CN112795535B - Composition for inducing mesenchymal stem cells to release exosomes with specific functions for promoting differentiation of skin epithelial cells and application of composition - Google Patents
Composition for inducing mesenchymal stem cells to release exosomes with specific functions for promoting differentiation of skin epithelial cells and application of composition Download PDFInfo
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- CN112795535B CN112795535B CN202110046906.7A CN202110046906A CN112795535B CN 112795535 B CN112795535 B CN 112795535B CN 202110046906 A CN202110046906 A CN 202110046906A CN 112795535 B CN112795535 B CN 112795535B
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Abstract
The invention discloses a composition for inducing mesenchymal stem cells to release exosomes with specific functions for promoting differentiation of skin epithelial cells and application thereof. The composition comprises a ROCK inhibitor, an epidermal growth factor and an active peptide. The specific function exosome has at least one of the following functions: 1) promoting differentiation of skin fibroblasts into epithelial cells, 2) promoting repair of photodamaged skin and/or promoting repair of photoaging damaged skin, 3) resisting photodamage and/or photoaging damage of skin, and 4) improving oxidation resistance of skin. The exosome with the specific function can be non-invasively introduced into injured skin cells and tissues by skin epidermis smearing or needleless injection, participates in skin repair, has the effects of preventing and/or treating photodamage aging, and can become a biological medicine for cell-free replacement therapy of skin rehabilitation.
Description
Technical Field
The invention relates to the field of biomedicine, and relates to a composition for inducing mesenchymal stem cells to release exosomes with specific functions for promoting differentiation of skin epithelial cells and application thereof.
Background
Exosomes (exosomes) are end products of the endocytic process of cells, structured by organelles formed by lipid bilayer membranes. All cells in the body have the function of producing and secreting exosomes. Generally, cells form endocytic vesicles from exogenous substances into cytoplasm through endocytosis, and form early endosomes (early endosomes) through budding under the action of organelles such as golgi, etc., selectively receive bioactive components such as proteins, nucleic acids, lipids, etc. in cells, budding again to form late endosomes (late endosomes), finally form multivesicular bodies (MVB), fuse with cell membranes, discharge the vesicles in the form of exocytosis, and are called exosomes with the diameter of about 30-150 nm. The exosome contains components similar to the source cell and is rich in protein, active polypeptide, lipid and nucleic acid molecules such as mRNAs, miRNAs, lncRNAs and the like. The exosome can mediate intracellular signal regulation through communication among cells, directly activates target cells and enables the target cells to generate a series of biological effects. Exosomes have a homing mechanism to change the microenvironment of damaged tissues and repair the damage.
Mesenchymal Stem Cells (MSCs) come from a variety of tissues including bone marrow, adipose tissue, dental pulp, cord blood, amniotic fluid, placenta, amniotic membrane, umbilical cord Wharton's jelly, and the like. Mesenchymal stem cells of various origins have the capacity of clonal growth, self-renewal and pluripotent differentiation. The mesenchymal stem cells can be differentiated into cells of corresponding tissues to repair damaged tissues and have a substitution effect. The mesenchymal stem cells also have the effect of paracrine factors, and can promote the self-repair of damaged tissues. The mesenchymal stem cells have low immunogenicity and immunoregulation function, and have clinical application safety. Therefore, there have been a lot of research reports on the treatment of skin injury by mesenchymal stem cells from various sources, including skin wound healing, skin cut wound healing, treatment of skin burn, and the like. However, since the size of the mesenchymal stem cells is between 10-25 μm, it is impossible to non-invasively penetrate the membrane, and there is a limitation in the application process of the skin.
The exposed surface of the skin is easily damaged by environmental factors such as dryness, ultraviolet rays and toxic substances, and the quality of life is affected by skin problems such as aging, dermatitis and eczema caused by damaged skin. Exosomes secreted by mesenchymal stem cells are based on nanoscale (30-150nm) size characteristics, non-immunogenicity, non-tumorigenicity, biological functions similar to those of stem cells and the like, and a large number of research reports are also available. The research reports of obtaining the cell release exosome with specific functions by adopting methods such as transgenosis, hypoxia, small molecule induction and the like are not repeated.
Although the skin is an organ with strong regeneration capability, the intervention and treatment of stem cell-derived exosomes with specific functions are still needed for a plurality of problems such as photoaging damage, age-related skin thinning and relaxation, telangiectasia, wrinkle formation and the like. The exosome can be non-invasively introduced into injured skin cells and tissues through needle-free injection to participate in the repair of the skin, and is expected to become a cell-free therapeutic biological medicine for skin rehabilitation.
Disclosure of Invention
The technical problem to be solved by the invention is how to induce the mesenchymal stem cells to release exosomes with specific functions. The specific function exosome has at least one of the following functions: 1) promoting differentiation of skin fibroblasts into epithelial cells, 2) promoting repair of photodamaged skin and/or promoting repair of photoaging damaged skin, 3) resisting photodamage and/or photoaging damage of skin, and 4) improving oxidation resistance of skin.
In order to solve the above technical problems, the present invention provides a composition for inducing mesenchymal stem cells to release exosomes.
The composition for inducing the mesenchymal stem cells to release exosomes comprises a selective Rho-associated coiled coil-forming protein kinase (ROCK) inhibitor, an Epidermal Growth Factor (EGF) and an active peptide.
The above composition for inducing the release of exosomes from mesenchymal stem cells may further comprise basic fibroblast growth factor (bFGF) and/or Polyvinyl alcohol (PVA).
The composition for inducing the mesenchymal stem cells to release exosomes can be an adherent culture composition or a suspension culture composition; the adherent culture composition can be adherent culture composition a1 or adherent culture composition a2, the adherent culture composition a1 consisting of the ROCK inhibitor, epidermal growth factor and the active peptide, the adherent culture composition a2 consisting of the adherent culture composition a1 and polyvinyl alcohol;
the suspension culture composition can be suspension culture composition B1 or suspension culture composition B2 or suspension culture composition B3, the suspension culture composition B1 is composed of the ROCK inhibitor, the epidermal growth factor, the basic fibroblast growth factor and the active peptide, the suspension culture composition B2 is composed of the suspension culture composition B1 and polyvinyl alcohol, and the suspension culture composition B3 is composed of the suspension culture compositions B2 and B27.
In the above composition for inducing the release of exosomes from mesenchymal stem cells, the active peptide may be at least one of: palmitoyl pentapeptide-3 (palmitoyl pentapeptide-3, palmitoyl-KTTKS, pal-KTTKS), Argireline (Argireline), and ceruloplasmin (Copper Peptide, GHK-Cu), and/or the ROCK inhibitor may be Y-27632, and/or the polyvinyl alcohol may be a polyvinyl alcohol having a degree of hydrolysis of 87-90%.
In the composition for inducing the release of exosomes from the mesenchymal stem cells, the ratio of each component can be determined by those skilled in the art according to the effect of inducing the release of exosomes from the mesenchymal stem cells, for example, the ratio of the ROCK inhibitor, EGF, bFGF, the active peptide and the polyvinyl alcohol can be 1-10 μmol or 5 μmol of the ROCK inhibitor: EGF 10-100 μ g or 20-50 μ g or 50 μ g: 10-100 μ g or 20-50 μ g or 20 μ g of bFGF: said active peptide is 0.01-100 μmol, 0.1-50 μmol, 0.1-10 μmol, 0.1-7 μmol, 0.5-5 μmol, 7 μmol, 5 μmol or 1 μmol: 50-8000mg, 100-8000mg, 1000mg or 8000mg of the polyvinyl alcohol; the proportion of the ROCK inhibitor, the EGF, the bFGF, the active peptide and the polyvinyl alcohol can also be 1-10 mu mol or 5 mu mol: EGF 10-100 μ g or 20-50 μ g or 50 μ g: bFGF10-100ng or 20-50ng or 20 ng: said active peptide is 0.01-100 μmol, 0.1-50 μmol, 0.1-10 μmol, 0.1-7 μmol, 0.5-5 μmol, 7 μmol, 5 μmol or 1 μmol: the polyvinyl alcohol accounts for 1g or 8 g.
The composition for inducing the mesenchymal stem cells to release exosomes may be any one of the following compositions:
c1 and the adherent culture composition A1, wherein the proportion of the ROCK inhibitor, the EGF and the active peptide is 1-10 mu mol or 5 mu mol of the ROCK inhibitor: EGF 10-100 μ g or 20-50 μ g or 50 μ g: the active peptide is 0.01-100 mu mol, 0.1-50 mu mol, 0.1-10 mu mol, 0.1-7 mu mol, 0.5-5 mu mol, 7 mu mol, 5 mu mol or 1 mu mol;
c2 and the adherent culture composition A1, wherein the proportion of the ROCK inhibitor, the EGF and the active peptide is that the ROCK inhibitor is 5 mu mol: EGF 10-100 μ g or 20-50 μ g or 50 μ g: said active peptide is 0.01-100 μmol, 0.1-50 μmol, 0.1-10 μmol, 0.1-7 μmol, 0.5-5 μmol, 7 μmol, 5 μmol or 1 μmol;
c3 and the adherent culture composition A2, wherein the ratio of the adherent culture composition A1 to the polyvinyl alcohol meets the following requirements that the ROCK inhibitor is 1-10 mu mol or 5 mu mol: 50-8000mg, 100-1000mg or 1000mg of polyvinyl alcohol;
c4 and the adherent culture composition A2, wherein the proportion of the adherent culture composition A1 to the polyvinyl alcohol meets the requirement that the ROCK inhibitor is 5 mu mol: 50-5000mg, 100-25000mg or 1000mg of polyvinyl alcohol; c5 and the suspension culture composition B1, wherein the mixture ratio of the ROCK inhibitor, the EGF, the bFGF and the active peptide is 1-10 mu mol or 5 mu mol of the ROCK inhibitor: EGF 10-100 μ g or 20-50 μ g or 50 μ g: 10-100ng or 20-50ng or 20ng of bFGF: said active peptide is 0.01-100. mu. mol, 0.1-50. mu. mol, 0.1-10. mu. mol, 0.1-7. mu. mol, 0.5-5. mu. mol, 7. mu. mol, 5. mu. mol or 1. mu. mol;
c6 and the suspension culture composition B1, wherein the proportion of the ROCK inhibitor, the EGF, the bFGF and the active peptide is that the ROCK inhibitor is 5 mu mol: EGF 10-100 μ g or 20-50 μ g or 50 μ g: 10-100 μ g or 20-50 μ g or 20 μ g of bFGF: said active peptide is 0.01-100 μmol, 0.1-50 μmol, 0.1-10 μmol, 0.1-7 μmol, 0.5-5 μmol, 7 μmol, 5 μmol or 1 μmol;
c7 and the suspension culture composition B2, wherein the proportion of the suspension culture composition B1 to the polyvinyl alcohol meets the following conditions that the ROCK inhibitor is 1-10 mu mol or 5 mu mol: 8g of polyvinyl alcohol;
c8 and the suspension culture composition B2, wherein the proportion of the suspension culture composition B1 to the polyvinyl alcohol meets the following requirement that the ROCK inhibitor is 5 mu mol: 8g of polyvinyl alcohol;
c9 and the suspension culture composition B3, wherein the proportion of the suspension culture compositions B2 and B27 meets the following conditions that the ROCK inhibitor is 1-10 mu mol or 5 mu mol: the B2720 mL-100 mL;
c10 and the suspension culture composition B3, wherein the proportion of the suspension culture compositions B2 and B27 meets the requirement that the ROCK inhibitor is 5 mu mol: the B2720 ml.
In the composition for inducing the mesenchymal stem cells to release exosomes, PVA is required to be packaged separately, and other components can be mixed together or can be packaged separately.
In order to solve the above technical problems, the present invention provides a composition for preparing exosomes.
The composition for preparing exosome provided by the invention contains the composition for inducing the mesenchymal stem cell to release exosome and the isolated mesenchymal stem cell.
The composition for preparing exosome further comprises a mesenchymal stem cell basic culture medium. In one embodiment of the invention, the mesenchymal stem cell basal medium is adherent mesenchymal stem cell culture basal liquid-a. The adherent mesenchymal stem cell culture base liquid-A consists of a serum-free mesenchymal stem cell base culture liquid (Lonza, the code number of 12-725F) and a serum substitute (Ultroser G, the code number of Pall 15950-107). In the adherent mesenchymal stem cell culture base solution-A, the content of the serum substitute is 20 ml/L.
In another embodiment of the present invention, the mesenchymal stem cell basic medium is suspension mesenchymal stem cell culture base liquid B. The suspension mesenchymal stem cell culture base solution B consists of serum-free mesenchymal stem cell base culture solution (Lonza, the commodity number is 12-725F), serum substitute (Ultroser G, the commodity number is Pall 15950-107), B-27 and polyvinyl alcohol with the hydrolysis degree of 87-90%. In the suspension mesenchymal stem cell culture base solution B, the content of a serum substitute is 20ml/L, the content of B-27 is 20ml/L, and the content of polyvinyl alcohol with the hydrolysis degree of 87-90% is 8 g/L.
In the composition for preparing exosomes, the composition for preparing exosomes may be composed of the composition for inducing the mesenchymal stem cells to release exosomes, a mesenchymal stem cell basic culture medium and isolated mesenchymal stem cells.
In the composition for preparing exosomes, the composition for inducing the mesenchymal stem cells to release exosomes, the mesenchymal stem cell basic culture medium and the isolated mesenchymal stem cells are respectively and independently packaged.
Any of the following applications also fall within the scope of the present invention:
the application of P1, the composition for inducing the mesenchymal stem cells to release exosomes (including the composition used in combination with a mesenchymal stem cell culture base solution) or the composition for preparing exosomes in preparing products for preventing and/or treating photo-damage and/or photo-aging damage of skin;
p2, the application of the composition for inducing the mesenchymal stem cells to release the exosomes (including the composition used in combination with the mesenchymal stem cell culture base solution) or the composition for preparing the exosomes in preparing the medicines for the cell-free substitution treatment of skin rehabilitation,
p3, the application of the composition for inducing the mesenchymal stem cells to release the exosomes or the composition for preparing the exosomes in preparing the exosomes,
p4, the application of the composition for inducing the mesenchymal stem cells to release the exosome or the composition for preparing the exosome in promoting the differentiation of the skin fibroblasts to the epithelial cells,
the application of P5, the composition for inducing the mesenchymal stem cells to release the exosomes or the composition for preparing the exosomes in preparing products for promoting the differentiation of the skin fibroblasts into the epithelial cells.
In order to solve the above technical problems, the present invention provides a method for preparing exosomes.
The method for preparing the exosome comprises the step of culturing the isolated mesenchymal stem cells by using a culture medium containing the composition for inducing the mesenchymal stem cells to release the exosome to obtain the exosome.
In the above method, the composition is the adherent culture composition, and the culture is adherent culture. The composition is the suspension culture composition, and the culture is suspension culture. The culturing includes culturing the isolated mesenchymal stem cells with a medium containing the composition for 1-24 hours or 24 hours (induction culture). The culturing further comprises culturing the isolated mesenchymal stem cells with a medium containing the composition, followed by culturing with a medium not containing the composition for 24-72 hours.
In the above method, the method further comprises collecting a supernatant from the culture broth, and obtaining the exosome from the supernatant.
In the above method, obtaining exosomes from the supernatant comprises enriching, isolating and purifying exosomes.
In the above method, the culture medium containing the composition and the culture medium not containing the composition can be serum-free culture media.
In the above method, obtaining exosomes from the supernatant comprises collecting exosomes using an ultracentrifugation method.
Hereinbefore, the exosome has at least one of the following functions:
1) promoting the differentiation of skin fibroblasts to epithelial cells,
2) promoting the repair of photodamaged skin and/or promoting the repair of photoaging damaged skin,
3) against photodamage and/or photoaging damage of the skin,
4) improving the oxidation resistance of the skin.
The improving the skin antioxidant capacity can be improving the activity of skin antioxidant enzyme. The antioxidant enzyme can be Superoxide Dismutase (SOD), Catalase (CATalase), and/or Glutathione peroxidase (GSH-PX).
The exosomes prepared by the above method also belong to the protection scope of the present invention.
The product may be a skin care product, a cosmetic product and/or a pharmaceutical product, as hereinbefore described.
As described above, the mesenchymal stem cells may be derived from mesenchymal stem cells of an accessory structure (including umbilical cord, placenta, amniotic fluid) delivered from a fetus, adipose tissue, subcutaneous connective tissue, and the like. That is, the mesenchymal stem cell may be an umbilical cord mesenchymal stem cell, a placental mesenchymal stem cell, an amniotic fluid mesenchymal stem cell, an adipose mesenchymal stem cell, or a mesenchymal stem cell derived from a subcutaneous connective tissue.
As above, the ROCK inhibitor may be Y-27632. Y-27632 can be used at a concentration of 1-10. mu. mol/L or 5. mu. mol/L.
As above, EGF may be used at a concentration of 10ng/ml to 100ng/ml, or 20ng/ml to 50ng/ml, or 50 ng/ml.
Hereinbefore, bFGF can be used in a concentration of 10ng/ml to 100ng/ml or 20ng/ml to 50ng/ml or 20 ng/ml.
As described above, palmitoyl pentapeptide-3 may be used at a concentration of 1-5. mu. mol/L or 5. mu. mol/L.
As described above, the hexapeptide may be used at a concentration of 0.01 to 100. mu. mol/L, 0.1 to 10. mu. mol/L, 0.5 to 5. mu. mol/L, or 1. mu. mol/L.
As described above, the use concentration of the bluecopper peptide may be 0.1 to 10. mu. mol/L, 0.5 to 5. mu. mol/L or 1. mu. mol/L.
As mentioned above, the Polyvinyl alcohol (PVA) may be a Polyvinyl alcohol having a degree of hydrolysis of 87 to 90%, abbreviated as 87 to 90% hydrolyzed Polyvinyl alcohol. The 87-90% hydrolyzed polyvinyl alcohol can be used at a concentration of 0.1% -1.0%, 0.1% -0.8%, 0.1%, or 0.8%.
Experiments prove that the composition for inducing the mesenchymal stem cells to release exosomes plays an important induction role in culturing the mesenchymal stem cells to generate and release exosomes for promoting the differentiation of skin epithelial cells and repairing skin injury. After the mesenchymal stem cells are induced for 24-48 hours, the mesenchymal stem cells can be activated to transdifferentiate into epithelial cells, so that the secretion of relevant cell active substances such as COL17A1, CK14, p63, integrin-beta 1 and the like is promoted. The composition for inducing the mesenchymal stem cells to release the exosomes of the invention realizes the production and secretion of the exosomes with specific functions by promoting the mesenchymal stem cells, and the exosomes with specific functions have at least one of the following functions: 1) promoting differentiation of skin fibroblasts into epithelial cells, 2) promoting repair of photodamaged skin and/or promoting repair of photoaging damaged skin, 3) resisting photodamage and/or photoaging damage of skin, and 4) improving oxidation resistance of skin. The invention aims at the characteristic that the skin has a compact barrier structure, and molecules generally larger than 1,500Da can not be absorbed by the skin, adopts a specific composition to induce mesenchymal stem cells to release exosomes with specific functions, the exosomes with specific functions not only have the particle size of 30-150nm, can be non-invasively introduced into damaged skin cells and tissues (non-invasively permeate the skin barrier) by skin epidermis smearing or needleless injection, participate in the repair of the skin, are not easy to cause immunoreaction, have the common advantages of biological activity of similar source cells and the like, but also have the functions of promoting the differentiation of skin epithelium, inducing the transdifferentiation of skin fibroblasts into skin epithelial-like cells, improving the microenvironment of the damaged skin, improving the activity of skin antioxidase, repairing the damaged skin, preventing and/or treating photodamage aging, and can become biological medicines for cell-free substitution treatment of skin rehabilitation, has important application prospect in the field of regenerative medicine.
Drawings
Fig. 1 is a ZetaView nanoparticle tracking analyzer (left panel) and transmission electron microscope image (right panel, arrows show exosomes are disc-shaped) of exosomes in cell culture supernatant after adherent mesenchymal stem cell induction of step 1.2 of example 1.
Fig. 2 shows the results of ZetaView nanoparticle tracking analyzer for detecting the exosome particle size (left) and the exosome morphology (right, arrow shows exosome is disc-shaped) in the induced suspension mesenchymal stem cell culture supernatant of step 1.2 of example 2 and transmitted electron ytterbium (right).
Fig. 3 is the results of Western Blot (WB) detection of inducing adherent mesenchymal stem cell exosomes in example 1 and inducing suspension mesenchymal stem cell exosomes to express their marker proteins HSP70, TSG101 and CD63 in example 2. In the figure, 1 is induced adherence mesenchymal stem cell protein, 2 is induced suspension mesenchymal stem cell protein, 3 is induced adherence mesenchymal stem cell exosome protein, and 4 is induced suspension mesenchymal stem cell exosome protein.
Fig. 4 is WB detection results of Mouse Embryonic Fibroblasts (MEFs) cultured in vitro treated by the adherent mesenchymal stem cell exosomes and the suspension mesenchymal stem cell exosomes induced to up-regulate expression of epithelial cell differentiation markers in example 4. In the figure, 1 is fibroblast before exosome induction, 2 is fibroblast after exosome treatment for inducing adherent mesenchymal stem cells, and 3 is fibroblast after exosome treatment for inducing suspension mesenchymal stem cells.
FIG. 5 shows mRNA expression of type I collagen and type III collagen in SD rats treated with skin photodamage by exosomes detected by qRT-PCR. In the figure, 1 is rat skin of a skin photoaging model, 2 is SD rat skin treated by inducing adherent mesenchymal stem cell exosome, and 3 is SD rat skin treated by inducing suspension mesenchymal stem cell exosome.
FIG. 6 shows that SOD activity detection exosomes prevent SD rat skin photodamage. Blank control represents control group treatment, photodamage represents skin photoaging model treatment, adherent Exo represents induced adherent mesenchymal stem cell exosome prevention treatment, and suspended Exo represents induced suspended mesenchymal stem cell exosome prevention treatment.
FIG. 7 shows that CAT activity assay exosomes prevent SD rat skin photodamage. Blank control represents control group treatment, photodamage represents skin photoaging model treatment, adherent Exo represents induced adherent mesenchymal stem cell exosome prevention treatment, and suspended Exo represents induced suspended mesenchymal stem cell exosome prevention treatment.
FIG. 8 shows that GSH-PX activity detection exosomes prevent SD rat skin photodamage. Blank control represents control group treatment, photodamage represents skin photoaging model treatment, adherent Exo represents induced adherent mesenchymal stem cell exosome prevention treatment, and suspended Exo represents induced suspended mesenchymal stem cell exosome prevention treatment.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Y-27632 in the following examples is Sigma-Aldrich, Cat. Y0503; EGF (epidermal growth factor) is a product of Perotech company, and the product number is AF-100-15; bFGF (basic fibroblast growth factor) is a product of Perotech company, and the product number is ZIKER-2235R; palmitoyl pentapeptide-3 (palmitoyl pentapeptide-3, palmitoyl-KTTKS, pal-KTTKS), hexapeptide (Argiline), and Copper-coated Peptide (conjugate Peptide, GHK-Cu) are products of Jile Biochemical (Shanghai) Limited; poly (vinyl alcohol) having a degree of hydrolysis of 87 to 90% is a product of Sigma-Aldrich, under the trade name P8136.
The serum-free mesenchymal stem cell basic culture solution in the following examples is a product of LONZA company, and has a product number of 12-725F.
The serum replacement in the following examples is Ultroser G serum replacement, a product of Pall corporation, cat # 15950-107.
In the following examples B-27 is B-27TMSupplement (50X), minus vitamin A, is a product of Gibco, Inc. under the product designation 12587010.
The mesenchymal stem cells in the following examples are human umbilical cord mesenchymal stem cells.
The PBS in the following examples is 0.01M phosphate buffer pH7.4, and is prepared by the following steps: 1.9734g of disodium hydrogen phosphate and 0.2245g of monopotassium phosphate are dissolved by adding deionized water, the pH value is adjusted to 7.4 by NaOH and HCl, and the volume is adjusted to 1000ml by using the deionized water, so that 0.01M phosphate buffer solution with the pH value of 7.4 is obtained.
D-PBS in the following examples is Dulbecco's phosphate buffer solution, and the composition of the D-PBS is as follows: the solute and its concentration are 2.67mM KCl and 1.47mM KH2PO4，138mM NaCl，8.10mM Na2HPO4(ii) a Deionized water as solvent. The formula of the D-PBS is as follows: 8g of NaCl, 0.2g of KCl, KH2PO4 0.2g,Na2HPO41.15g, adding 800ml of deionized water, stirring for dissolving, adjusting the pH value to 7.4 by HCl, adding deionized water to dissolve the solution to 1000ml, and sterilizing at high temperature (121.3 ℃) and high pressure (103.4kPa) for 30 minutes. Used for cell recovery and passage.
Example 1 isolation, purification and Collection of exosomes released by cells following Induction of adherent culture mesenchymal Stem cells
The composition for inducing the mesenchymal stem cell to release the exosome provided in this embodiment is two adherent culture compositions for inducing the mesenchymal stem cell to release the exosome, which are respectively an adherent culture composition a1 for inducing the mesenchymal stem cell to release the exosome and an adherent culture composition a2 for inducing the mesenchymal stem cell to release the exosome. Adherent culture composition A1 for inducing the release of exosomes from mesenchymal stem cells consists of Y-27632, EGF and palmitoyl pentapeptide-3. In an adherent culture composition A1 for inducing mesenchymal stem cells to release exosomes, the proportion of Y-27632, EGF and palmitoyl pentapeptide-3 is Y-276325 mu mol: EGF 50. mu.g: palmitoyl pentapeptide-35. mu. mol.
The adherent culture composition A2 for inducing the mesenchymal stem cells to release the exosomes consists of an adherent culture composition A1 for inducing the mesenchymal stem cells to release the exosomes and polyvinyl alcohol with the hydrolysis degree of 87-90%. In an adherent culture composition A2 for inducing mesenchymal stem cells to release exosomes, the proportion of Y-27632, EGF, palmitoyl pentapeptide-3 and polyvinyl alcohol with the hydrolysis degree of 87-90% is Y-276325 mu mol: EGF 50. mu.g: palmitoyl pentapeptide-35 μmol: 1g of polyvinyl alcohol having a degree of hydrolysis of 87 to 90%.
Serum-free mesenchymal stem cell basic culture solution (Lonza, Cat. No. 12-725F) and serum substitute (Ultroser G, Cat. No. Pall 15950-. In the adherent mesenchymal stem cell culture base solution-A, the content of the serum substitute is 20 ml/L.
The embodiment provides a composition for preparing exosome, which consists of adherent mesenchymal stem cell culture base solution-A, an adherent culture composition for inducing mesenchymal stem cells to release exosome and isolated mesenchymal stem cells. Wherein the adherent mesenchymal stem cell culture base solution-A and the adherent culture composition for inducing the mesenchymal stem cells to release the exosomes form a culture solution for preparing the exosomes by adherent culture. This example provides two culture solutions for the preparation of exosomes by adherent culture, namely, adherent induction culture solution-A1 and adherent induction culture solution-A2. The adherent induction culture solution-A1 comprises an adherent culture composition A1 for inducing mesenchymal stem cells to release exosomes and an adherent mesenchymal stem cell culture base solution-A, wherein in the adherent induction culture solution-A1, the content of Y-27632 is 5 mu mol/L, EGF is 50ng/ml, and the content of palmitoyl pentapeptide-3 is 5 mu mol/L. The adherent induction culture solution-A2 comprises an adherent culture composition A2 for inducing mesenchymal stem cells to release exosomes and an adherent mesenchymal stem cell culture base solution-A, wherein in the adherent induction culture solution-A2, the content of Y-27632 is 5 mu mol/L, EGF is 50ng/ml, the content of palmitoyl pentapeptide-3 is 5 mu mol/L, and the content of polyvinyl alcohol with the hydrolysis degree of 87-90% is 1 g/L.
The induced mesenchymal stem cells are cultured as adherent culture cells, and the specific experimental steps are as follows:
1. preparation of exosomes
The mesenchymal stem cells are cultured by adherent mesenchymal stem cell culture base liquid-A, then induced for 24 hours by adherent culture composition A1 for inducing the mesenchymal stem cells to release exosomes, induced for 24 hours by adherent culture composition A2 for inducing the mesenchymal stem cells to release exosomes, and then changed into adherent mesenchymal stem cell culture base liquid-A for 72 hours, culture supernatant is collected, and exosomes are obtained by separation. The specific method comprises the following steps:
1.1 resuscitating cryopreserved mesenchymal stem cells at 5X 106Inoculating T25 plastic culture bottle to each cell/bottle, adding adherent mesenchymal stem cell culture base liquid-A at 37 deg.C and 5% CO2Incubate for 12 hours.
1.2 induction culture: replacing culture solution with adherent induction culture solution-A1 for inducing adherent mesenchymal stem cells at 37 deg.C and 5% CO2Incubate 24 hours in incubator (i.e. induced with adherent culture composition a1 that induces exosome release from mesenchymal stem cells); replacing culture solution with adherent induction culture solution-A237 deg.C, 5% CO2The incubator was continued for 24 hours (i.e. induced with adherent culture composition a2 inducing the release of exosomes from mesenchymal stem cells); replacing culture solution with adherent mesenchymal stem cell culture base solution-A5 ml/T25 bottle at 37 deg.C and 5% CO2And (4) incubating the culture box for 72 hours, collecting culture supernatant (namely cell culture supernatant after induction of the adherent mesenchymal stem cells), and separating, purifying and collecting exosomes. The mesenchymal stem cells remaining after collecting the culture supernatant are called induced adherent mesenchymal stem cells for short.
2. Separating, purifying and collecting exosome
And (3) respectively collecting cell culture supernatants collected in the step 1.2 after the induction of the adherent mesenchymal stem cells into 25ml centrifuge tubes. Centrifuging for the first time at 500 Xg for 10min, discarding the precipitate, and taking supernatant to remove exfoliated cells; centrifuging for a second time at 2,000 Xg for 10min at 4 deg.C, discarding the precipitate and collecting the supernatant to remove cell debris; centrifuging for the third time at 10,000 Xg for 30min at 4 deg.C, removing the precipitate, and collecting supernatant to remove large vesicles; and (3) performing ultracentrifugation for the fourth time, concentrating the precipitate at 4 ℃ for 70 minutes at a volume ratio of the precipitate volume to D-PBS of 1:10, and collecting the precipitate, wherein the precipitate is a purified exosome, and the purified exosome is hereinafter referred to as an induced adherent mesenchymal stem cell exosome. Diluting with D-PBS, packaging, and storing at-80 deg.C. And collecting 40ml of cell culture supernatant dry ice logistics after the induction of the adherent mesenchymal stem cells in the step 1.2, sending the cell culture supernatant dry ice logistics to Shanghai Xiaopeng Biotech limited company for particle size analysis and transmission electron microscope images of the detected exosomes.
The result of the hydrodynamic Particle size analysis and the transmission electron microscope (fig. 1) for detecting exosomes by using the Nanoparticle Tracking Analysis (NTA) instrument (Particle diameter, ZetaView, germany) for inducing exosomes of adherent mesenchymal stem cells shows that in the cell culture supernatant after the adherent mesenchymal stem cells are induced in the step 1.2, 92.9% or more of the exosomes have the diameter of 113nm and the concentration of 1.68 × 108Number per ml (the optimal detection interval for NTA is 10)7Particles/ml. 60 times of the test sample is diluted and tested on a computer, and the exosome concentration of the left graph of the figure 1 needs to be multiplied by 60).
BCA (BCA protein concentration determination reagent, Beijing Feimer Biotech Co., Ltd., 7010004-B) exosome protein quantification: resuspending the above-mentioned adherent mesenchymal stem cell exosome with PBS, taking 50. mu.l, adding 20. mu.l RIPA-PMSF (100:1) lysate (prepared by mixing RIPA lysate and PMSF according to the volume ratio of 100:1), and the rest steps are as per the instruction. The result of 20 mul of lysis supernatant by enzyme labeling instrument (reading at 562nm wavelength) shows that the content of exosome in the cell culture supernatant after the above adherent mesenchymal stem cell induction is 8.66 mug/ml calculated by protein. The protein loading amount is used as a basis for calculating protein immunoblotting (Western Blot, WB) detection.
Example 2 isolation, purification and Collection of exosomes released by cells following Induction of mesenchymal Stem cells in suspension culture
The composition for inducing the release of exosomes from the mesenchymal stem cells provided in this embodiment is suspension culture composition B for inducing the release of exosomes from the mesenchymal stem cells. The suspension culture composition B for inducing the mesenchymal stem cells to release exosomes consists of Y-27632, EGF, bFGF, palmitoyl pentapeptide-3, hexapeptide and ceruloplasmin. In the suspension culture composition B for inducing the mesenchymal stem cells to release exosomes, the proportion of Y-27632, EGF, bFGF, palmitoyl pentapeptide-3, hexapeptide and bluecopper peptide is Y-276325 mu mol: EGF 50 ng: 20ng of bFGF: palmitoyl pentapeptide-35 μmol: hexakistan 1. mu. mol: 1 mu mol of blue copper peptide.
Serum-free mesenchymal stem cell basic culture solution (Lonza, with the stock number of 12-725F), serum substitute (Ultroser G, with the stock number of Pall 15950-. In the suspension mesenchymal stem cell culture base solution B, the content of a serum substitute is 20ml/L, the content of B-27 is 20ml/L, and the content of polyvinyl alcohol with the hydrolysis degree of 87-90% is 8 g/L.
The embodiment provides a composition for preparing exosome, which consists of suspension mesenchymal stem cell culture base solution B, suspension culture composition B for inducing the mesenchymal stem cells to release exosome and isolated mesenchymal stem cells.
And mixing the suspension mesenchymal stem cell culture base solution B and the suspension culture composition B for inducing the mesenchymal stem cells to release the exosomes to obtain a liquid serving as a culture solution for preparing the exosomes by suspension culture, which is called suspension induction culture solution-B for short. The suspension induction culture solution-B consists of a suspension mesenchymal stem cell culture base solution B and a suspension culture composition B for inducing the mesenchymal stem cells to release exosomes. In the suspension induction culture solution-B, the content of Y-27632 is 5 mu mol/L, EGF is 50ng/ml, the content of bFGF is 20ng/ml, the content of palmitoyl pentapeptide-3 is 5 mu mol/L, the content of hexapeptide is 1 mu mol/L, the content of copper blue peptide is 1 mu mol/L, B-27 is 20ml/L, and the content of polyvinyl alcohol with the hydrolysis degree of 87-90% is 8 g/L.
The induced mesenchymal stem cells are suspension-forming cell ball-like growing cells, and the specific experimental steps are as follows:
1. preparation of exosomes
Culturing the mesenchymal stem cells by suspension mesenchymal stem cell culture base solution B, inducing by a suspension culture composition B for inducing the mesenchymal stem cells to release exosomes for 24 hours, culturing by suspension mesenchymal stem cell culture base solution-B for 72 hours, collecting culture supernatant, and separating to obtain exosomes. The specific method comprises the following steps:
1.1 resuscitating cryopreserved mesenchymal stem cells at 1X 107Inoculating 10cm Corning non-treated culture dish to each cell/dish, suspending in mesenchymal stem cell culture base liquid B at 37 deg.C and 5% CO2And incubating the culture box for 12 hours to obtain the suspension mesenchymal stem cells growing like cell spheres.
1.2 induction culture: and (4) replacing the culture solution with a suspension induction culture solution-B to induce the mesenchymal stem cells.
Transferring the suspension mesenchymal stem cells of 1.1 into a 25ml centrifuge tube, centrifuging at 300 Xg for 5 minutes, collecting the suspension cultured cells, and changing into suspension induction culture solution-B induced mesenchymal stem cells at 37 ℃ and 5% CO2Incubating in an incubator for 24 hours; transferring the cultured suspension cells to a 25ml centrifuge tube, centrifuging at 300 Xg for 5 min, collecting the suspension cultured cells, and changing to suspension mesenchymal stem cell culture base solution B, 20ml/10cm corning non-treated culture dish, 37 deg.C, 5% CO2And (3) incubating the culture box for 72 hours, transferring the culture box to a 15ml centrifuge tube, centrifuging the centrifuge tube at 300 Xg for 5 minutes, collecting culture cell supernatant (namely culture supernatant of the induced suspension mesenchymal stem cells), and separating, purifying and collecting exosomes. The mesenchymal stem cells remaining after collecting the culture supernatant are abbreviated as induced suspension mesenchymal stem cells.
2. Separating, purifying and collecting exosome
Exosomes were isolated, purified, and collected as in step 2 of example 1. The purified exosomes are called induced suspension mesenchymal stem cell exosomes. Diluting with D-PBS, packaging, and storing at-80 deg.C. And collecting 40ml of the induced suspension mesenchymal stem cell culture supernatant dry ice material flow obtained in the step 1.2, and sending the induced suspension mesenchymal stem cell culture supernatant dry ice material flow to Shanghai Xiaopeng Biotech Limited company for particle size analysis and transmission electron microscope images of the detected exosomes.
Hydrodynamic particle size analysis and transmission electron microscopy results of the induced suspension mesenchymal stem cell exosomes (figure 2),shows that more than 97.9 percent of the exosomes in the culture supernatant of the induced suspension mesenchymal stem cells in the step 1.2 have the diameter of 117.2nm and the concentration of 5.9 multiplied by 108One/ml (the optimum detection interval for NTA is 10)7Particles/ml. The test sample is diluted by 200 times and tested on a machine, and the exosome concentration of the left graph of the graph of FIG. 2 needs to be multiplied by 200).
BCA (BCA protein concentration determination reagent, Beijing Feimer Biotech Co., Ltd., 7010004-B) exosome protein quantification: resuspending the above-mentioned induced suspension mesenchymal stem cell exosomes with PBS, taking 50. mu.l, adding 20. mu.l RIPA-PMSF (100:1) lysate (prepared by mixing RIPA lysate and PMSF according to the volume ratio of 100:1), and the rest steps are as described in the specification. The result of 20 mul of lysis supernatant being processed by a microplate reader (reading at 562nm wavelength) shows that the content of exosome in the culture supernatant of the induced suspension mesenchymal stem cells is 11.92 mug/ml calculated by protein. The protein loading amount is used as a basis for calculating protein immunoblotting (Western Blot, WB) detection.
Example 3 detection of Induction of expression of exosome marker protein by adherent mesenchymal Stem cells
1.1. Preparing an exosome protein for inducing adherent mesenchymal stem cells: and adding 20 mu L of precooled protein lysate (adding PSMF with the final concentration of 0.1mmol/L and protease inhibitor with the final concentration of 0.1mmol/L into the protein lysate) into 20 mu L of the adherent mesenchymal stem cell exosome inducing suspension of the step 2 in the embodiment 1, and uniformly mixing on a vortex device to obtain the adherent mesenchymal stem cell exosome inducing protein, wherein the protein concentration is 20 mu g/ml.
1.2. Preparing an exosome protein of the induced suspension mesenchymal stem cells: and 20 mu L of the suspension-inducing mesenchymal stem cell exosome suspension obtained in the step 2 of the embodiment 2, adding 20 mu L of precooled protein lysate (the protein lysate is added with PSMF with the final concentration of 0.1mmol/L and protease inhibitor with the final concentration of 0.1 mmol/L), and uniformly mixing on a vortex apparatus to obtain the suspension-inducing mesenchymal stem cell exosome protein with the protein concentration of 20 mu g/ml.
1.3. Preparing the induced adherent mesenchymal stem cell protein:
washing induced adherent mesenchymal stem cells obtained in the step 1.2 of the example 1 with D-PBS for 3 times, adding 2ml of precooled protein lysate, repeatedly blowing and beating a cell scraper and a tip pipette (operating on ice), transferring the cells to a 15ml centrifuge tube, uniformly mixing the cells on a vortex apparatus, and obtaining the induced adherent mesenchymal stem cell protein, wherein the protein concentration is 20 mu g/ml (the protein concentration is quantitatively determined by BCA).
1.4. Preparing induced suspension mesenchymal stem cell protein: washing induced suspension mesenchymal stem cells obtained in the step 1.2 of the example 1 with D-PBS for 3 times, adding 2ml of precooled protein lysate, repeatedly blowing and beating a cell scraper and a sharp pipette (ice operation), transferring the cells to a 15ml centrifuge tube, and uniformly mixing the cells on a vortex apparatus to obtain induced suspension mesenchymal stem cell protein with the protein concentration of 20 mu g/ml (protein concentration is quantitatively determined by BCA).
1.5. Protein sample preparation: respectively taking 1.1-1.4 protein samples, adding 1/4 sample volume of 5 Xloading buffer into the protein samples, boiling for 3 minutes at 100 ℃, cooling to room temperature, centrifuging for 10min at 1000rpm/min, 4 ℃. And placing on ice for later use.
1.6. Preparing glue, pouring glue, loading and performing electrophoresis: sequentially loading the prepared protein samples, wherein each hole is 20 microliters, switching on a power supply, starting electrophoresis, and performing gel concentration constant pressure 75V for 30 min; the separation gel has a constant pressure of 120V for 2-3 hours.
1.7. Film transfer: pretreating the PVDF membrane, performing constant current of 200mA, and rotating the membrane for 2 hours.
1.8. Immunoblotting: TBST buffer was washed 1 time, transferred to 5% BSA in TBST blocking solution, and blocked on a shaker (20rpm/min) for 1 hour at room temperature. TBST dilution primary (1:1000) HSP70(abcam, ab69413), TSG101(abcam, ab30871), CD63(abcam, ab68418), shaker (20rpm/min), and incubation at 4 ℃ overnight. Horseradish peroxidase conjugated secondary antibodies (Abcam, ab205718,1:2000) were shaken (20rpm/min) for 1 hour at room temperature. And (3) carrying out color development by a chemiluminescence method, and placing the PVDF film in an exposure instrument to visualize a strip.
The results showed that the adherent mesenchymal stem cell exosomes and the suspension mesenchymal stem cell exosomes were induced to express their marker proteins HSP70, TSG101 and CD63 (fig. 3).
Example 4 Induction of adherent and suspended mesenchymal Stem cell exosomes to promote differentiation of skin fibroblasts into epithelial cells
In the experiment for detecting the mouse embryonic fibroblasts cultured in vitro by the exosome for inducing adherent mesenchymal stem cells of example 1 and the exosome for inducing suspended mesenchymal stem cells of example 2, the exosome for inducing adherent mesenchymal stem cells has the capability of promoting the fibroblasts to differentiate into epithelial-like cells, and the specific experimental steps are as follows:
(1) mouse Embryo Fibroblast (MEF) is obtained from tissues of a head, viscera, trunk skeleton and limbs of a 15-day pregnant mouse embryo, and is subjected to digestion and culture of the fibroblast.
(2) 10%, 5% and 2% fetal bovine serum MEF: a liquid medium obtained by adding L-glutamate, penicillin, streptomycin and fetal calf serum to a DMEM/F12 high-sugar medium. In 10% fetal calf serum MEF culture solution, the content of L-glutamate is 2mmol/L, the content of penicillin is 100 units/ml, the content of streptomycin is 100ng/ml, and the volume content of fetal calf serum is 10%. In 5% fetal calf serum MEF culture solution, the content of L-glutamate is 2mmol/L, the content of penicillin is 100 units/ml, the content of streptomycin is 100ng/ml, and the volume content of fetal calf serum is 5%. In the 2% fetal calf serum MEF culture solution, the content of L-glutamate is 2mmol/L, the content of penicillin is 100 units/ml, the content of streptomycin is 100ng/ml, and the volume content of fetal calf serum is 2%.
Induced differentiation culture solution 1: an induced differentiation culture solution was obtained by adding ultraser G and exosomes (the induced adherent mesenchymal stem cell exosomes of example 1 or the induced suspension mesenchymal stem cell exosomes of example 2) to a 5% fetal bovine serum MEF culture solution. In the induced differentiation culture solution 1, the content of Ultroser G was 1%, and the content of exosomes was 50. mu.g/ml in terms of protein.
Induced differentiation culture solution 2: ultroser G and exosomes (the exosomes for inducing adherent mesenchymal stem cells of example 1 or the exosomes for inducing suspended mesenchymal stem cells of example 2) are added into a 2% fetal bovine serum MEF culture solution to obtain an induced differentiation culture solution 2. In the induced differentiation culture solution 2, the content of Ultroser G is 2%, and the content of exosome is 50 mu G/ml calculated by protein.
(3) The frozen MEF cells were revived on day 1 at 1X 106Individual cells/flask and 1X 106Inoculating each cell/dish into T25 plastic culture bottle and 3.5cm laser confocal culture dish, adding 10% fetal calf serum MEF culture solution, 37 deg.C, and 5% CO2Incubate overnight in incubator to obtain pre-induction fibroblasts.
(4) On day 2, the differentiation medium was changed to 1 for fibroblast differentiation at 37 ℃ with 5% CO2Incubate for 2 days.
(5) Culturing on day 4, changing induced differentiation culture solution 2, continuously inducing fibroblast differentiation, at 37 deg.C and 5% CO2And (5) incubating the culture box for 3 days to obtain the induced fibroblasts. And finishing the fibroblast oriented induction and differentiation process, and detecting the cultured cells.
(6) The molecular markers COL17A1 and integrin beta 1 (integrin-beta 1) of the differentiation of the skin epithelial cells expressed by the fibroblasts before and after induction are detected by a Western blot method. In the Western blot detection, beta-actin is used as an internal reference, and the expression conditions of COL17A1 and integrin beta 1 in the induced pre-fibroblast and the induced post-fibroblast are detected by using integrin beta 1 antibody (Santa Cruz), COL17A1 antibody (mouse monoclonal antibody COL17A1 antibody (Santa Cruz) and beta-actin antibody. the results show that the expressions of COL17A1 and integrin beta 1 are respectively found in the induced pre-fibroblast, the induced post-fibroblast induced by the induced adherent mesenchymal stem cell exosome and the induced post fibroblast induced by the induced suspension mesenchymal stem cell exosome, and the expression levels of COL17A1 and integrin beta 1 in the induced post fibroblast induced by the induced adherent mesenchymal stem cell exosome are higher than those in the pre-fibroblast (FIG. 4), and the results also show that the fibroblast treated by the suspension mesenchymal stem cell exosome is remarkably increased by the total fibroblast and integrin beta-17A 3850 g The exosome/ml can be induced for 5 days, can be differentiated to an epithelial-like cell, and expresses skin epithelial cell specific markers COL17A1 and integrin beta 1.
Example 5 Induction of adherent and suspended mesenchymal Stem cell exosomes for treatment of skin photoaging in rats
The experimental study for promoting rat skin photoaging repair by detecting the adherent mesenchymal stem cell-inducing exosomes of example 1 and the suspended mesenchymal stem cell-inducing exosomes of example 2 includes the following specific experimental steps:
1. preparation of Experimental rats
32 SD rats (body weight 210 + -20 g) were randomly divided into 2 groups, a control group of rats and a skin photoaging model group, 8 control groups, and 24 skin photoaging model groups, and the specific treatment was as follows:
preparation of skin photoaging model rats: after 10% chloral hydrate (3.5ml/kg) of SD rats (body weight 210 +/-20 g) is anesthetized, animals are fixed, the back of the SD rats is depilated and treated in a 5 x 6cm area, the SD rats are cleaned and disinfected, 15W UVA and UVB ultraviolet lamps are placed 35cm above the exposed skin area of the back of the rats, 2 hours are taken each day for 60 days, new hairs in the rat irradiation area are removed every other day, and the SD rats are raised in cages to obtain skin photoaging model rats.
Control rat preparation: SD rats (210 +/-20 g in weight) are anesthetized with 10% chloral hydrate (3.5ml/kg), the animals are fixed, the area of 5 x 6cm is subjected to dorsal depilating treatment, the animals are cleaned and disinfected, a 15W incandescent lamp is placed 35cm above the bare skin area of the back of the rats, the time is 2 hours per day for 60 days, new hairs in the rat irradiation area are removed every other day, and the rats are raised in cages to obtain a control group of rats (not subjected to ultraviolet irradiation).
Compared with the control group of rats, the skin photoaging model rats have rough skin, lack elasticity, obvious deep and large wrinkles and thickened epidermis. The control rat had smooth, normal color, elasticity, and no significant wrinkles.
2. Exosomes treat photodamaged skin.
The four treatments described below were parallel experiments that differed only in the application of the drug to the bare skin area of the back.
The method comprises the following specific steps:
control group rat treatment: each of the 8 control rats was smeared with 500. mu. l D-PBS on the exposed skin area of the back once a day for 7 days.
Skin photoaging model rat treatment: 8 rats with photoaging model of skin were smeared with 500 μ l D-PBS on each of the exposed skin areas of the back once a day for 7 days.
Treating rats by inducing adherent mesenchymal stem cell exosome treatment: each dorsal bare skin area of 8 skin photoaging model rats was smeared with 500. mu.l of exosomes once a day for 7 days. The dosage of each exosome was 150 μ g protein/protein. The exosome liquid was obtained by suspending the adherent mesenchymal stem cell-inducing exosome of example 1 in D-PBS.
Treating rats by inducing suspension mesenchymal stem cell exosome treatment: each dorsal bare skin area of 8 skin photoaging model rats was smeared with 500. mu.l of exosomes once a day for 7 days. The dosage of each exosome was 150 μ g protein/protein. The exosome liquid was obtained by suspending the induced suspension mesenchymal stem cell exosome of example 2 in D-PBS.
The apparent wrinkle determination scoring method (J Funt Foods,2013,5: 527-: the sensory scores were divided into 5 grades of I, II, III, IV and V, and the total sensory score was 15. The sensory score of grade I is 5, and the appearance of the skin is that fine skin lines parallel to the long axis of the body are visible on the surface of the skin, appear and disappear along with movement, and no wrinkles exist; sensory score of grade II is 4, and the appearance of the skin is that fine skin lines distributed in parallel along the long axis of the spinal column disappear, a small amount of transverse shallow wrinkles appear, and the fine skin lines can appear and disappear along with movement; grade III sensory score is 3, the appearance of the skin is that fine skin lines distributed in parallel along the long axis of the spinal column completely disappear, a small amount of transverse deep wrinkles appear, the transverse deep wrinkles do not disappear along with movement, and the skin appearance is continuous; the sensory score of the IV grade is 2, the appearance of the skin is that fine skin lines distributed in parallel along the long axis of the spinal column completely disappear, more transverse coarse wrinkles appear, the wrinkles do not disappear along with movement, and the wrinkles continuously exist; the sensory score of the V grade is 1, and the appearance of the skin is that a large number of transverse coarse deep wrinkles are distributed on the surface of the skin, do not disappear along with movement, and exist continuously. The degree of photoaging and the improvement effect were scored according to the above scoring method, as shown in table 1. The results showed that 8 rats treated the control group with a sensory score of 5 and a total score of 40. In the treatment of 8 rats, 1 of 8 rats had a sensory score of 3,6 had a sensory score of 2, 1 had a sensory score of 1, and the total score was 16. In the treatment of rats with the exosome-induced adherent mesenchymal stem cells, 5 of 8 rats had a sensory score of 4,3 had a sensory score of 3, and the total score was 29. In the treatment of rats with the induction of suspension mesenchymal stem cell exosomes, 6 out of 8 rats had a sensory score of 4,2 had a sensory score of 3, and the total score was 30 (table 1). The total score of rat treatment by inducing adherent mesenchymal stem cell exosome and rat treatment by inducing suspension mesenchymal stem cell exosome is obviously higher than that of rat treatment of skin photoaging model, which shows that both the adherent mesenchymal stem cell exosome induced in example 1 and the suspension mesenchymal stem cell exosome induced in example 2 can treat skin photoaging injury.
TABLE 1 Induction of mesenchymal Stem cell exosomes for treating SD rat skin photodamage wrinkle grade and sensory evaluation table
3. Effect of exosomes on type I and type III collagen expression
The results show that compared with the treatment of rats with skin photoaging model, the expression level of type I collagen mRNA is reduced and the expression level of type III collagen mRNA is increased in the treatment of rats with the induction of adherent mesenchymal stem cell exosome treatment and in the treatment of rats with the induction of suspension mesenchymal stem cell exosome treatment (fig. 5).
Example 6 prevention of rat skin photoaging by inducing adherent and suspended mesenchymal Stem cell exosomes
1. Experimental study to test exosomes against rat skin photoaging, the following four treatments were parallel experiments that differed only in the agents applied to the bare skin area of the back. The 48 SD rats (body weight 210 + -20 g) were divided randomly into 4 groups, and the specific treatment was as follows:
treatment of the control group: SD rats (body weight 210 + -20 g) were depilated from the back of 5X 6cm area, cleaned and disinfected. 12 rats were each smeared with 500 μ l D-PBS on the bare skin area of the back once a day for 7 days. After anesthesia with 10% chloral hydrate (3.5ml/kg), the animals were fixed and placed 35cm directly above the bare skin area on the back of the rat with a 15W incandescent lamp for 2 hours a day for 60 days, and during the irradiation period, 500. mu. l D-PBS was applied to each bare skin area on the back of the rat once a day to obtain control-treated rats (not irradiated with ultraviolet rays).
Skin photoaging model treatment: SD rats (body weight 210 + -20 g) were dehaired on the back of 5X 6cm area, cleaned and disinfected. 12 rats were each smeared with 500 μ l D-PBS on the bare skin area of the back once a day for 7 days. After 10% chloral hydrate (3.5ml/kg) was anesthetized, the animals were fixed, 15W UVA and UVB ultraviolet lamps were placed 35cm above the bare skin area on the back of the rat for 2 hours a day for 60 days, and 500. mu. l D-PBS was applied to the bare skin area on the back of each rat once a day during irradiation to obtain skin photoaging model-treated rats.
And (3) inducing the adherent mesenchymal stem cell exosome prevention treatment: SD rats (body weight 210 + -20 g) were dehaired on the back of 5X 6cm area, cleaned and disinfected. Each of the 12 rats was coated with 500 μ l of exosomes on the bare skin area of the back once a day for 7 days. After 10% chloral hydrate (3.5ml/kg) is anesthetized, animals are fixed, 15W UVA and UVB ultraviolet lamps are placed 35cm above the naked skin area of the back of a rat for 2 hours every day for 60 days, 500 mu l of exosome liquid is smeared on the naked skin area of the back of each rat during irradiation, and the rat subjected to the exosome prevention treatment for inducing the adherent mesenchymal stem cells is obtained once a day. The dosage of each exosome was 150 μ g protein/protein. The exosome liquid is obtained by suspending the exosome inducing adherent mesenchymal stem cells of example 1 in D-PBS.
And (3) inducing suspension mesenchymal stem cell exosome prevention treatment: SD rats (body weight 210 + -20 g) were dehaired on the back of 5X 6cm area, cleaned and disinfected. Each of the 12 rats was coated with 500 μ l of exosomes on the bare skin area of the back once a day for 7 days. After 10% chloral hydrate (3.5ml/kg) is anesthetized, animals are fixed, 15W UVA and UVB ultraviolet lamps are placed 35cm above the exposed skin area of the back of the rat, 2 hours are taken each day for 60 days, 500 mu l of exosome liquid is smeared on the exposed skin area of the back of each rat during irradiation, and the rat for preventing and treating the exosome of the induced suspension mesenchymal stem cells is obtained once a day. The dosage of each exosome was 150 μ g protein/protein. The above exosome liquid was obtained by suspending the induced suspension mesenchymal stem cell exosome of example 2 in D-PBS.
The degree of photoaging and the improvement effect were scored according to the skin transverse wrinkles using the apparent wrinkle determination scoring method (J Funt Foods,2013,5: 527-.
The results indicated that 12 rats treated the control group had a sensory score of 5 and a total score of 60. In the skin photoaging model treatment, 1 rat out of 12 rats had a sensory score of 3,8 rats had a sensory score of 2, and 3 rats had a sensory score of 1, with a total score of 22. In the treatment for inducing the prevention of adherent mesenchymal stem cell exosomes, 10 out of 12 rats had a sensory score of 4,2 had a sensory score of 3, and the total score was 46. In the treatment for inducing suspension mesenchymal stem cell exosome prevention, 11 out of 12 rats had sensory scores of 4 and 1 had sensory score of 3, and the total score was 47 (table 2). Therefore, the total score of the adherent mesenchymal stem cell exosome prevention treatment and the suspension mesenchymal stem cell exosome prevention treatment is significantly higher than that of the skin photoaging model treatment, which shows that the adherent mesenchymal stem cell exosome in the embodiment 1 and the suspension mesenchymal stem cell exosome in the embodiment 2 can resist the photodamage of the skin, promote the photodamage repair and prevent the photoaging damage of the skin.
TABLE 2 Induction of mesenchymal Stem cell exosomes to prevent SD rat skin photodamage wrinkle grade and sensory evaluation table
2. Inducing wall-attached mesenchymal stem cell exosome and inducing suspension mesenchymal stem cell exosome to have antioxidant capacity
Animals were sacrificed by cervical dislocation after scoring. Taking 1cm of skin in back irradiation area2Skin tissue, detecting activities of Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione peroxidase (GSH-PX), performing according to the kit specification of Nanjing-built bioengineering company, setting a control hole, a control blank hole, a measurement blank hole and a measurement hole in an experiment, and measuring by using an enzyme-labeling instrument with a wavelength of 450nm, wherein the result shows that the adherent mesenchymal stem cell exosome induced in the example 1 and the suspended mesenchymal stem cell exosome induced in the example 2 have the antioxidant capacity, the activities of the antioxidant enzymes (SOD, CAT and GSH-PX) are improved, and the skin light-mediated oxidative stress damage is repaired (figures 6-8).
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (12)
1. A composition for inducing the release of exosomes from mesenchymal stem cells, characterized by: the composition is an adherent culture composition or a suspension culture composition; the adherent culture composition is an adherent culture composition A1 or an adherent culture composition A2,
the adherent culture composition A1 consists of Y-27632, EGF and palmitoyl pentapeptide-3, and the proportion of the Y-27632, the EGF and the palmitoyl pentapeptide-3 in the adherent culture composition A1 is Y-276325 mu mol: EGF 50 microgram: palmitoyl pentapeptide-35 μmol;
the adherent culture composition A2 consists of the adherent culture composition A1 and polyvinyl alcohol with hydrolysis degree of 87-90%, and in the adherent culture composition A2, the mixture ratio of Y-27632, EGF, palmitoyl pentapeptide-3 and the polyvinyl alcohol with hydrolysis degree of 87-90% is Y-276325 μmol: EGF 50 microgram: palmitoyl pentapeptide-35 μmol: 1g of polyvinyl alcohol with the hydrolysis degree of 87-90 percent;
the suspension culture composition consists of Y-27632, EGF, bFGF, palmitoyl pentapeptide-3, hexapeptide and ceruloplasmin, and the proportion of the Y-27632, the EGF, the bFGF, palmitoyl pentapeptide-3, the hexapeptide and the ceruloplasmin in the suspension culture composition is Y-276325 [ mu ] mol: EGF 50 ng: 20ng of bFGF: palmitoyl pentapeptide-35 μmol: 1 mu mol of hexapeptide: 1 mu mol of blue copper peptide.
2. The composition of claim 1, wherein: the exosome has at least one of the following functions:
1) promoting the differentiation of skin fibroblasts to epithelial cells,
2) promoting the repair of photodamaged skin and/or promoting the repair of photoaging damaged skin,
3) against photodamage and/or photoaging damage to the skin,
4) improving the oxidation resistance of the skin.
3. Composition for the preparation of exosomes, characterized in that: the composition for preparing exosome comprises the composition of claim 1 and mesenchymal stem cells ex vivo.
4. A composition for the preparation of exosomes according to claim 3, characterized in that: the composition for preparing exosome consists of the composition of claim 1, mesenchymal stem cell basal medium and mesenchymal stem cells ex vivo.
5. A composition for the preparation of exosomes according to claim 3 or 4, characterized in that: the exosome has at least one of the following functions:
1) promoting the differentiation of skin fibroblasts to epithelial cells,
2) promoting the repair of photodamaged skin and/or promoting the repair of photoaging damaged skin,
3) against photodamage and/or photoaging damage of the skin,
4) improving the oxidation resistance of the skin.
6. Use of a composition according to claim 1 or 2 or a composition according to any one of claims 3 to 5 for the preparation of an exosome for the preparation of a product for preventing and/or treating photodamage and/or photoaging damage of the skin.
7. Use of a composition according to claim 1 or 2 or a composition according to any one of claims 3 to 5 for the preparation of an exosome for the preparation of a medicament for cell-free replacement therapy for skin healing.
8. Use of a composition according to claim 1 or 2 or a composition according to any one of claims 3-5 for the preparation of an exosome.
9. Use according to claim 8, characterized in that: the exosome has at least one of the following functions:
1) promoting the differentiation of skin fibroblasts to epithelial cells,
2) promoting the repair of photodamaged skin and/or promoting the repair of photoaging damaged skin,
3) against photodamage and/or photoaging damage of the skin,
4) improving the oxidation resistance of the skin.
10. Use of a composition according to claim 1 or 2 or a composition according to any one of claims 3 to 5 for the preparation of exosomes for the preparation of a product for promoting differentiation of skin fibroblasts into epithelial cells.
11. A method for preparing exosomes, comprising culturing ex vivo mesenchymal stem cells with a medium comprising the composition of claim 1 or 2 to obtain exosomes.
12. The method of claim 11, wherein: the exosome has at least one of the following functions:
1) promoting the differentiation of skin fibroblasts to epithelial cells,
2) promoting the repair of photodamaged skin and/or promoting the repair of photoaging damaged skin,
3) against photodamage and/or photoaging damage of the skin,
4) improving the oxidation resistance of the skin.
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