10 8 |
| D | PDGF-BB | 901±262μg/10 8 |
| E | SCF | 656±229μg/10 8 |
| F | IFN-γ | 256±134μg/10 8 |
| G | EGF+PDGF-BB | 2507±233μg/10 8 |
5.4
The Elisa method is used for measuring the content of factors such as VEGF, IL-6, TGF, bFGF and the like, and determining the optimal stimulating factors as EGF and PDGF-BB.
TABLE 4 Table 4
| Group of | Stimulant | VEGF content | bFGF content | TGF content | IL-6 content |
| A | Non-stimulated group | 1165±171pg/mL | 1296±187pg/mL | 1301±194pg/mL | 2271±128pg/mL |
| B | EGF | 1888±263pg/mL | 1778±219pg/mL | 1969±275pg/mL | 3630±265pg/mL |
| C | IGF-1 | 1514±231pg/mL | 1579±209pg/mL | 1692±236pg/mL | 2589±135pg/mL |
| D | PDGF-BB | 2218±372pg/mL | 2015±294pg/mL | 2560±213pg/mL | 3781±177pg/mL |
| E | SCF | 1609±182pg/mL | 1701±124pg/mL | 1757±165pg/mL | 3856±187pg/mL |
| F | IFN-γ | 1232±208pg/mL | 1374±124pg/mL | 2012±128pg/mL | 3002±134pg/mL |
| G | EGF+PDGF-BB | 2912±397pg/mL | 2809±625pg/mL | 3011±462pg/mL | 4505±729pg/mL |
The statistical results of the VEGF, IL-6, TGF and bFGF factor contents of each group are shown in Table 4, when a certain stimulator is used alone, the VEGF, IL-6, TGF and bFGF factor contents are obviously higher than those of the B group and the D group, namely the EGF, PDGF-BB group and the exosome protein contents, meanwhile, the VEGF, IL-6, TGF and bFGF factor contents of the G group are tested, and the result is obviously higher than those of the A-F group, and still is positively correlated with the exosome protein contents.
5.5 verification that each of the stimulatory factors EGF, IGF-1, PDGF-BB, SCF, IFN-gamma, EGF+PDGF-BB stimulated cultured pulp stem cell exosomes have better fibroblast (HF-1) proliferation stimulating function.
FIG. 6 is a graph showing the effect of different cytokines on proliferation of fibroblasts, wherein each of the stimulating factors Untreated, EGF, IGF-1, PDGF-BB, SCF, IFN-gamma and EGF+PDGF-BB was added to the fibroblasts (HF-1) to stimulate the cultured pulp stem cell exosomes, and the proliferation was observed in the MTT assay, with the ordinate OD490nm. Untreated is an experimental group without the addition of the stimulator, and the result shows that the OD value of the addition factor group is obviously higher than that of the Untreated group (P < 0.05), and the treatment group of the mixed factor group (EGF+PDGF-BB) is slightly higher than that of the single factor treatment. The results show that the exosomes released by the dental pulp stem cells after being treated by adding the factors EGF, IGF-1, PDGF-BB, SCF, IFN-gamma and EGF+PDGF-BB can obviously promote the proliferation of fibroblasts (HF-1) in vitro, and the exosomes released by the dental pulp stem cells after being stimulated by EGF+PDGF-BB has stronger HF-1 proliferation promoting capacity.
5.6 effects of different cytokines on vascular endothelial cells to form capillary-like structures by exosomes secreted by dental pulp stem cells.
FIG. 7 shows the effect of exosomes stimulated by different cytokines on vascular endothelial cells to form capillary-like structures, showing that endothelial cells of the group without any stimulation (Untreated group), EGF, IGF-1, PDGF-BB, SCF, IFN-gamma, EGF+PDGF-BB have a number of reticulocytes per field of view of 2.3.+ -. 1.2, 9.4.+ -. 0.89, 7.+ -. 1.42, 8.3.+ -. 1.33, 7.+ -. 0.54, 8.+ -. 1.15, 10.+ -. 1.06, respectively, with limited lumen forming capacity without any stimulation (Untreated group); the endothelial cells forming lumen were significantly higher in the cell growth factor treated group than in the untreated group, and the mixed factor (EGF+PDGF-BB) treated group was higher than in the single factor treated group. The results show that the exosomes released by the dental pulp stem cells after EGF+PDGF-BB combined stimulation have stronger angiogenesis promoting capacity.
In conclusion, the high-concentration exosomes (the content of the basic fibroblast growth factor is 2000 ng/mL) produced by the invention can directly act on the skin. The invention takes the cell factor as the stimulator, obviously improves the secretion of the exosomes of the stem cells, and the obtained exosomes can be used for anti-aging or other medical purposes and have wide market value.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. Use of a cytokine in the preparation of a stimulant, wherein the stimulant is used to promote secretion of exosomes by dental pulp stem cells; wherein the cytokine is insulin-like growth factor I.
2. A method for preparing exosomes, comprising the steps of: adding cell factors into a culture medium to prepare a culture medium containing the cell factors, and adding dental pulp stem cells into the culture medium for culture to obtain exosomes secreted by the dental pulp stem cells; wherein the cytokine is insulin-like growth factor I.
3. The method according to claim 2, wherein the concentration of the insulin-like growth factor I in the medium is 1 to 100ng/mL.
4. The method of claim 2, wherein the medium comprises a medium for promoting growth of dental pulp stem cells and physiological saline.
5. The method according to claim 2, wherein the exosomes are obtained by collecting the supernatant after the completion of the culture and centrifuging.
6. An exosome produced by the production method according to any one of claims 2 to 5.
7. A formulation comprising the exosome of claim 6, wherein the formulation is for promoting fibroblast proliferation and/or promoting angiogenesis.
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