CN106983704A - Use of mesenchymal stem cell culture or its culture supernatant - Google Patents

Abstract

The present invention relates to the use of mesenchymal stem cell cultures or culture supernatants thereof. Specifically, the invention relates to a mesenchymal stem cell culture, a culture supernatant in the mesenchymal stem cell culture, a secretion of mesenchymal stem cells, a freeze-dried powder prepared from the culture or the culture supernatant or the secretion of mesenchymal stem cells and an application of a solution obtained after dissolving the freeze-dried powder in the preparation of a cosmetic composition. The invention also relates to cosmetic compositions containing TNF-alpha, IL-6 and IFN-gamma. The cosmetic composition prepared by the invention can obviously improve skin, promote skin tissue repair and slow down skin aging, and the mesenchymal stem cells have rich sources, can be used by self, are safer and more convenient, and have good application prospects.

Description

Use of mesenchymal stem cell culture or its culture supernatant

technical field

The present invention relates to the use of mesenchymal stem cells, specifically, the present invention relates to mesenchymal stem cell cultures, culture supernatants in said mesenchymal stem cell cultures, secretions of mesenchymal stem cells, cultured or cultured The lyophilized powder prepared from the supernatant or the secretion of the mesenchymal stem cells and the solution obtained after the lyophilized powder is dissolved are used for preparing a cosmetic composition. The present invention also relates to cosmetic compositions containing TNF-alpha, IL-6 and IFN-gamma.

Background technique

Stem cells are a type of undifferentiated cells with self-replication ability and multi-directional differentiation potential, and have the potential function of regenerating various tissues and organs of the human body, so they are also called "universal cells" by the medical field. A number of studies have confirmed that stem cells can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, heart muscle, endothelial and other tissue cells under specific induction conditions. Not only that, stem cells are continuously subcultured and frozen After preservation, the initial multi-directional differentiation potential can still be preserved, and it can be used as an ideal seed cell for the repair of tissue and organ damage caused by aging and pathological changes.

Current stem cell research mainly focuses on anti-aging, organ transplantation, disease treatment and self-repair of the immune system. The U.S. Food and Drug Administration has approved more than 60 stem cell clinical trials, involving: 1. Enhancement of hematopoietic function, promotion of hematopoietic stem cell transplantation and treatment of graft-versus-host disease. 2. Repair of tissue damage such as bone, cartilage, joint damage, heart damage, liver damage, spinal cord damage and nervous system damage. 3. Treatment of autoimmune diseases such as systemic lupus erythematosus, scleroderma, inflammatory bowel disease and other diseases. In recent years, my country has also begun to use stem cells to treat some refractory diseases clinically, such as spinal cord injury, cerebral palsy, amyotrophic lateral sclerosis, systemic lupus erythematosus, systemic sclerosis, Crohn's disease, stroke, diabetes, diabetes Foot, liver cirrhosis, etc., have achieved significant curative effect.

There have also been some studies on the role of stem cells in skin regeneration and anti-aging. For example, Blue Powder International Global R&D Center started research in this field a long time ago, and established an independent experimental department dedicated to the study of human skin stem cells, and has discovered this new source of life: dermal stem cells. It is located in the deepest layer of the skin and plays a vital role in improving the vitality of the cells in the lower dermis.

In addition, in 1962, Dr. Cohen, an American scientist, discovered the "epidermal growth factor" that determines the age of the skin. It is further confirmed that the lack of epidermal growth factor is the root cause of skin aging, and supplementing the skin with epidermal growth factor can make the aging skin younger again. Since then, the mystery of skin youthfulness and aging has been uncovered, and the human dream of being younger has become a reality. On December 10, 1986, Dr. Cohen won the Nobel Prize, one of the highest scientific awards in the world.

However, there is no report on the use of mesenchymal stem cells, especially oral tissue-derived mesenchymal stem cells, for repairing skin tissue, slowing down skin aging, or preparing cosmetics.

Contents of the invention

The inventors of the present invention surprisingly found that the cell culture of mesenchymal stem cells, especially oral cavity tissue-derived mesenchymal stem cells or its culture supernatant, or the secretion of mesenchymal stem cells can promote skin tissue repair, alleviate and/or Or lighten scars, slow down skin aging, and obviously improve skin texture, so it is especially suitable for preparing cosmetics, thus completing the present invention.

The first aspect of the present invention relates to the use of mesenchymal stem cell culture or its culture supernatant for preparing a cosmetic composition.

The second aspect of the present invention relates to the use of mesenchymal stem cell culture or its culture supernatant for preparing preparations for improving skin quality, promoting skin tissue repair, reducing and/or reducing scars, or slowing down skin aging.

The third aspect of the present invention relates to the use of the secretion of mesenchymal stem cells for the preparation of cosmetic compositions.

The fourth aspect of the present invention relates to the use of the secretion of mesenchymal stem cells for preparing preparations for improving skin quality, promoting skin tissue repair, reducing and/or reducing scars, or slowing down skin aging.

The fifth aspect of the present invention relates to a method for improving skin quality, promoting skin tissue repair, reducing and/or lightening scars, or slowing down skin aging, the method comprising administering an effective amount of mesenchymal stem cells to a subject in need Culture or culture supernatant thereof, or secretion of mesenchymal stem cells, or cosmetic composition or preparation containing culture of mesenchymal stem cells or culture supernatant thereof, or cosmetic composition containing secretion of mesenchymal stem cells or preparation steps.

The above five aspects are further described below.

In one embodiment of the present invention, the culture of mesenchymal stem cells is a culture obtained by culturing mesenchymal stem cells in a medium suitable for culturing mesenchymal stem cells.

In one embodiment of the present invention, wherein the culture supernatant of the mesenchymal stem cell culture refers to the culture supernatant of a culture obtained by culturing mesenchymal stem cells in a medium suitable for culturing mesenchymal stem cells, that is The supernatant obtained after removing the mesenchymal stem cells in the mesenchymal stem cell culture; methods for removing cells in the culture are known in the art, such as centrifugation, filtration and the like.

In one embodiment of the present invention, the mesenchymal stem cell culture contains mesenchymal stem cells, a medium suitable for culturing mesenchymal stem cells, and substances secreted by cells during cell culture (i.e. secretions, such as cytokines, etc.).

In one embodiment of the present invention, the secretion of mesenchymal stem cells is the secretion obtained from culturing mesenchymal stem cells in a medium suitable for culturing mesenchymal stem cells.

In the present invention, the secretion of mesenchymal stem cells refers to substances secreted by mesenchymal stem cells during the culture process, such as secreted proteins, wherein the proteins include, for example, enzymes, antibodies, hormones and cytokines, etc. .

In one embodiment of the present invention, the secretion of the mesenchymal stem cells contains cytokines such as TNF-α, IL-6 and IFN-γ.

In one embodiment of the present invention, the secretion of the mesenchymal stem cells does not contain growth hormone (GH), epidermal growth factor (EGF) and endothelial cell growth factor (ECGF).

In one embodiment of the present invention, the medium suitable for culturing mesenchymal stem cells does not contain phenol red.

It is well known in the art that phenol red is commonly used in cell culture medium as an acid-base indicator; Mesenchymal stem cell cultures also do not contain phenol red. Similarly, those skilled in the art can understand that if the cell culture medium contains substances that are not suitable for cosmetics, and removing the substance will not affect the growth of mesenchymal stem cells, then when preparing a cosmetic composition, the cell culture Substances such as antibiotics should not be present in the base.

In one embodiment of the present invention, the culture obtained by culturing mesenchymal stem cells in a medium suitable for culturing mesenchymal stem cells or its culture supernatant is a culture medium obtained by culturing mesenchymal stem cells in a medium suitable for culturing mesenchymal stem cells. The culture obtained from the third or fourth passage of mesenchymal stem cells or its culture supernatant.

In one embodiment of the present invention, the secretion obtained from culturing mesenchymal stem cells in a medium suitable for culturing mesenchymal stem cells is the third Secretions from the first or fourth generation.

In the present invention, the mesenchymal stem cells that have just been isolated are primary mesenchymal stem cells, that is, the zeroth generation mesenchymal stem cells. The fourth generation was obtained after four passages. In an embodiment of the present invention, the third-generation or fourth-generation mesenchymal stem cell culture or its culture supernatant has the best effect for preparing cosmetics.

In one embodiment of the present invention, wherein said mesenchymal stem cells are selected from gingival stem cells (gingival mesenchymal stem cells, GMSC), periodontal ligament stem cells (periodontal ligament stem cells, PDLSC), dental pulp stem cells (post-nataldental pulp stem cells (DPSC), stem cells from apical papilla (SCAP), deciduous tooth pulp stem cells (stem cells from exfoliateddeciduous teeth, SHED), dental follicle precursor cells (dental follicle precursor cells, DFPC), bone marrow Mesenchymal stem cells (BMMSCs) and adipose-derived mesenchymal stem cells (ADSCs). In a specific embodiment of the present invention, the bone marrow mesenchymal stem cells are human jaw bone marrow mesenchymal stem cells hABMMSCs (human alveolar bonemarrow mesenchymal stem cells).

In the present invention, the medium suitable for culturing mesenchymal stem cells is well known in the art, such as being selected from MEM medium, DMEM medium and DMEM:F12 medium. In one embodiment of the present invention, the medium suitable for culturing mesenchymal stem cells is α-MEM medium.

It is well known in the art that the formula of MEM medium is not fixed, and may have different additions and subtractions according to different culture requirements, for example, refer to http://www.thermofisher.com/cn/zh/home.html A description of the related product.

In a specific embodiment of the present invention, the phenol red-free MEM medium is a GBICO product of Thermo Fisher Company, the article number is: 41061-029.

In one embodiment of the present invention, the medium suitable for culturing mesenchymal stem cells further contains glutamine, the concentration of which is, for example, 1-3 mM.

In one embodiment of the present invention, the medium suitable for culturing mesenchymal stem cells further contains serum (such as fetal calf serum), the concentration of which is, for example, 5%-20%.

In one embodiment of the present invention, the medium suitable for culturing mesenchymal stem cells does not contain antibiotics.

In one embodiment of the present invention, the mesenchymal stem cell culture or its culture supernatant contains TNF-α, IL-6 and IFN-γ.

In one embodiment of the present invention, wherein said mesenchymal stem cell secretion contains TNF-α, IL-6 and IFN-γ.

In one embodiment of the present invention, the mesenchymal stem cell culture or its culture supernatant is a freeze-dried powder obtained after freeze-drying.

In one embodiment of the present invention, the secretion of the mesenchymal stem cells is freeze-dried powder.

In one embodiment of the present invention, wherein said mesenchymal stem cell culture or its culture supernatant is a solution, said solution is prepared by the following method: said mesenchymal stem cell culture or its culture supernatant The prepared lyophilized powder is dissolved in a solvent.

In one embodiment of the present invention, wherein the secretion of the mesenchymal stem cells is a solution, the solution is prepared by the following method: dissolving the lyophilized powder prepared from the secretion of the mesenchymal stem cells in in solvent.

In one embodiment of the present invention, wherein the solvent can be any solvent that can dissolve the above-mentioned lyophilized powder and is suitable for pharmaceutical or cosmetic use, such as being selected from serum, normal saline, glucose solution (such as isotonic glucose solution) and glycerin.

In one embodiment of the present invention, the cosmetic composition or preparation further contains acceptable auxiliary materials (such as carriers or excipients) in cosmetic compositions.

In one embodiment of the present invention, the improvement of skin texture is selected from the group consisting of reducing wrinkles, lightening spots, making skin firmer and making skin more rosy.

The invention also relates to compositions comprising TNF-alpha, IL-6 and IFN-gamma.

In one embodiment of the present invention, wherein said composition is a cosmetic composition.

In one embodiment of the present invention, the cosmetic composition further contains cosmetically acceptable excipients.

In the present invention, the acceptable excipients in the cosmetics are well known in the art. Those skilled in the art can properly select and mix the raw materials used in cosmetics according to the type of cosmetics, such as squalene, liquid paraffin, isoparaffin, vaseline, microcrystalline wax, ozokerite, refined ozokerite, myristic acid , palmitic acid, stearic acid, oleic acid, isostearic acid, cetyl alcohol, stearyl alcohol, cetyl alcohol, oleyl alcohol, cetyl-2-ethylhexanoate, 2-ethylhexyl palmitic acid ester, 2-octyldodecyl myristate, 2-octyldodecyl gum ester, neopentyl glycol-2-ethylhexanoate, isooctyl triglyceride, 2-octyldodecyl Alkyl Oleate, Isopropyl Myristate, Isopropyl Palmitate, Isostearate Triglyceride, Coconut Fatty Acid Triglyceride, Olive Oil, Avocado Oil, Beeswax, Carmine Myristate Cardamom butter, mink oil, lanolin and other hydrocarbons, higher fatty acids, oils, esters, higher alcohols, waxes, silicone oil and other oils, acetone, toluene, butyl acetate, ethyl acetate and other organic solvents, alkyd resins , acrylic resin, urea resin and other resins, plasticizers such as camphor and acetyl tributyl citrate, ultraviolet absorbers, antioxidants, preservatives, surfactants, humectants, fragrances, water, alcohol, tackifiers, etc. . In addition, as powders used in cosmetics, for example, talc, kaolin, sericite, muscovite, synthetic mica, phlogopite, red mica, biotite, phlogopite, vermiculite, magnesium carbonate, calcium carbonate, diatomaceous earth, , magnesium silicate, calcium silicate, aluminum silicate, barium silicate, barium sulfate, strontium silicate, metal tungstate, silicon dioxide, hydroxyapatite, zeolite, boron nitride, ceramic powder and other inorganic powders, Nylon powder, polyethylene powder, benzomelamine powder, tetrafluoroethylene powder, stilbene phenyl pinhole polymer powder, microcrystalline cellulose powder and other organic powders, silicone rubber, silicone resin powder, titanium oxide, zinc oxide Inorganic white pigments such as iron oxide (iron red), iron titanate and other inorganic red pigments, γ-iron oxide and other inorganic brown pigments, yellow iron oxide, loess and other inorganic yellow pigments, black iron oxide, carbon black and other inorganic black pigments Pigments, inorganic purple pigments such as mango violet and cobalt violet, inorganic green pigments such as chromium oxide, chromium hydroxide, and cobalt titanate, inorganic blue pigments such as ultramarine blue and Prussian blue, mica coated with titanium oxide, bismuth oxychloride coated with titanium oxide, Bismuth oxychloride, titanium oxide coated talc, fish scale foil, dyed titanium oxide coated mica and other pearlescent pigments, aluminum powder, copper powder and other metal powder pigments, red 201, red 202, red 204, red 205, red 220 No., red No. 226, red No. 228, red No. 405, orange No. 203, orange No. 204, yellow No. 205, yellow No. 401, blue No. 404, etc., red No. 3, red No. 104, red No. 106 Zirconium, barium or Organic pigments such as aluminum lakes, natural pigments such as chlorophyll and β-carotene, but are not limited to these.

In one embodiment of the present invention, wherein said cosmetic composition contains water, glycerol polymethacrylate (and) propylene glycol, petrolatum, dioctyl ether, PEG-5 glyceryl stearate, glycerin, poly Dimethiconol, Cetyl Alcohol, Sweet Almond Oil, Acrylic Acid/C10-30 Alkyl Acrylic Polymer, Tocopherol Acetate, Phenoxyethanol, Benzyl Alcohol, EDTA, Hydrogen Sodium Oxide and Lactic Acid.

The invention also relates to the use of a composition according to any one of the invention for the preparation of a cosmetic composition.

In one embodiment of the present invention, wherein said cosmetic composition is used to improve skin texture, promote skin tissue repair, reduce and/or fade scars, or slow down skin aging, preferably, wherein said improvement of skin texture is selected from Self-reducing wrinkles, lightening dark spots, making skin firmer and making skin more rosy.

The present invention also relates to the use of any one of the compositions of the present invention in the preparation of preparations or cosmetic compositions for improving skin quality, promoting skin tissue repair, alleviating and/or lightening scars, or slowing down skin aging.

In one embodiment of the present invention, the improvement of skin texture is selected from the group consisting of reducing wrinkles, lightening spots, making skin firmer and making skin more rosy.

In one embodiment of the present invention, the expression levels of cytokines TNF-α, IL-6 and IFN-γ in the cell culture supernatant after using cell culture medium to culture mesenchymal stem cells are significantly higher than those in simple cell culture medium. Therefore, it can be used as an active ingredient to improve skin quality and promote skin tissue repair.

In the present invention, the mesenchymal stem cells are derived from mammals, preferably humans. The mesenchymal stem cell culture or its culture supernatant can be used for autologous or allogeneic, preferably autologous.

In the present invention, the mesenchymal stem cell culture or its culture supernatant can be injected or applied to the skin, or the culture or its culture supernatant can be freeze-dried and then dissolved with an appropriate solvent, and then injected or applied to the skin skin. In an embodiment of the present invention, the mesenchymal stem cell culture or its culture supernatant may be mixed with a cosmetic base or other cosmetic base ingredients, and then injected or smeared.

In the present invention, the scar is also called scar, which is a general term for the appearance and histopathological changes of normal skin tissue caused by various traumas, including superficial scars, hypertrophic scars, atrophic scars and scars. Pimples and many other types.

In the present invention, the lightening and/or lightening of scars refers to reducing, lowering or flattening scars, lightening their color and making them closer to normal skin.

Beneficial Effects of the Invention

The inventors of the present invention have proved through experiments that mesenchymal stem cell culture or its culture supernatant can effectively promote skin tissue repair, slow down skin aging, improve skin quality, and prove that TNF-α in culture or culture supernatant , IL-6 and IFN-γ played a key role.

The present invention uses mesenchymal stem cells, especially oral stem cells, which have sufficient tissue sources, are less affected by environmental factors, are convenient and quick to obtain materials, and can extract a large number of oral stem cells without major trauma. In particular, the extraction process of gingival stem cells is extremely simple, and its tissue source is close to epithelial cells, and the gingiva itself has the ability to repair itself after trauma, so it is closer to the skin and has better absorption effect.

The present invention can adopt autologous or allogeneic oral stem cells without worrying about immune rejection and inflammation, and the product has high stability and safety.

Description of drawings

Fig.1 The supernatant after culturing gingival stem cells (GMSC) and apical papilla stem cells (SCAP) with phenol red-free α-MEM medium to stimulate dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC) and jaw stem cells The images under the microscope of cells grown from bone marrow stem cells (BMSC) stained by BrdU cell immunohistochemistry, where the scale bar represents 500 μm; the vehicle group represents the control group.

Fig.2 The supernatant after culturing gingival stem cells (GMSC) and apical papilla stem cells (SCAP) with phenol red-free α-MEM medium to stimulate dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC) and jaw stem cells The growth and proliferation rate obtained from the growth of bone marrow stem cells (BMSC), wherein the ordinate represents 5-bromodeoxyuridine positive cells, * represents P<0.05, ** represents P<0.01, *** represents P<0.001; where The vehicle group represents the control group.

Figure 3 After culturing gingival stem cells in phenol red-free α-MEM medium, the contents of TNF-α, IL-6 and IFN-γ detected in the cell supernatant were lower than those of phenol red-free α-MEM without cultured cells. The content in the MEM medium was significantly increased, where * represents P<0.05 compared with the control, and ** represents P<0.01 compared with the control.

Photo (left) and photo (right) after freeze-drying of cell supernatant in Fig. 4

Figure 5 is a diagram of the effects of cosmetic bases and cosmetics added with cell culture supernatants of the present invention, where the left pictures of A and B are cosmetic bases, and the right pictures are cosmetics added with cell culture supernatants of the present invention, where A is left Side face, B is the right face.

Figure 6 is a diagram of the effects of the cosmetics before using the cosmetics and 30 days after using the cell culture supernatant of the present invention, wherein Figure A is before using the cosmetics, and Figure B is 30 days after using the cosmetics using the cell culture supernatant of the present invention.

Figure 7 Microscopic images of primary cultured gingival stem cells, periodontal ligament stem cells, apical papilla stem cells, dental pulp stem cells and deciduous tooth pulp stem cells

Figure 8 Cellular immunophenotype identification of dental pulp stem cells

Figure 9 Identification of adipogenic and osteogenic differentiation of dental pulp stem cells

Figure 10 Cellular immunophenotype identification of periodontal ligament stem cells

Figure 11 Identification of adipogenic and osteogenic differentiation of periodontal ligament stem cells

Figure 12 Cellular immunophenotype identification of gingival stem cells

Figure 13 Identification of adipogenic and osteogenic differentiation of gingival stem cells

Figure 14 Cellular immunophenotype identification of root canine papilla stem cells

Figure 15 Identification of adipogenic and osteogenic differentiation of root canine papilla stem cells

Figure 16 Cellular immunophenotype identification of dental pulp stem cells in deciduous teeth

Figure 17 Identification of adipogenic and osteogenic differentiation of deciduous dental pulp stem cells

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

α-MEM medium was purchased from Gbico brand of Thermo Fisher Company, product number 11095-080. The phenol red-free a-MEM medium used is the GBICO brand of Thermo Fisher Company, the article number is: 41061-029.

Example 1 The cultivation of oral cavity-related stem cells

The cell culture medium used in this example is α-MEM medium without phenol red (product number: 41061-029).

1. Cell passage method

Oral tissue stem cells to be primary extracted (see Example 4, including gingival stem cells, apical papilla stem cells, dental pulp stem cells, periodontal ligament stem cells, deciduous dental pulp stem cells and dental follicle precursor stem cells, etc.), cell growth fusion rate If it is more than 90%, it can be subcultured. The subculture method is a method commonly used in the art, specifically:

1.1 Take out the cells to be treated from the incubator, observe their growth status under an inverted microscope, confirm that they are in the logarithmic growth phase, and then put them into the ultra-clean bench.

1.2 After inserting the aspirator head into the yellow gun head, turn on the vacuum pump and suck up the supernatant.

1.3 Add an appropriate amount of PBS along the inner wall of the container, shake the plate slightly, rinse the residual medium, and then repeat the above step 1.2.

1.4 According to the container specifications, add 8-10ml trypsin along the inner wall of the 150mm cell culture dish for digestion, transfer the cells to a microscope to observe the degree of digestion, during which time they can be placed in a 37°C incubator to speed up their digestion (the time depends on Different cell types and reagents should be explored), during which the medium was constantly shaken to accelerate cell digestion. After observing about 80%-90% of the cells floating under the microscope, an equal volume of medium was immediately added to terminate the digestion reaction.

1.5 Prepare the centrifuge tube in advance, repeatedly blow every corner of the bottom of the medium with a pipette, transfer the digested cells to the centrifuge tube, put the medium under an inverted microscope to observe the residual amount of cells before balancing, and then centrifuge at 1200rpm/7min .

1.6 After the centrifugation is complete, take out the centrifuge tube, discard the supernatant, and use the tip of a pipette to blot up the remaining supernatant as much as possible. Add 4ml of medium to resuspend the precipitate according to the passage ratio of 1:4, and mix well. After homogenization, inoculate 1ml of cell suspension in each required medium, and then add 20-25ml of medium to a 150mm cell culture dish.

1.7 Mark the cell name, generation number, subculture date, and operator name on the surface of the medium, shake the medium sufficiently to ensure uniform inoculation, and then transfer the cells to an incubator at 37°C, 5% CO ₂ for culture.

After passage, observe the cells every 2-3 days. If the cells do not grow to the logarithmic growth phase, replace the medium. If the cells grow to the logarithmic growth phase, follow the passage operation, usually after the third or fourth passage It can be used in the preparation of cosmetics, wherein in the cell proliferation experiment in Example 2, the cells were cultured and passed to the third generation, and the cells used in the production of cosmetics in Example 3 were the cells were cultured and passed to the fourth generation.

2. Cell replacement method

2.1 Take out the cells that have reached the normal passage number from the incubator, and observe their growth status under an inverted microscope. If the exponential growth has not reached the logarithmic growth phase, the medium needs to be replaced.

2.4 Refer to the method of aspirating and discarding the supernatant in step 1.2 of digestion and passage, and add an appropriate amount of fresh and complete medium according to the medium. Add an appropriate amount of medium according to the medium; ② When observed under the microscope, no dead cells and debris are produced, but only a simple change in pH value, and only the medium in the medium needs to be discarded, and an appropriate amount of medium is added according to the needs of the medium.

2.5 Transfer the medium after the liquid exchange to the incubator at 37°C, 5% CO ₂ to continue culturing.

Example 2 Stimulation of the culture supernatant of oral cavity-related stem cells on the growth and proliferation of other cells

1. Brdu proliferation experiment:

According to the method in Example 1, the primary (i.e. zeroth generation) gingival stem cells and apical papilla stem cells were cultured to the third generation, and the cell supernatant after 2 days of culture was taken and transferred to the third generation of cells just seeded on a 6-well plate. Human periodontal ligament stem cells (PDLSC), jaw bone marrow stem cells (BMSC), and dental pulp stem cells (DPSC) were further cultured (the medium is phenol red-free medium, Cat. No. 41061-029, 37°C, 5% CO ₂ ) Each well contains 1ml of cell suspension and 2ml of cell supernatant transferred from gingival stem cells and apical papilla stem cells, and then detects the proliferation ratio of human periodontal ligament stem cells, jaw bone marrow stem cells, and dental pulp stem cells. Among them, the periodontal ligament stem cells and dental pulp stem cells were cultured to the third generation according to the method in Example 1, and the bone marrow stem cells of the jaw were obtained from the bone slices taken out of the alveolar fossa during tooth extraction, collected by centrifugation, inoculated and cultured, and passed to the third generation . Among them, the control group (vehicle) continued to culture the above-mentioned third generation human periodontal ligament stem cells (PDLSC), jaw bone marrow stem cells (BMSC) and dental pulp with the α-MEM medium (product number 11095-080) that had not been cultured. stem cells (DPSCs).

1. Inoculate the cells to be tested on a 6-well plate containing coverslips and grow for 24 hours;

2. Prepare BrdU reagent and cell culture medium at a ratio of 1:100, and preheat at 37°C in advance;

3. Discard the cell supernatant in step 1, and add the BrdU-containing medium prepared in step 2;

4. Continue to culture the cells for 12-16 hours;

5. Discard the cell supernatant and wash twice with phosphate buffered saline;

6. Fix with 70% ethanol at 4°C for 30 minutes;

7. Wash 3 times with phosphate buffered saline, 2 minutes each time;

8. Stain the cells according to the BrdU staining kit (the staining kit is from Thermo Fisher Company, product number 93-3944; BrdU is from Thermo Fisher Company, product number 00-0103);

9. Mix reagent 2 with pure water 1:1, incubate the cells for 30 minutes, then wash with phosphate buffer 3 times, 2 minutes each time;

10. Add reagent 3 and incubate at room temperature for 10 minutes, then do not wash;

11. Add reagent 4 and incubate at room temperature for 60 minutes, then wash with phosphate buffer 3 times, 2 minutes each time;

12. Add reagent 5 and incubate at room temperature for 10 minutes, then wash with phosphate buffer 3 times, 2 minutes each time;

13. To prepare the chromogenic solution, take one drop each of 6A, 6B, and 6C into 1ml of pure water, and vortex;

14. Cover the cell surface with the chromogenic solution, observe the color change under a microscope, and determine the color development time according to the color change, usually for 5 minutes, and then stop the color reaction with pure water;

15. Wash the cells with ultrapure water, add reagent 7 to return to blue for 2 minutes, and then wash with pure water;

16. Dehydrate the cells in 70%, 80%, 90%, 95%, 100%-1, 100%-2 ethanol in sequence for 1 minute, then clear the cells with xylene-1, xylene-2 minute;

17. Take out the cell cover slip, drop reagent 8 on the slide, and mount the slide;

18. Randomly intercept 4 fields of view from each film under the microscope;

19. Count the number of positive cells in the 4 fields of view, and then conduct statistical analysis. The experimental results are shown in Figure 1 and Figure 2. It can be seen that the culture supernatant of apical papilla stem cells and gingival stem cells can significantly stimulate the proliferation of human periodontal ligament stem cells, jaw bone marrow stem cells and dental pulp stem cells.

2. ELISA detection of cell supernatant components experiment:

According to the method of Example 1, the primary (i.e. zeroth generation) gingival stem cells were cultured to the third generation, and the cell supernatant after 2 days of culture was taken, and human growth hormone (GH) was detected using an R&D Systems imported subpackaging kit. Epidermal growth factor (EGF), endothelial cell growth factor (ECGF), tumor necrosis factor (TNF-a), interleukin-6 (IL-6) and gamma-interferon (IFN-gamma) content. The control was the original phenol red-free α-MEM medium (Cat. No. 41061-029) that had not been cultured.

The steps of this kit are the same except that the horseradish peroxidase (HRP)-labeled detection antibody used is itself and the concentration of the standard is different.

1. Take out the required strips from the aluminum foil bag after equilibrating at room temperature for 20 minutes.

2. Set standard wells, add 50 μl of different concentrations of standard wells to each well

3. Add 10 μl of the sample to be tested first in the sample well, then add 40 μl of the sample diluent; do not add to the blank well

4. Add 100 μl of horseradish peroxidase (HRP)-labeled detection antibody to each well except the blank well, seal the reaction well with a sealing film, and incubate in a 37-degree water bath or incubator 60min.

5. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 5 times.

6. Add 50 μl of substrates A and B to each well, and incubate at 37 degrees for 15 minutes in the dark.

7. Add 50 μl of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

8. Draw a standard curve: In the Excel worksheet, use the concentration of the standard as the abscissa and the OD value as the ordinate to draw the linear regression curve of the standard, and calculate the concentration of each sample according to the curve equation. The results are shown in Figure 3.

It can be seen from Figure 3 that compared with the original α-MEM medium without phenol red, TNF-α in the culture supernatant of cells cultured in the culture supernatant obtained by culturing cells in α-MEM medium without phenol red , IL-6 and IFN-γ content significantly increased (compared with the culture medium of uncultured cells, TNF-α, 37.5pg/ml vs. 30.5pg/ml; IL-6, 485pg/ml vs. 360pg /ml; IFN-γ, 213pg/ml vs.175pg/ml), while growth hormone (GH), epidermal growth factor (EGF) and endothelial growth factor (ECGF) contents had no significant difference, indicating that the cultured stem cells The medium secreted TNF-α, IL-6 and IFN-γ.

Example 3 The effect experiment of the culture supernatant of oral cavity-related stem cells being used to improve the skin

1) Lyophilization of cell culture supernatant

Take the culture supernatant of the fourth passage gingival stem cells. Transfer the culture supernatant to a new 150mm petri dish with a pipette, and store it in a freezer at -80°C for 24 hours or more, then put the frozen supernatant into a lyophilizer, and store it at -50°C Freeze-dried until the sample was dry powder, the results are shown in Figure 4.

2) Dissolution of cell culture supernatant lyophilized powder and mixing with cosmetic matrix

Get the freeze-dried powder in a 150mm petri dish after freeze-drying, add 5ml of serum to dissolve, then mix with 100g of cosmetic base, and said base is Cetaphil Shute skin moisturizing cream. The ingredients in Cetaphil Soothe Body Balm include: Water, Glyceryl Methacrylate (and) Propylene Glycol, Petrolatum, Dicapryl Ether, PEG-5 Glyceryl Stearate, Glycerin, Dimethicone Alcohol, Cetyl Alcohol, Sweet Almond Oil, Acrylic/C10-30 Alkyl Acrylic Polymer, Ascopheryl Acetate, Phenoxyethanol, Benzyl Alcohol, EDTA, Sodium Hydroxide, Lactic Acid.

3) The effect comparison between the cosmetics containing cell culture supernatant prepared by the present invention and ordinary cosmetics

Comparing the cosmetic containing cell supernatant solution in the above 2) with the cosmetic matrix itself, the supernatant of gingival stem cells is used, which has no special requirements for human skin. Apply it once a day in the morning and evening, and use it continuously for 30 days. The effect picture See Figure 5 and Figure 6, respectively.

Among them, the upper left picture and the lower left picture of Figure 5 are the effect pictures of the left and right faces after using the cosmetic matrix for 20 days, and the right picture of the upper picture and the lower right picture are the cell-containing pictures after using the cosmetic matrix for 30 days. Left and right face renderings of cultured supernatant cosmetic matrix.

It can be seen from Fig. 5 that compared with the cosmetic matrix, the cosmetic containing cell culture supernatant of the present invention can significantly improve skin quality, including reducing or even eliminating wrinkles, lightening spots, and making the skin firmer and ruddy.

The left picture in Fig. 6 is before using the cosmetic, and the right picture is the effect picture after using the cosmetic containing the cell culture supernatant of the present invention for 30 days, and the application method is the same as above. It can be seen that compared with before use, the bulge of the scar is significantly reduced, and the color is obviously lightened.

Example 4 Isolation and preparation method of oral cavity-related stem cells of the present invention

The oral cavity-related stem cells of the present invention can be isolated and prepared by well-known methods in the art, for example, the enzymatic digestion method after mixing type I collagenase and dispase at a ratio of 1:1 is usually used for extraction, and the digestion time is 1 h, or for example, refer to Lan Ma, Yusuke Makino, Haruyoshi Yamaza, et al. Cryopreserved Dental Pulp Tissues of Exfoliated Deciduous Teeth Is a Feasible Stem Cell Resource for Regenerative Medicine., PLoS One. 2012;7(12):e51777.

The growth status of oral cavity-related stem cells (including gingival stem cells, periodontal ligament stem cells, dental pulp stem cells, apical tooth papilla stem cells, deciduous tooth pulp stem cells and dental follicle precursor stem cells) isolated and prepared by the present invention under a microscope after primary culture As shown in Figure 7.

For cell phenotype identification methods and adipogenic and osteogenic differentiation identification methods, please refer to the following literature:

GANG DING, WEI WANG, YI LIU, et al. Effect of Cryopreservation on Biological and Immunological Properties of Stem Cells From Apical Papilla. JOURNAL OF CELLULAR PHYSIOLOGY, J. Cell. Physiol. 223:415–422, 2010.

F Feng1, K Akiyama2, Y Liu, et al, Utility of PDL progenitors for in vivotissue regeneration: a report of 3 cases. Oral Diseases (2010) 16, 20–28.

Takayoshi Yamaza, Akiyama Kentaro, Chider Chen, et al.

Immunomodulatory properties of stem cells from human exfoliated deciduous teeth. Stem Cell Research & Therapy 2010,1:5.

The results of cell immunophenotype identification and adipogenic and osteogenic differentiation identification of dental pulp stem cells are shown in Figures 8 and 9, respectively;

The results of cell immunophenotype identification and adipogenic and osteogenic differentiation identification of periodontal ligament stem cells are shown in Figures 10 and 11, respectively;

The results of cell immunophenotype identification and adipogenic and osteogenic differentiation identification of gingival stem cells are shown in Figures 12 and 13, respectively;

The results of cell immunophenotype identification and adipogenic and osteogenic differentiation identification of apical papilla stem cells are shown in Figures 14 and 15, respectively;

The results of cell immunophenotype identification and adipogenic and osteogenic differentiation identification of deciduous dental pulp stem cells are shown in Figures 16 and 17, respectively.

It can be seen that the isolated and cultured dental pulp stem cells, periodontal ligament stem cells, gingival stem cells, apical tooth papilla stem cells and deciduous tooth pulp stem cells conform to the expression characteristics of mesenchymal stem cells (for example: mesenchymal stem cells highly express CD44, CD59, CD146, STRO-1; do not express CD34, CD45), and have multiple differentiation capabilities, can differentiate into bone tissue and adipose tissue, and thus belong to mesenchymal stem cells.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (24)

1. Use of mesenchymal stem cell culture or its culture supernatant for preparing a cosmetic composition. 2. The use of the mesenchymal stem cell culture or its culture supernatant for preparing preparations for improving skin quality, promoting skin tissue repair, reducing and/or reducing scars, or slowing down skin aging. 3. Use of the secretion of mesenchymal stem cells for the preparation of a cosmetic composition. 4. Use of the secretion of mesenchymal stem cells for preparing preparations for improving skin quality, promoting skin tissue repair, reducing and/or reducing scars, or slowing down skin aging. 5. A method for improving skin quality, promoting skin tissue repair, alleviating and/or desalinating scars, or slowing down skin aging, said method comprising giving an effective amount of mesenchymal stem cell culture or its The step of culture supernatant, or secretion of mesenchymal stem cells, or cosmetic composition or preparation containing mesenchymal stem cell culture or its culture supernatant, or cosmetic composition or preparation containing secretion of mesenchymal stem cells . 6. The use according to claim 1 or 2, or the method according to claim 5, wherein said mesenchymal stem cell culture is a culture obtained by culturing mesenchymal stem cells in a medium suitable for culturing mesenchymal stem cells. 7. The use according to claim 3 or 4, or the method according to claim 5, wherein the secretion of mesenchymal stem cells is the secretion obtained by culturing mesenchymal stem cells in a medium suitable for culturing mesenchymal stem cells. 8. The use or method according to claim 6 or 7, wherein said medium suitable for culturing mesenchymal stem cells does not contain phenol red. 9. The use or method according to claim 6 or 7, wherein said culture culture or its culture supernatant obtained by culturing mesenchymal stem cells in a medium suitable for culturing mesenchymal stem cells, or the obtained secretion is a culture medium suitable for culturing mesenchymal stem cells. The medium for mesenchymal stem cells is a culture obtained by culturing mesenchymal stem cells at the third or fourth passage, or its culture supernatant, or the obtained secretion. 10. The use or method according to any one of claims 1-9, wherein said mesenchymal stem cells are selected from the group consisting of gingival stem cells, periodontal ligament stem cells, dental pulp stem cells, apical tooth papilla stem cells, deciduous tooth pulp stem cells and dental follicles Precursor stem cells, bone marrow mesenchymal stem cells (such as jaw bone marrow mesenchymal stem cells) and adipose mesenchymal stem cells. 11. The use or method according to any one of claims 6-9, wherein the medium suitable for culturing mesenchymal stem cells is selected from MEM medium, DMEM medium and DMEM:F12 medium. 12. The use or method according to claim 11, wherein said medium suitable for culturing mesenchymal stem cells further contains serum. 13. The use or method according to claim 11 or 12, wherein said medium suitable for culturing mesenchymal stem cells does not contain antibiotics. 14. The use or method of any one of claims 1-13, wherein said mesenchymal stem cell culture or its culture supernatant, or said mesenchymal stem cell secretion contains TNF-α, IL-6 and IFN-γ. 15. The use or method according to any one of claims 1-14, wherein the mesenchymal stem cell culture or its culture supernatant, or the secretion of mesenchymal stem cells is freeze-dried powder. 16. The use or method of any one of claims 1-14, wherein the mesenchymal stem cell culture or its culture supernatant, or the secretion of mesenchymal stem cells is a solution, and the solution is prepared by the following method : dissolving the lyophilized powder obtained from the mesenchymal stem cell culture or its culture supernatant, or the secretion of the mesenchymal stem cells in a solvent. 17. The use or method of claim 16, wherein the solvent is selected from serum, saline, glucose solution (eg isotonic glucose solution) and glycerol. 18. The use or method according to any one of claims 1-17, wherein said cosmetic composition or preparation further comprises cosmetically acceptable excipients. 19. The use of claim 2 or 4 or the method of claim 5, wherein said improving skin texture is selected from the group consisting of reducing wrinkles, lightening pigmentation, making skin firmer and making skin more rosy. 20. A composition comprising TNF-alpha, IL-6 and IFN-gamma. 21. The composition of claim 20 which is a cosmetic composition. 22. Use of the composition according to claim 20 for the preparation of a cosmetic composition. 23. The purposes of claim 22, wherein said cosmetic composition is used to improve skin texture, promote skin tissue repair, reduce and/or fade scars, or slow down skin aging, preferably, wherein said improvement of skin texture is selected from Reduces wrinkles, fades dark spots, makes skin firmer and more rosy. 24. The composition of claim 20 is used in the preparation of preparations or cosmetic compositions for improving skin quality, promoting skin tissue repair, reducing and/or reducing scars, or slowing down skin aging.