CN116650394A - Stem cell anti-aging mask - Google Patents
Stem cell anti-aging mask Download PDFInfo
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- CN116650394A CN116650394A CN202310836882.4A CN202310836882A CN116650394A CN 116650394 A CN116650394 A CN 116650394A CN 202310836882 A CN202310836882 A CN 202310836882A CN 116650394 A CN116650394 A CN 116650394A
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- A—HUMAN NECESSITIES
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/0212—Face masks
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K8/00—Cosmetics or similar toiletry preparations
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Abstract
The invention provides a stem cell anti-aging facial mask, and belongs to the technical field of skin aging. The facial mask for resisting aging of the stem cells comprises facial mask liquid and facial mask paper, wherein the core component of the facial mask liquid is adipose-derived mesenchymal stem cells culture supernatant, and when the supernatant is prepared, P3 generation adipose-derived mesenchymal stem cells are inoculated into a culture dish; after the cells are fully attached, replacing the culture medium with a complete culture medium containing 10% FBS and Feleucoin-K3 polypeptide; after 24 hours of treatment, the culture medium is replaced by a serum-free culture medium, and the culture is continued for 48 hours; collecting culture medium, centrifuging at 4deg.C at 200g, discarding precipitate, and filtering with 0.22 μm filter membrane to obtain the culture supernatant of mesenchymal stem cells. The adipose mesenchymal stem cell culture supernatant after pretreatment of the Feleucoin-K3 polypeptide more obviously reverses the decrease of the activity of skin fibroblasts and aging caused by ultraviolet, thereby more effectively realizing the aging resistance of the skin.
Description
Technical Field
The invention belongs to the technical field of skin aging, and particularly relates to a stem cell anti-aging mask.
Background
Skin aging is a series of natural changes that occur with age, including skin sagging, fine lines, wrinkles, spots, pigmentation, loss of elasticity, and the like. During skin aging, collagen and elastic fibers of the dermis layer gradually decrease, resulting in skin becoming loose and thin. The reduction in the number of keratinocytes in the epidermis results in a decrease in the skin's ability to retain water and the increased tendency to dryness and wrinkles. At the same time, a reduced number of pigment cells leads to skin pigmentation and spots, while the generation of free radicals leads to an accelerated skin ageing.
Stem cell supernatant is a common skin care product ingredient containing growth factors and cytokines that stimulate skin cell regeneration and repair, thereby delaying skin aging. However, the effect of using stem cell supernatant alone to inhibit skin aging is general, and therefore, how to effectively improve the effect of stem cell supernatant is a problem that needs to be solved at present.
Disclosure of Invention
The invention aims to provide a stem cell anti-aging facial mask prepared from stem cell supernatant with more excellent effect.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides a stem cell anti-aging facial mask which consists of facial mask paper and facial mask liquid, wherein the core component of the facial mask liquid is the supernatant of a adipose-derived mesenchymal stem cell culture liquid.
Preferably, the preparation method of the adipose-derived mesenchymal stem cell culture supernatant comprises the following steps:
(1) Inoculating the P3 generation adipose-derived mesenchymal stem cells into a culture dish;
(2) After the cells are fully attached, replacing the culture medium with a complete culture medium containing 10% FBS and Feleucoin-K3 polypeptide;
(3) After 24 hours of treatment, the culture medium is replaced by a serum-free culture medium, and the culture is continued for 48 hours;
(4) Collecting culture medium, centrifuging at 4deg.C at 200g, discarding precipitate, and filtering with 0.22 μm filter membrane to obtain the culture supernatant of mesenchymal stem cells.
Preferably, the concentration of Feleucoin-K3 polypeptide in the complete medium is 25-50. Mu.g/mL.
Preferably, the mask liquid further comprises a mask liquid auxiliary material, wherein the mask liquid auxiliary material comprises sterile water, rose essential oil, trehalose or aloe extract.
Secondly, the invention provides application of a polypeptide in preparation of a preparation for promoting adipose-derived mesenchymal stem cells to secrete VEGF and bFGF, wherein the polypeptide is Feleucoin-K3 polypeptide.
Secondly, the invention provides application of a polypeptide in preparation of a preparation for improving the promotion of the expression of a Collagen I gene in a skin fibroblast by using a culture supernatant of adipose tissue-derived stem cells, wherein the polypeptide is Feleucodin-K3 polypeptide.
Secondly, the invention provides application of the polypeptide in improving the inhibition of the adipose-derived mesenchymal stem cell culture supernatant on skin aging, which is characterized in that the polypeptide is Feleucoin-K3 polypeptide.
In addition, the invention provides application of a polypeptide in preparing a preparation for improving the ultraviolet aging resistance of adipose-derived mesenchymal stem cell culture supernatant, and is characterized in that the polypeptide is Feleucoin-K3 polypeptide.
Preferably, the polypeptide increases the ability of adipose mesenchymal stem cell culture supernatant to reverse the reduction in skin fibroblast activity caused by ultraviolet light.
Preferably, the polypeptide increases the ability of adipose mesenchymal stem cell culture supernatant to reverse aging of skin fibroblasts caused by ultraviolet light.
The invention has the beneficial effects that:
the invention utilizes Feleucoin-K3 polypeptide to effectively improve the secretion of VEGF and bFGF of adipose-derived mesenchymal stem cells.
Meanwhile, the invention utilizes the adipose-derived mesenchymal stem cell culture supernatant after Felein-K3 polypeptide pretreatment to improve the gene expression of the culture supernatant on Collagen I, and the invention more obviously reverses the reduction of the activity and aging of skin fibroblasts caused by ultraviolet.
Therefore, the adipose mesenchymal stem cell culture supernatant treated by the Feleucoin-K3 polypeptide is used for preparing the efficient stem cell anti-aging mask, so that the skin anti-aging effect is realized more effectively.
Drawings
FIG. 1 shows VEGF content of different experimental treatment groups;
FIG. 2 shows bFGF content of different experimental treatment groups;
FIG. 3 shows the relative expression level differences of the CollagenI gene in different experimental treatment groups;
FIG. 4 shows the differences in the A values of skin fibroblasts from different treatment groups;
FIG. 5 shows the protein expression differences of senescence key protein P21 in skin fibroblasts of different treatment groups;
in the above figures, P < 0.05, P < 0.01, and P < 0.001.
Detailed Description
The polypeptide has rich and various structural and functional characteristics and is widely applied to the fields of medicines, foods, cosmetics and the like, so that the application of the polypeptide in improving stem cell aging resistance is researched. Felein-K3 is a polypeptide consisting of 9 amino acids, the amino acid sequence of the polypeptide is FLKLLKKLL, and the Felein-K3 polypeptide has various biological activities and particularly has obvious effects on immunoregulation and antibiosis. However, the effect of Feleucodin-K3 on stem cell regulation is not clear at present, and thus the present invention has been studied.
Example 1 polypeptide treatment of adipose mesenchymal Stem cells for the detection of Vascular Endothelial Growth Factor (VEGF) and basic fibroblast growth factor (bFGF)
The experimental method comprises the following steps:
resuscitating the frozen adipose-derived mesenchymal stem cells of the company and culturing until the generation of P3; the P3 generation adipose mesenchymal stem cells are digested by pancreatin and prepared into single cell suspension according to the proportion of 5 multiplied by 10 per well 4 Is inoculated in a 6-well culture plate; after the cells were completely adherent, the blank control group was supplemented with DMEM medium (containing serum), the experimental group 1 was supplemented with DMEM medium (containing 10% FBS and 25 μg/mL felucin-K3), the experimental group 2 was supplemented with DMEM medium (containing 10% FBS and 50 μg/mL felucin-K3), and 3 replicates were set per group; after 24 hours of treatment, the culture medium of each group is sucked and removed, and replaced by a serum-free DMEM culture medium, and the culture is continued for 48 hours; collecting culture medium, centrifuging at 4deg.C for 15min at 200g, discarding precipitate, and filtering with 0.22 μm filter membrane to obtain culture supernatant of desired fat stem cells; the contents of VEGF and bFGF in the culture supernatant were detected using ELISA kits for VEGF and bFGF, thereby determining whether Felein-K3 polypeptide treatment can effectively promote the secretion of VEGF and bFGF by adipose-derived mesenchymal stem cells.
Experimental results:
the results of example 1 are shown in fig. 1 and 2, and it can be seen from fig. 1 and 2 that adipose-derived mesenchymal stem cells pretreated with the polypeptide can secrete more VEGF and bFGF, and the effect becomes more remarkable with increasing concentration.
Example 2: effect of culture supernatants of polypeptide-treated adipose Stem cells on Collagen I (Collagen I) expression in skin fibroblasts
The experimental steps are as follows:
the experimental procedure of reference example 1 was followed to collect a fat stem cell culture supernatant that was not treated with the polypeptide (culture supernatant 1) and a fat stem cell culture supernatant treated with 50. Mu.g/mL of Feleucoin-K3 polypeptide (culture supernatant 2); human skin fibroblast HSF was inoculated into 6-well plates, and when the cell density reached about 80%, a blank control group was added with new DMEM medium (containing 10% FBS), an experimental group 1 was added with serum-added culture supernatant 1 (containing 10% FBS), an experimental group 2 was added with serum-added culture supernatant 2 (containing 10% FBS), and 3 replicates were set for each group; after cell culture for 24 hours, adding TRIZOL reagent to extract RNA of each group, and carrying out reverse transcription on the RNA into cDNA by using a reverse transcription kit after the quality detection of the RNA is qualified; the relative expression level of Collagen I was detected by fluorescent quantitative PCR.
Experimental results:
as shown in FIG. 3, the experimental results of example 2 show that the expression level of the Collagen I mRNA in human skin fibroblasts treated with culture supernatant 2 is significantly higher than that in human skin fibroblasts treated with culture supernatant 1, indicating that the ability of the adipose tissue stem cell supernatant to promote the expression of the Collagen I gene in skin fibroblasts after pretreatment with Feleucon-K3 polypeptide is improved.
Example 3: effect of treated adipose mesenchymal Stem cell supernatant on skin fibroblast Activity
The experimental procedure of reference example 1 was followed to collect a fat stem cell culture supernatant that was not treated with the polypeptide (culture supernatant 1) and a fat stem cell culture supernatant treated with 50. Mu.g/mL of Feleucoin-K3 polypeptide (culture supernatant 2); 2000 human dermal fibroblast HSF/well were inoculated into 96-well plates, and when cells were fully adherent, the blank group was supplemented with fresh DMEM medium (containing 10% FBS), the experimental group 1 was supplemented with fresh DMEM medium (containing 10% FBS) and UVB 80mJ/cm was irradiated under sterile conditions 2 Experiment group 2 serum-added culture supernatant 1 (containing 10% FBS) was added and UVB 80mJ/cm was irradiated under sterile culture hood 2 Experiment group 3 culture supernatant 2 (containing 10% FBS) added with serum was added and UVB 80mJ/cm was irradiated under sterile culture hood 2 The method comprises the steps of carrying out a first treatment on the surface of the Each group was examined for absorbance at 450nm using CCK-8 48h post-irradiation.
Experimental results:
the decrease in proliferation activity and aging of skin cells caused by ultraviolet rays are the main types of skin aging, so that the influence of the adipose mesenchymal stem cell culture fluid treated by Feleucoin-K3 polypeptide on the inhibition of skin fibroblast aging caused by ultraviolet rays is examined. The results of the experiment are shown in fig. 4, and it can be seen from fig. 4 that the absorbance of the experiment group 1 is obviously reduced compared with that of the blank control group, which indicates that the ultraviolet injury simulation of the invention is successful. The increase in absorbance of experiment group 2 compared to experiment group 1 suggests that inhibition of cell activity by ultraviolet rays can be reduced when skin fibroblasts are treated with a culture solution of adipose-derived mesenchymal stem cells. Compared with the experimental group 2, the absorbance of the experimental group 3 is higher, which shows that the adipose-derived mesenchymal stem cell culture solution treated by the eleucin-K3 polypeptide has better effect on the reduction of the activity of the skin fibroblasts caused by ultraviolet rays.
Example 4
The experimental method comprises the following steps:
the experimental procedure of reference example 1 was followed to collect a fat stem cell culture supernatant that was not treated with the polypeptide (culture supernatant 1) and a fat stem cell culture supernatant treated with 50. Mu.g/mL of Feleucoin-K3 polypeptide (culture supernatant 2); human skin fibroblast HSF was inoculated into 6-well culture plates, when cells were fully adherent, a blank control group was added with fresh DMEM medium (containing 10% FBS), test group 1 was added with fresh DMEM medium (containing 10% FBS) and irradiated with UVB 80mJ/cm 2 under a sterile culture hood, test group 2 was added with serum-added culture supernatant 1 (containing 10% FBS) and irradiated with UVB 80mJ/cm 2 under a sterile culture hood, test group 3 was added with serum-added culture supernatant 2 (containing 10% FBS) and irradiated with UVB 80mJ/cm under a sterile culture hood 2 The method comprises the steps of carrying out a first treatment on the surface of the After 48 hours of irradiation, the culture medium is removed, after rinsing by PBS, protein lysate is added to extract protein samples of each group; preparing SDS electrophoresis gel, mounting an electrophoresis frame, carrying out electrophoresis after sample loading, and stopping electrophoresis when bromophenol blue reaches the bottom; assembling an electric rotating clamp according to a classical sandwich model, and adding an electric rotating buffer solution and an ice box to perform electric rotation; placing the membrane into skimmed milk powder after the electric conversion is finished, and sealing for 1h at room temperature; adding P21 and GAPDH primary antibodies, incubating for 1h at room temperature, and washing the membrane; adding a secondary antibody, incubating for 1.5h at room temperature, and washing the membrane; developing exposure is performed.
Experimental results:
the results obtained in example 4 are shown in fig. 5, and from fig. 5 we can see that the expression of senescence-critical protein P21 in experimental group 1 is significantly increased compared with that in control group, indicating that the successful uv treatment adopted in the present invention resulted in senescence of skin fibroblasts; compared with the experimental group 1, the expression of the senescence-critical protein P21 in the experimental group 2 is reduced, which indicates that the aging caused by ultraviolet can be effectively reduced by using the adipose mesenchymal stem cell culture supernatant to treat the skin fibroblasts; compared with the experimental group 2, the decrease of the senescence-critical protein P21 of the experimental group 3 is more remarkable, which indicates that the supernatant of the adipose-derived stem cell culture treated by the Felein-K3 polypeptide can more effectively reduce the skin cell senescence caused by ultraviolet.
In view of the above examples, it can be seen that pretreatment of adipose mesenchymal stem cells with felein-K3 polypeptide can significantly increase the secretion of VEGF and bFGF, and can increase the gene expression level of adipose mesenchymal stem cell culture supernatant on collagen I in skin fibroblasts to some extent. Meanwhile, compared with untreated cells, the adipose-derived mesenchymal stem cell culture supernatant after the treatment of the Felein-K3 polypeptide can remarkably reduce the reduction of the activity and aging of skin cells caused by ultraviolet. Therefore, by utilizing the above characteristics, we can prepare an anti-aging cosmetic having more excellent effects.
Claims (10)
1. The stem cell anti-aging facial mask is characterized by comprising facial mask paper and facial mask liquid, wherein the core component of the facial mask liquid is the supernatant of the adipose-derived mesenchymal stem cell culture liquid.
2. The stem cell anti-aging facial mask of claim 1, wherein the preparation method of the adipose-derived mesenchymal stem cell culture supernatant comprises the following steps:
(1) Inoculating the P3 generation adipose-derived mesenchymal stem cells into a culture dish;
(2) After the cells are fully attached, replacing the culture medium with a complete culture medium containing 10% FBS and Feleucoin-K3 polypeptide;
(3) After 24 hours of treatment, the culture medium is replaced by a serum-free culture medium, and the culture is continued for 48 hours;
(4) Collecting culture medium, centrifuging at 4deg.C at 200g, discarding precipitate, and filtering with 0.22 μm filter membrane to obtain the culture supernatant of mesenchymal stem cells.
3. The stem cell anti-aging facial mask of claim 2, wherein the concentration of Feleucin-K3 polypeptide in the complete medium is 25-50 μg/mL.
4. The stem cell anti-aging facial mask of claim 3, wherein the facial mask liquid further comprises a facial mask liquid auxiliary material comprising sterile water, rose essential oil, trehalose or aloe vera extract.
5. An application of a polypeptide in preparing a preparation for promoting adipose-derived stem cells to secrete VEGF and bFGF, wherein the polypeptide is a Felein-K3 polypeptide.
6. An application of a polypeptide in preparing a preparation for improving the promotion of the expression of a collagani gene in a skin fibroblast by using a culture supernatant of adipose tissue-derived stem cells, which is characterized in that the polypeptide is a Felein-K3 polypeptide.
7. An application of a polypeptide in improving the inhibition of skin aging of adipose-derived mesenchymal stem cell culture supernatant, which is characterized in that the polypeptide is a Felein-K3 polypeptide.
8. An application of a polypeptide in preparing a preparation for improving the ultraviolet aging resistance of a culture supernatant of adipose-derived mesenchymal stem cells, which is characterized in that the polypeptide is a Feleucoin-K3 polypeptide.
9. The use according to claim 8, wherein the polypeptide increases the ability of adipose mesenchymal stem cell culture supernatant to reverse the decrease in skin fibroblast activity caused by ultraviolet light.
10. The use according to claim 8, wherein the polypeptide increases the ability of adipose mesenchymal stem cell culture supernatant to reverse skin fibroblast senescence induced by ultraviolet light.
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