CN116987151A - Preparation method and application of umbilical cord mesenchymal stem cell supernatant in skin repair and anti-aging skin care products - Google Patents
Preparation method and application of umbilical cord mesenchymal stem cell supernatant in skin repair and anti-aging skin care products Download PDFInfo
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- CN116987151A CN116987151A CN202310965180.6A CN202310965180A CN116987151A CN 116987151 A CN116987151 A CN 116987151A CN 202310965180 A CN202310965180 A CN 202310965180A CN 116987151 A CN116987151 A CN 116987151A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/59—Mixtures
- A61K2800/592—Mixtures of compounds complementing their respective functions
- A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The application relates to a preparation method and application of umbilical cord mesenchymal stem cell supernatant in skin repair and anti-aging skin care products. The application is based on the characteristic that the placenta is rich in antioxidant polypeptide,the polypeptide D25-F7 with strong antioxidation property is obtained by separating and identifying from deer placenta, and has strong DPPH scavenging ability and ABTS scavenging ability on H 2 O 2 The induced HDF cell damage has a protective effect. The polypeptide and the supernatant of the umbilical cord mesenchymal stem cells are prepared into a composition, so that the hydroxyproline content in skin can be effectively improved after the composition is used, the P53 protein expression reduction can be obviously reduced, the antioxidation effect is effectively realized, and the market application value is good.
Description
Technical Field
The application relates to the field of biological treatment, in particular to a preparation method and application of umbilical cord mesenchymal stem cell supernatant in skin repair and anti-aging skin care products.
Background
Aging refers to the gradual decline of physiological functions of the body with age. As an important risk factor, the process of aging is accompanied by the development of a number of chronic diseases including diabetes, heart disease, cancer, and Alzheimer's disease. With the continuous improvement of living standard and medical level, the average life of modern human beings is greatly prolonged. It is estimated that by 2050, the global population of elderly people over 60 years old or more than 20 billion. With the increase of the aging population, the incidence rate of the aging-related chronic diseases is increased year by year, which not only seriously threatens the life health of the aged, but also brings heavy social, medical and economic burdens to countries around the world. The pursuit of longevity is not limited to the prolongation of survival time, but the pursuit of life cycle maximization is also focused on how to improve the quality of life of the aged, thereby realizing healthy aging.
Skin aging refers to the aging and sexual damage of skin function, and the protective ability, regulation ability and the like of the skin on the body are reduced, so that the skin cannot adapt to the change of internal and external environments, and the overall appearance conditions such as color, luster, shape, texture and the like are changed. Skin aging is also caused by aging marks on everyone, such as loss of moisture from the skin, dry skin, wrinkles, pigmentation, hypomnesis, hair loss, decreased resistance, etc., which occur gradually. The intrinsic cause is loss of elastin in the superficial fascia layers, weakening of the pulling function, and the web breaks to form wrinkles.
At present, for the treatment of skin aging, the following common therapeutic substances are mainly used. Polyphenols, such as stilbenes, flavonoids and chalcones, are effective in improving a variety of aging-related phenotypes in vitro and in vivo, including oxidative stress, inflammation, impaired protein homeostasis and cellular aging. Resveratrol is a natural nutrient substance rich in grape, and can activate AMPK and Sirt1 signal paths, remove active oxygen and free radicals, relieve oxidative stress, reduce the level of pro-inflammatory factors, regulate and control the function of monocytes, and exert anti-inflammatory and antioxidant activities, thereby positively affecting the improvement of skin aging-related diseases. Curcumin is a polyphenol substance extracted from turmeric, has various activities such as antioxidation, anti-inflammatory, anti-tumor and the like, and mainly plays a role in regulating and controlling skin aging through a plurality of signal paths. Quercetin is a flavonoid compound widely existing in fruits, leaves, stems and roots of plants, has good capabilities of resisting oxidative stress and reducing ROS (reactive oxygen species) generation in various modes of organisms, and can inhibit skin aging.
Bioactive peptides (BAP) are peptide compounds which are beneficial to the life activities of organisms or have physiological effects, and are polypeptides with relative molecular mass less than 6000Da and multiple biological functions. The molecular structure is a molecular polymer between amino acids and proteins, is small enough to be composed of two amino acids, is large enough to be formed by connecting dozens of amino acids through peptide bonds, and can be modified through phosphorylation, glycosylation or acylation. Most bioactive peptides exist in the long chain of the protein in an inactive state, and when hydrolyzed with an appropriate protease, their molecular fragments and activity are released. At present, the idea that oxidation is an important cause of skin aging phenomenon has been confirmed. Healthy skin also needs to have a self-protection mechanism of stabilizing and scavenging free radicals, namely an antioxidant enzyme system, but the activity and content level of the antioxidant enzyme system are easy to be reduced due to the interference of various factors inside and outside a receptor, if enough antioxidant substances cannot be timely supplemented from food, free radicals are accumulated, excessive free radicals are oxidized, and the excessive free radicals are easy to become a molecular basis of cell death, so that the aging process of human skin is accelerated, and various diseases are caused. The antioxidant mechanism of the polypeptide comprises providing hydrogen for antioxidant enzyme, chelating metal ion, capturing free radical, etc. Many substances with antioxidant activity in organisms belong to proteins, and because the proteins have excellent emulsifying property and can play a role in mediating an oil-water interface, the substances have important significance in removing excessive free radicals in organisms and inhibiting skin peroxidation.
Stem cells are cells that have self-renewal capacity and multipotency. According to the source and differentiation potential of stem cells, stem cells can be classified into totipotent stem cells and specific tissue stem cells (adult stem cells are derived from adult tissues such as adipose tissue, peripheral blood, bone, skin and liver, etc.), the former can be developmentally differentiated into a complete body, and the latter is the originating cell of one or several tissues, the differentiation of which is classified into totipotent stem cells, pluripotent stem cells, multipotent stem cells, autologous serum based on the plasma technology enriched in growth factors has been shown to have remarkable photoaging resistance, various serum growth factors such as transforming growth factor beta 1 (TGF beta 1) and Epidermal Growth Factor (EGF) can promote and participate in transcription and synthesis of proteins regenerated from skin, regenerative medicine developed around the stem cell technology, the stem cells can reduce oxidative stress, inhibit apoptosis and aging, promote synthesis of extracellular matrix (ECM) and regeneration of skin, regulate inflammatory processes, have anti-wrinkle and whitening effects. Exosomes in stem cell conditioned medium have also been shown to have an anti-photoaging effect. Compared with other types of stem cells, human umbilical cord mesenchymal stem cells (hUC-MSCs) can be collected in a non-invasive manner, have lower immunogenicity and faster proliferation speed, and are the first choice for regenerative medicine.
Disclosure of Invention
Based on the characteristic that the placenta is rich in antioxidant polypeptide, the polypeptide D25-F3 with strong antioxidant property is obtained from the deer placenta by separation and identification, and the polypeptide has strong DPPH clearance ability and ABTS clearance ability.
Specifically, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.
Further, the polypeptide was verified to have a polypeptide sequence corresponding to H 2 O 2 The induced HDF cell damage has a protective effect.
In one aspect of the application, the use of a polypeptide D25-F3 having strong antioxidant properties for the preparation of an antioxidant pharmaceutical composition is provided.
Furthermore, the application also provides application of the strong antioxidant characteristic polypeptide D25-F3 and umbilical cord mesenchymal stem cell culture supernatant in preparing an antioxidant pharmaceutical composition, wherein the umbilical cord mesenchymal stem cell culture supernatant is obtained after the umbilical cord mesenchymal stem cells are cultured under the condition of containing the strong antioxidant characteristic polypeptide D25-F3.
Furthermore, the application also provides application of the strong antioxidant characteristic polypeptide D25-F3 and umbilical cord mesenchymal stem cell culture supernatant in preparing antioxidant and anti-aging cosmetics, wherein the umbilical cord mesenchymal stem cell culture supernatant is obtained by culturing the umbilical cord mesenchymal stem cells under the condition of containing the strong antioxidant characteristic polypeptide D25-F3.
Specifically, the polypeptide and the supernatant of the umbilical cord mesenchymal stem cells are prepared into a composition, so that the hydroxyproline content in skin can be effectively improved after the composition is used, the P53 protein expression reduction can be obviously reduced, and the effects of oxidation resistance and aging resistance are effectively realized.
In another aspect, the application provides a composition, e.g., a pharmaceutical composition, comprising one of the polypeptides or combinations described above formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the application may also be administered in combination therapy, i.e. in combination with other active agents. Examples of therapeutic agents that may be used in combination therapy are described in more detail below in the section on the use of the polypeptides of the application.
Further, the pharmaceutical composition of the present application further comprises a pharmaceutically acceptable carrier.
The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which a therapeutic agent is administered. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The compositions may be formulated as suppositories using conventional binders and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are described in "Remington's pharmaceutical sciences" of e.w. martin.
Advantageous effects
Based on the characteristic that placenta is rich in antioxidant polypeptide, the application separates and identifies polypeptide D25-F7 with strong antioxidant property from deer placenta, and the polypeptide has strong DPPH scavenging ability and ABTS scavenging ability on H 2 O 2 The induced HDF cell damage has a protective effect. The polypeptide and the supernatant of the umbilical cord mesenchymal stem cells are prepared into a composition, so that the hydroxyproline content in skin can be effectively improved after the composition is used, the P53 protein expression reduction can be obviously reduced, the antioxidation effect is effectively realized, and the market application value is good.
Drawings
Fig. 1: mean fluorescence intensity plots for each group;
fig. 2: ratio graphs of beta-galactosidase positive cells for each group.
Detailed Description
Specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the application are shown in the drawings, it should be understood that the application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.
Example 1 preparation and Activity characterization of antioxidant Polypeptides
Accurately weighing deer placenta of a certain mass, chopping into small pieces, freezing with liquid nitrogen, rapidly grinding and crushing (completed within 1 min), rapidly adding deionized water to prepare a substrate solution of 20g/L, rapidly heating to 90 ℃, keeping the temperature for 10min, cooling to 50 ℃, adjusting the pH to be 6.5, adding papain for enzymolysis to 100 min, adding 6000U/g of enzyme, heating to 95 ℃ after the hydrolysis is finished, keeping for 15min for enzyme deactivation treatment, cooling to room temperature, adjusting the pH to be neutral, centrifuging at a low temperature of 8000r/min for 20min, collecting supernatant, extracting fat by adopting normal hexane, and freeze-drying to obtain crude peptide.
The crude peptide was dissolved in PBS and then separated by ultrafiltration membrane with a molecular weight cut-off of 5 kDa. The ultrafiltration conditions are as follows: pressure 0.5mpa, ph=7.0, temperature 25 ℃. The retentate was collected and concentrated on a reverse osmosis membrane. Then, 10g/L of polypeptide aqueous solution was prepared, and the polypeptide aqueous solution was separated by Sephadex G-10 sephadex gel chromatography (2.6 cm. Times.50 cm), the eluent was deionized water, the elution rate was 0.5mL/min, the ultraviolet detection wavelength was 220nm, and each peak component was collected and DPPH-clearance of each component was compared. The peak component D25 with higher DPPH clearance is enriched, and further liquid phase separation is carried out: filtration was performed using a 0.45 μm syringe filter and 4mL of the sample was introduced into the preparation liquid phase. The preparation conditions of the liquid phase are as follows: the UltimateAQ-C18 (250×20mm,5 μm) column contains 0.1% (v/v) trifluoroacetic acid, the flow rate is 8mL/min, the detection wavelength is 280nm,1 min/tube is used for collecting mobile phase, each chromatographic peak is respectively mixed with corresponding test tube, DPPH of each chromatographic peak is measured, 2 chromatographic peaks with the strongest activity are determined, the two components are dried by a nitrogen blowing instrument and sent to Shanghai chemical company, and the polypeptide sequences are respectively D25-F3 and D25-F7 by Nano-LC-ESI-MS/MS analysis, wherein the amino acid sequences are respectively shown as SEQ ID NO:1 and SEQ ID NO: 2.
2 polypeptides shown in SEQ ID No. 1 and SEQ ID No. 2 were synthesized, the polypeptide powders were added to distilled water respectively to prepare solutions with a concentration of 0.01-5mg, and the IC50 of DPPH and ABTS clearance rates of the 2 polypeptides were detected by a measurement method common in the art, and the results are shown in Table 1.
Determination of antioxidant Capacity of Polypeptides of Table 1
As can be seen from Table 1, the polypeptide D25-F3 of SEQ ID NO. 1 has stronger antioxidant property, and compared with the polypeptide D25-F7 of SEQ ID NO. 2, the clearance ability against DPPH and the clearance ability against ABTS are both obviously improved (P < 0.01).
EXAMPLE 2 Re-characterization of D25-F3 antioxidant Properties
HDF cells were resuscitated and then lysed at 37℃in 5% CO 2 The culture was performed under conditions using DMEM complete medium containing 5% horse serum, 10% fetal bovine serum and 1% penicillin and streptomycin diabody solution. The culture environment is 37 ℃ and 5 percent CO 2 Cells were changed every 2-3 days. According to the growth state and density of the cells, the cells are passaged at proper time, the culture medium is discarded, 2mL of pancreatin with 0.25% EDTA is added for digestion, and 4mL of complete culture medium is added for stopping digestion immediately after the cells are rounded. Cells were collected by slow blowing and transferred to a 15mL centrifuge tube by centrifugation at 1000 rpm. Passaging experiments were performed at a ratio of 1:3 to select cells in the logarithmic growth phase.
HDF cells were grown in 2X 10 cells 5 After the cells were completely adhered, the experimental groups were each pre-incubated with the D25-F3 polypeptide containing 10, 50, 100, 200. Mu.g/mL of the pre-incubated HDF cells for 24h, and the model groups and the blank groups were each incubated with an equal amount of complete medium. Then, the medium was aspirated and removed, and the experimental group and the mold were usedGroup addition of H containing 0.4mmol/L 2 O 2 Is added to the complete medium of the culture medium (C) without H 2 O 2 The cells were incubated for an additional 4h in complete medium. Then, H-containing 2 O 2 After one PBS wash, cells were harvested by digestion. And the DCFH-DA probe is loaded by a suspension cell probe loading method. The serum-free DMEM culture medium is adopted to prepare DCFH-DA working solution with the concentration of 10 mu mol/L. After centrifuging the cells, pouring out the culture medium, adding 0.5 mM CFH-DA working solution, gently blowing and mixing uniformly, incubating in a cell culture box at 37 ℃ for 20min, and reversing and mixing uniformly every 5min during the period, so that the cells and the DCFH-DA probe are fully and uniformly mixed. Cells were washed 2 times with PBS, centrifuged at 1000rpm, resuspended in PBS containing 1% serum, and placed on ice for measurement. Selecting FITC channel to detect DCF by using Bruce Cytoflex flow cytometer, and detecting total cell number of 1×10 4 And each. The instrument parameters were set as follows, SSC voltage 100Volt, FSC voltage 120Volt, FITC voltage 5V. The levels of reactive oxygen species in the cells under corresponding treatment were reflected by analysis of the FITC mean fluorescence intensity of the cell population. The collected histograms were processed using FlowJoV10 software and the average fluorescence intensity for each treatment group was counted.
As a result, as shown in FIG. 1, the average fluorescence intensity in cells of the model group is significantly improved compared with that of the blank group, and according to the principle of the DCFH-CA kit, the ROS level in cells of the model group is significantly higher than that of the blank group, and the experimental model is established. Meanwhile, the intracellular ROS level of the HDF cells incubated by the polypeptide is obviously reduced compared with that of a model group. Experimental results show that the polypeptide has the function of inhibiting H 2 O 2 The induced HDF cell damage has a protective effect, the protective effect is gradually improved along with the increase of the concentration, the dose dependency is realized, and the fluorescence intensity is as low as (18.26+/-1.43)% and is obviously lower than that of the model group (99.86+/-5.97)% at the polypeptide concentration of 100 mug/mL.
Example 3 preparation and efficacy validation of high Activity umbilical mesenchymal Stem cell supernatant
The umbilical cord is treated within 6 hours after collection, the parts with clamp marks and blood stasis on the two sides are excised, and the umbilical cord is fully washed by PBS buffer solution containing double antibodiesPeripheral and umbilical vein lumens, the umbilical cord tissue was cut into 1.0mm sections 3 Directly adding 1.5g/L collagenase II into the small blocks to incubate for 20 hours at 37 ℃, washing the small blocks by PBS, adding 25g/L pancreatin into the small blocks to incubate for 0.5 hour at 37 ℃; adding culture medium containing 20% fetal bovine serum to neutralize pancreatin, filtering cells with 100 μm filter, and collecting filtrate; after washing with PBS and discarding the supernatant, the supernatant was resuspended in complete medium at 1X 10 6 /cm 2 Inoculating in T25 culture flask, and changing liquid completely after 3d, and changing liquid 2 times per week. P3 cells were digested with trypsin, washed 2 times with phosphate buffer and divided into 5X 10 tubes 5 Cells were incubated with mouse anti-human PE or FITC labeled mab CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD105, CD106, at 4deg.C for 30min in the absence of light, washed with phosphate buffer, fixed with 10g/L paraformaldehyde, and analyzed by flow cytometry. The results show that isolated umbilical cord mesenchymal stem cells highly express CD13, CD29, CD105, CD44, weakly express CD106, and do not substantially express CD11a, CD14, CD31, CD34, CD45.
Supernatant group well-grown P3 generation umbilical cord mesenchymal stem cells at 37 ℃ and 5% CO 2 Culturing in DMEM/F12 culture medium containing 100 μg/mLD25-F3 polypeptide for 72 hr, centrifuging at 3000r/min for 10min, collecting supernatant, filtering with 0.22 μm filter, packaging, and preserving at-80deg.C.
Supernatant control group is prepared by culturing well-grown P3 generation umbilical cord mesenchymal stem cells at 37deg.C and 5% CO 2 Culturing in DMEM/F12 culture medium for 72h, centrifuging at 3000r/min for 10min, collecting supernatant, filtering with 0.22 μm filter, packaging, and preserving at-80deg.C.
The HDF suspension was plated in 12-well plates and incubated with 10% fetal bovine serum DMEM/F12 medium, 5% CO2, at 37 ℃. When the cell density is as high as 80%, the cells are divided into 4 groups, namely a control group and H 2 O 2 Group, supernatant group, H 2 O 2 +supernatant group, H 2 O 2 +supernatant control. Wherein H is 2 O 2 Group, H 2 O 2 +culture supernatant group, H 2 O 2 200. Mu. Mol/LH for cells in the +supernatant control group 2 O 2 Serum-free DMEM/F12 medium treatment; control group, supernatant group stillTreated with serum-free DMEM/F12 medium, cultured for 4 hr, and induced to age with HDF. Thereafter, the medium was removed and washed 2 times with PBS. Control group and H 2 O 2 The group was cultured with DMEM/F12 medium containing 10% fetal bovine serum, while H 2 O 2 +culture supernatant group, H 2 O 2 The +supernatant control groups were each incubated with a mixture of 10% fetal bovine serum DMEM/F12 and the umbilical cord mesenchymal stem cell culture supernatant (1:1) described above. After the liquid exchange, each group of cells was further cultured for 48 hours. Beta-galactosidase staining was performed according to the kit instructions. Discarding the culture medium, washing with PBS for 1 time, adding 0.5mL of fixing solution, and fixing at room temperature for 15min; removing the fixed liquid, washing the cells with PBS for 3 times for 3min each time; discarding PBS, and adding 0.5mL of dyeing working solution into each hole; incubate overnight at 37℃and seal the 12-well plate with preservative film to prevent evaporation. The cells were observed under an optical microscope for staining. Photographs were taken under a mirror, and the percentage of staining positive cells out of 100 cells was counted. The results are shown in FIG. 2.
As shown in FIG. 2, H compared with the control group 2 O 2 The positive cells of the group beta-galactosidase are obviously increased; and H is 2 O 2 The +supernatant group of beta-galactosidase positive cells were significantly reduced (P < 0.05). The increased expression of beta-galactosidase is characteristic of cell senescence, and the percentage of beta-galactosidase positive cells related to the senescence of HDF cells is reduced by the treatment of the culture supernatant of umbilical cord mesenchymal stem cells, which indicates that the culture supernatant of umbilical cord mesenchymal stem cells can inhibit H 2 O 2 The induced fibroblast aging process can be seen from FIG. 2, and the supernatant obtained by culturing umbilical cord mesenchymal stem cells with the D25-F3 polypeptide has stronger characteristic of inhibiting HDF aging.
Example 4 preparation and efficacy validation of an anti-skin aging composition
Table 1 composition of each group
The preparation method comprises the following specific preparation steps: adding the component 1 into deionized water, heating to 90 ℃, maintaining for 15min for sterilization, then cooling to 75 ℃ (injection: hydroxyethyl cellulose and carbomer are independently prepared, dissolving in water, fully stirring at room temperature for dissolving, and then adding other components to enable the components to be uniformly swelled to prevent agglomeration); heating the component 2 to 75 ℃ under the condition of continuous stirring, dissolving uniformly, and preserving the heat for 10min for later use; slowly adding the component 2 into the component 1 at the rotating speed of 120r/min, and stirring for 20min; during the period, when the temperature is reduced to below 40 ℃, adding the components of the component 3 one by one, stirring uniformly, and homogenizing for 5min to obtain the skin aging resisting composition.
The Kunming mice were randomly divided into control groups and model administration groups, 10 mice each, and the mice were fed with the same feed under the conditions of cleaning, ventilation, drying, and room temperature for 25 mice, and 200mg/kg of 2% D-galactose was subcutaneously injected into the nuchal of the mice for 8 consecutive weeks to create subacute aging models. Normal control group was injected with the same volume of physiological saline. The back of the mouse is dehaired by 10cm by using dehairing cream before experiment 2 Each group was coated with 3 compositions corresponding to table 1, 1 time a day, 0.5g each time. The blank control group and the model group are smeared with the same amount of physiological saline. After 45d of administration, taking the skin of the medicine action part after the cervical dislocation is killed, and measuring the hydroxyproline content of the skin with the same area.
Skin hydroxyproline content determination: the weighed skin was rinsed in cold saline, wiped dry with filter paper, 50mg was placed in a 10ml beaker, the skin was minced with small ophthalmic scissors, cold saline was added to 5ml, and homogenization was performed with an endo-type tissue homogenizer (homogenization time 10 s/time, gap 30s, 6 consecutive times, in ice water). The prepared homogenate is measured by using a skin hydroxyproline measuring kit. The results are shown in Table 3.
TABLE 3 results of hydroxyproline content in skin
| Group of | Hydroxyproline content (μg/mg) |
| Control group | 6.34±0.36 |
| Experiment group 1 | 7.26±0.51 |
| Experiment group 2 | 5.67±0.24 |
| Positive control group | 6.20±0.47 |
After 3 experimental groups were treated with D-galactose, they formed a model of aging, and the corresponding hydroxyproline content in the skin was also significantly reduced. The experimental group 2 contained no therapeutic ingredient, and the hydroxyproline content was also smaller than that of the control group. The hydroxyproline content in both groups was also significantly increased after treatment in experimental group 1 and positive control group, especially in experimental group 1, the increase was most different and significant (P < 0.01) compared to experimental group 2.
In addition, by adopting western blotting to detect the expression condition of the P53 protein in the skin of each group, the experimental group 1 and the positive control group can obviously reduce the expression reduction of the P53 protein to 83.5% and 46.8%, and can inhibit the aging process of the skin of mice, while the expression condition of the P53 of the experimental group 2 is basically consistent with that of the blank control group, and the expression of the P53 of the model group is increased by 186.4% compared with that of the blank control group.
When practicing or testing embodiments of the application, optional methods and materials similar or equivalent to those described in the specification may be used, with the preferred methods, devices, materials described in the specification. However, before describing the materials and methods of the present application, it is to be understood that the specific size, shape, dimensions, materials, methods, means, etc. described in this specification may be changed according to conventional experimental methods and optimization purposes, and thus the present application is not limited thereto. And it is to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present application which will be limited only by the scope of the appended claims.
Claims (6)
1. A polypeptide D25-F3 with high antioxidizing power and high DPPH and ABTS eliminating power to H 2 O 2 The induced HDF cell injury has a protective effect, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.
2. Use of the polypeptide D25-F3 with strong antioxidant properties according to claim 1 for the preparation of an antioxidant and anti-aging pharmaceutical composition.
3. Use of the polypeptide D25-F3 with strong antioxidant properties according to claim 1 for the preparation of cosmetics with antioxidant and anti-aging properties.
4. Use of the strong antioxidant property polypeptide D25-F3 of claim 1 and umbilical cord mesenchymal stem cell culture supernatant in the preparation of an antioxidant pharmaceutical composition, wherein the umbilical cord mesenchymal stem cell culture supernatant is obtained after culturing the umbilical cord mesenchymal stem cell under the condition of containing the strong antioxidant property polypeptide D25-F3.
5. The use of the strong antioxidant property polypeptide D25-F3 and the umbilical cord mesenchymal stem cell culture supernatant according to claim 1 for preparing an antioxidant and anti-aging cosmetic, wherein the umbilical cord mesenchymal stem cell culture supernatant is obtained by culturing the umbilical cord mesenchymal stem cells under the condition of containing the strong antioxidant property polypeptide D25-F3.
6. The use of claim 2 or 4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
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