CN114432427B

CN114432427B - Application of anti-aging active peptide and bone marrow mesenchymal stem cells in preparation of anti-aging drugs

Info

Publication number: CN114432427B
Application number: CN202210215871.XA
Authority: CN
Inventors: 吕荣取; 杨国林
Original assignee: Zhengzhou Bain Biotechnology Co ltd; Zhengzhou Revo Gene Industry Co ltd; Henan Yuanchuang Life Stem Cell Bank Technology Co ltd
Current assignee: Henan Yuanchuang Life Stem Cell Bank Technology Co ltd; Zhengzhou Bain Biotechnology Co ltd; Zhengzhou Yuanchuang Gene Technology Co ltd
Priority date: 2022-03-07
Filing date: 2022-03-07
Publication date: 2022-09-30
Anticipated expiration: 2042-03-07
Also published as: CN114432427A

Abstract

The invention relates to application of anti-aging active peptide and mesenchymal stem cells in preparation of anti-aging drugs. The active peptide for specifically inhibiting Caspase-3 is obtained by screening the constructed Caspase-3 inhibitor screening model, the polypeptide effectively promotes the proliferation of the mesenchymal stem cells by inhibiting the activity of Caspase-3, meanwhile, the polypeptide has the advantages of better inhibiting the damage of the fibroblasts to ultraviolet rays, and the mesenchymal stem cells cultured by the inhibitor have better effect of treating and repairing skin damage.

Description

Application of anti-aging active peptide and bone marrow mesenchymal stem cells in preparation of anti-aging drugs

Technical Field

The invention relates to the field of biology, in particular to the field of stem cell aging prevention and stem cell activity improvement, and can be used for preparing an anti-aging medicament.

Background

Stem cells have the capabilities of self-renewal, multidirectional differentiation and paracrine factor secretion, are seeds for realizing cell regeneration, and can realize cell renewal through the proliferation and differentiation of stem cells of an organism, but with the increase of age, the number of stem cells of each tissue organ can be gradually reduced, the proliferation and differentiation capabilities are reduced, damaged tissue organs cannot be repaired in time, and wrinkles, color spots, dark skin and the like can be shown on skin. With the maturity of stem cell research, the application potential of stem cell technology in the field of skin aging resistance is huge.

Stem cell anti-aging is the repair of injury by stem cell self-differentiation or stem cell-derived secreted factors. Stem cells can secrete a large amount of cytokines, can improve the anti-free radical capacity of an organism, promote angiogenesis and cell proliferation and differentiation, inhibit inflammatory reaction and chemotaxis, stimulate regeneration, repair and the like of tissue cells, so that stem cell secretion products have the effects of resisting aging, beautifying and protecting skin, and the anti-aging mechanism of the skin of the stem cells mainly comprises the following forms: cell differentiation and senescence delaying: the stem cells can be differentiated into various types of cells due to the inherent differentiation capacity of the stem cells, and the stem cells in the skin are divided into epidermal stem cells, dermal stem cells and the like, which can be proliferated and differentiated to delay skin aging; in addition, other types of stem cells can also differentiate into skin cells, e.g., mesenchymal stem cells can differentiate into epidermal cells, fibroblasts, melanocytes, etc. Paracrine action: a large number of researches prove that the stem cells can promote cell proliferation, promote regeneration and repair tissue damage by secreting cell factors, such as superoxide dismutase (SOD), Vascular Endothelial Growth Factor (VEGF) and the like. DNA damage repair: maintenance of DNA integrity plays an important role in preventing aging, and stem cells have a double-stranded DNA damage repair mechanism. Among them, telomere damage is a special form of DNA damage, which cannot be easily repaired, and stem cells can produce telomerase, improve telomere shortening function, and delay aging process. Promoting collagen synthesis: stem cells may be involved in stimulating collagen synthesis in fibroblasts. The research shows that the stem cells can improve and increase the thickness and density of the skin and collagen fibers of the aged mice and reduce skin wrinkles. Anti-inflammatory action: proinflammatory mediators such as interleukin-6, tumor necrosis factor, etc. induce skin aging, and some animal studies indicate that intravenous MSCs can reduce the proinflammatory response.

The bone marrow mesenchymal stem cells have unique advantages and can be differentiated into various mesenchymal cells, and the multidirectional differentiation potential provides a new treatment means for cell regeneration and repair for a plurality of diseases. In the aspect of skin anti-aging, researches show that the BMSCs modified by the hPlGF-2 gene can be differentiated into fibroblasts to participate in skin wound repair and vascular endothelial cells to participate in the regeneration of blood vessels. In addition, research shows that the MSCs can participate in the reconstruction of dermal tissues in vitro and in vivo experiments, and the deposition of extracellular collagen microfibril is observed in the ultrastructure of the MSCs under the action of a dermal fibroblast induction system. MSCs can be induced and differentiated into epidermal cells and dermal cells in vivo and in vitro, and the induced and differentiated dermal fibroblasts have the function of secreting type I collagen. In addition, the mesenchymal stem cells can secrete various growth factors and cytokines to play an anti-aging role from multiple angles. The growth factors secreted by the mesenchymal stem cells of the bone marrow may be an important reason for accelerating the healing of skin wounds, and researches show that the mesenchymal stem cells of the bone marrow express transforming growth factors, epidermal growth factors, vascular endothelial growth factors, epidermal growth factors, fibroblast growth factors and the like, and the factors play important roles in regulating cell phenotype and reconstructing wound surfaces.

However, at present, the growth of the mesenchymal stem cells is slow in the culture process, and how to obtain high-quality and high-yield mesenchymal stem cells is the focus of the current research. Although there are many methods for increasing mesenchymal stem cells in the research, for example, a eukaryotic recombinant expression plasmid (bFGF-pcDNA3) is transfected into BMSCs cultured in vitro by using a liposome transfection method, a BMSCs cell line stably expressing bFGF is selected, and it is found that self-expressed bFGF can promote the proliferation of BMSCs. The transgenic technology is utilized to implant the growth factor Neuroglobin into BMSCs of experimental rabbits, and the result shows that the injured tissue of the rabbits is remodeled and the growth of fiber bundles is remarkably accelerated. At present, a human telomerase reverse transcriptase (hTERT) gene is successfully transferred into BMSCs of Chinese Guizhou piglets by utilizing a transgenic technology, the transfected BMSCs have the same proliferation activity as normally cultured BMSCs, and the service life of the transfected BMSCs is prolonged. However, the technology also faces some problems to be solved, such as low gene transfection efficiency, short in vivo expression time, whether malignant transformation is caused, whether the original biological characteristics can be maintained after transgenosis, and the like. At present, the transgenic technology is still in a preliminary research stage, and the incomplete technical conditions determine that the transgenic technology is difficult to be comprehensively popularized and applied in a short time.

Disclosure of Invention

The researchers of the invention find that the Caspase-3 inhibitor can promote the proliferation of the mesenchymal stem cells, but the effect of the inhibitor is still required to be further improved.

The invention overcomes the defects of the prior art and provides a novel method for screening a Caspase-3 inhibitor and a novel Caspase-3 inhibitor screened and obtained according to the method.

Specifically, the screening method comprises the steps of designing a primer according to a human Caspase-3 sequence of NCBI, carrying out PCR amplification on a target fragment by using human peripheral blood DNA as a template, then cutting the target fragment and a vector by using restriction enzymes NdeI and XhoI, and expressing and purifying Caspase-3 protein. Constructing a screening system: the reaction system contained Caspase-3 protein, a proper amount of reaction buffer (25mmol/L HEPES, 0.1% CHAPS, 100mmol/L NaCl, 10mmol/L LDTT, 10% sucrose, 1mmol/L EDTA, pH7.5), a sample, a blank well as reaction buffer and AC-DEVD-AMC (20 μ g/mL), and after shaking and mixing, the fluorescence intensity value at 460nm (F1) was measured at 355nm as excitation wavelength, once per minute. The last reading was set to F2 in the linear range. The formula for calculating the inhibition rate of the Caspase-3 enzyme by the sample is as follows: relative activity of enzyme ═ sample well (F2-F1) (F2-F1) blank |/[ F2-F1) control well (F2-F1) blank |, i.e. (1-relative activity of enzyme) × 100% ═ inhibition of sample. Wherein the sample is a polypeptide library constructed in a computer simulation mode. The target polypeptide can be obtained by high-throughput screening.

Further, the sequence of the screened Caspase-3 protein inhibitory peptide is shown as SEQ ID NO: 1 is shown.

Further, the invention provides a method for promoting the activity of the mesenchymal stem cells to inhibit apoptosis, which comprises the step of culturing the mesenchymal stem cells in a culture medium containing Caspase-3 protein inhibitory peptide.

Furthermore, the invention also provides application of the mesenchymal stem cells in preparing a pharmaceutical composition for promoting skin injury repair.

Further, the pharmaceutical compositions of the present disclosure may also include pharmaceutically acceptable carriers, excipients, or diluents commonly used to prepare pharmaceutical compositions, and the carriers may include non-naturally occurring carriers. Examples of the carrier, excipient and diluent include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, ethylparaben, propylparaben, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical composition may be formulated into tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, transdermal absorbents, gels, lotions, ointments, creams, patches, cataplasms, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, medicated bandages or suppositories according to conventional methods. In particular, when formulated, the pharmaceutical compositions may be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants and surfactants. Solid materials for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules. Such solid materials may be prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like.

Furthermore, the pharmaceutical composition also contains an antioxidant. Antioxidants of the present disclosure can include, but are not limited to, antioxidants that can be used in cell culture. They may include one or more selected from glutathione, cysteine, cysteamine, ubiquinol, -mercaptoethanol, and Ascorbic Acid (AA). When the medium is supplemented with an antioxidant, the antioxidant may be added at a concentration of 10-50g/ml, preferably 10-30g/ml, more preferably 25 g/ml.

The dosage of the pharmaceutical composition according to the present disclosure may be, for example, 1mg/kg to 1000mg/kg per day, but the dosage does not limit the scope of the present disclosure in any way.

Further, the invention also provides application of Caspase-3 protein inhibitory peptide in preparing a pharmaceutical composition for preventing skin from being damaged by ultraviolet rays, wherein the sequence of the inhibitory peptide is shown as SEQ ID NO: 1 is shown.

Further, the invention also provides application of Caspase-3 protein inhibitory peptide and bone marrow mesenchymal stem cells in preparing a pharmaceutical composition for treating skin injury and ultraviolet radiation, wherein the sequence of the inhibitory peptide is shown as SEQ ID NO: 1, the mesenchymal stem cells are obtained by adopting a culture medium containing Caspase-3 protein inhibitory peptide for culture.

When the composition of the present disclosure is included in a quasi-drug for preventing or improving ultraviolet ray damage of the skin, the composition may be used as it is, or may be used together with other quasi-drug ingredients, and may be suitably used according to a general method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use. The quasi-drug of the present disclosure is not particularly limited thereto, but may be prepared and used in the form of, for example, cream, lotion, aerosol, shampoo, gel, or pack. In the case of creams, lotions, aerosols, shampoos, gels or packs, substrates such as white petrolatum, yellow petrolatum, lanolin, bleached beeswax, cetyl alcohol, stearyl alcohol, stearic acid, hydrogenated oils, gelled hydrocarbons, polyethylene glycols, liquid paraffin and squalane; solvents and dissolution aids such as oleic acid, isopropyl myristate, glycerol triisooctanoate, tryptanthrin, diethyl sebacate, diisopropyl adipate, hexyl laurate, fatty acids, fatty acid esters, fatty alcohols and vegetable oils; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene and butylhydroxyanisole; preservatives such as parabens; humectants such as glycerol, propylene glycol and sodium hyaluronate; surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters and lecithin; including thickeners such as carboxyvinyl polymers, xanthan gum, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and the like.

The pharmaceutical composition can be administered orally or parenterally. Parenteral administration includes, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration, and includes administration, spraying or inhalation of the pharmaceutical composition to a target site, but is not limited thereto.

The dosage of the pharmaceutical composition of the present invention is the amount of cells that allows a therapeutic effect to be obtained in a patient or subject to whom the pharmaceutical composition is administered, as compared to a patient or subject to whom the pharmaceutical composition is not administered. The specific dose can be appropriately determined depending on the administration form, administration method, intended use, age, body weight, symptom and the like of the patient or subject. There is no particular limitation on a single dose of mesenchymal stem cells in human, for example, 10 ⁴ 10 cells/kg body weight or more ⁵ Cells/kg body weight or more or 10 ⁶ Cells/kg body weight or more. Also, there is no particular limitation on a single dose of mesenchymal stem cells for human, for example, 10 ⁹ Cells/kg body weight or less, 10 ⁸ Cells/kg body weight or less or 107 cells/kg body weight or less.

Advantageous effects

The active peptide for specifically inhibiting Caspase-3 is obtained by screening the constructed Caspase-3 inhibitor screening model, the polypeptide effectively promotes the proliferation of mesenchymal stem cells by inhibiting the activity of Caspase-3, and simultaneously, the polypeptide has the advantages of better inhibiting the fibroblast from being damaged by ultraviolet rays, and the mesenchymal stem cells cultured by the inhibitor have better effect of treating and repairing skin damage.

Drawings

FIG. 1 high throughput screening results

FIG. 2 is a graph showing the effect of polypeptides on the proliferation of mesenchymal stem cells

FIG. 3 protein expression results of caspase-3

FIG. 4 result chart of cell viability

FIG. 5 is a graph showing the results of bone marrow mesenchymal stem cells promoting wound healing

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 screening of Caspase-3 inhibitors

Designing a primer F according to a human Caspase-3 sequence of NCBI: 5'-ggcgggtcatatgtataaaatggattatcctgag-3', R: 5'-gcgctcgagaaaatagagttcttttgtgagcat-3', using human peripheral blood DNA as a template to perform PCR amplification on a target fragment, then cutting the target fragment and a vector by restriction enzymes NdeI and XhoI, recovering the fragments, connecting and transforming the fragments to DH 5-alpha, coating an LB/Amp (100mg/L) plate, screening positive clones, transforming BL21(DE3) after sequencing, oscillating the plates at 16 ℃ with a final concentration of 0.4mmol/L IPTG overnight, inducing expression, collecting thalli, washing the thalli by PBS, performing ultrasonic disruption, collecting supernatant, purifying the supernatant by using a Ni column to obtain purified Caspase-3 protein, and analyzing by SDS-PAGE to show that an obvious band is observed and is consistent with the predicted molecular weight. Enzyme activity is measured, and the enzyme activity is defined as: when the substrate was saturated, the amount of enzyme required to catalyze the decomposition of the substrate AC-DEVD-AMC to produce 1pmol AMC per minute at 37 ℃ was defined as one unit of enzyme activity (U). Through determination, the enzyme activity of the purified Caspase-3 protein is close to 9800U/g, and the activity is better.

Constructing a screening system: the reaction system contained Caspase-3 protein, a proper amount of reaction buffer (25mmol/L HEPES, 0.1% CHAPS, 100mmol/L NaCl, 10mmol/L LDTT, 10% sucrose, 1mmol/L EDTA, pH7.5), a sample, a blank well as reaction buffer and AC-DEVD-AMC (20 μ g/mL), and after shaking and mixing, the fluorescence intensity value at 460nm (F1) was measured at 355nm as excitation wavelength, once per minute. The last reading was set to F2 in the linear range.

The formula for calculating the inhibition rate of the Caspase-3 enzyme by the sample is as follows: relative activity of enzyme ═ sample well (F2-F1) (F2-F1) blank |/[ F2-F1) control well (F2-F1) blank |, i.e. (1-relative activity of enzyme) × 100% ═ inhibition of sample. The screening system is verified by using a positive inhibitor AC-DEVD-CHO of Caspase-3, the concentration and the inhibition effect of the positive inhibitor are proved to have dose dependence, and the model can be used for subsequent screening.

In the screening process, the sample is a polypeptide library constructed in a computer simulation mode. By high throughput screening, typical screening results are shown in FIG. 1.

As can be seen from FIG. 1, the two polypeptides CpY-201 and CpY-423 have the best inhibition effect on the activity of Caspase-3 protein, can inhibit the activity of Caspase-3 by more than 99%, and have better inhibition effect. Wherein, the amino acid sequence of CpY-201 is identified as SEQ ID NO: 1 is shown.

Example 2 isolation and identification of mesenchymal Stem cells

Putting the marrow blood into a sterile centrifugal tube for separating mesenchymal stem mL by combining a density gradient centrifugation method and an adherence screening method, uniformly mixing the marrow blood with an equal amount of PBS, centrifuging for 5min at room temperature for 1000min, removing a supernatant and a fat layer, and resuspending cells by an equal amount of DMEM/F12. The cell suspension is tightly attached to the tube wall and slowly added into a human lymphocyte separation solution (a centrifugal tube of Ficoll) which is pre-filled with the equal volume density of 1.077g/mL, so that the cell suspension is prevented from shaking and slowly superposed on the liquid surface of the Ficoll. Centrifuging at 2000r/min for 25min, taking out the centrifuge tube carefully, and dividing into 4 layers, wherein the middle layer is milky cloudy mononuclear cell layer. The mononuclear cell layer was aspirated into another centrifuge tube, and PBS was added and mixed well. Centrifuging at 1000r/min for 3min, discarding the supernatant, suspending the cells in a complete cell culture solution (DMEM/F12 containing 10% FBS by volume fraction), and pumping uniformly. According to 5xl0 ⁵ cm ² Inoculating at a depth of 25cm ² Cell culture flask, placed at 37 deg.C with 5% CO volume fraction ₂ And culturing in an incubator with saturated humidity. After 72h, the solution is changed for the first time, suspended non-adherent cells are discarded, and the solution is changed for 1 time every two or three days later. Observing the growth state of the cells under an inverted phase contrast microscope, and when about 80 percent of adherent cells climb to the bottom of a culture flask, the cells can be passaged, digested by 0.25 percent trypsin, and subjected to cell culture according to the following ratio of 1: 3, passage and amplification.

Detection of hMSCs cell surface markers the 3 rd generation cells cultured to grow well were washed 3 times with Phosphate Buffered Saline (PBS) and trypsinized to single fineCentrifuging at 1500r/min for 15min, collecting cell precipitate, washing with PBS for 3 times, and collecting l × 10 ⁵ Adding 10 mu L of fluorescence labeled antibody into 100 mu L of each cell suspension; the reaction is incubated at room temperature for 30min, the unlabeled antibody is washed by PBS, and the surface antigen expression of the cells CD44, CD34, CD90 and CD45 is detected by a flow cytometer. The results show that: the positive expression of CD44 is more than 95%, the positive expression of CD90 is more than 94%, and CD34 and CD45 are hardly expressed, which shows that the bone marrow mesenchymal stem cells with higher purity are obtained by the separation culture of the experiment.

EXAMPLE 3 Effect of CpY-201 Polypeptides on bone marrow mesenchymal Stem cells

The bone marrow mesenchymal stem cells identified in example 2 were cultured in F12-DMEM medium containing 10% fetal bovine serum, and bone marrow mesenchymal stem cells in the logarithmic phase and in good growth condition were taken and subjected to conventional digestion to prepare a single cell suspension. Inoculating into 96-well plate, regulating cell number to 1 × 10 per well ⁴ And (4) one cell. And culturing for 24 h. CpY-201 polypeptide and F12-DMEM medium were added at different concentrations, respectively. The relationship between the proliferation promoting effect and the concentration is revealed by using the polypeptide with the concentration of 0, 20, 100 and 500 mu g/mL. The culture was continued for 48h after addition of different concentrations of the polypeptide. The OD value of each well at 490nm is detected by a microplate reader, and the OD value is plotted on the horizontal axis and the OD value is plotted on the vertical axis. The results are shown in FIG. 2. The results are shown in FIG. 2, in which 0 concentration of the polypeptide was used as a negative control, and the positive control group was AC-DEVD-CHO at concentrations of 20, 100 and 500. mu.g/mL.

As can be seen from the results in FIG. 2, the MTT method for detecting the effect of 48h of polypeptide at different concentrations on cell proliferation shows that the CpY-201 polypeptide concentrations of 20, l00 and 500. mu.g/mL all have better proliferation promoting effect at 48h than the positive control group, and OD490 reaches 1.13. + -. 0.07 at 100. mu.g/mL, while the positive control at the same concentration is only 0.88. + -. 0.06.

EXAMPLE 4 detection of protein expression of caspase-3

Immunohistochemical detection of caspase-3 protein expression: the cells of each group cultured for 48h in example 3 were collected. Cells were fixed and washed 5min3 times with PBS and a volume fraction of 3% H ₂ O ₂ Incubation in a wet box at room temperature for 10min, washing with PBS for 5min3 times, and volume divisionSealing normal goat serum with the concentration of 1O% at room temperature for 30min, adding rabbit anti-rat polyclonal primary antibody (1: 50) at 4 ℃ overnight, washing with PBS for 2min3 times, washing with secondary antibody at room temperature for 30min, washing with PBS for 5min3 times, performing DAB color development, performing hematoxylin counterstaining for 2min, dehydrating with ethanol, clearing with xylene, sealing with neutral gum, and observing under an optical microscope. Image pro plus Image analysis software calculates the average absorbance. Caspase-3 expression mean absorbance-cumulative absorbance/area in 200-fold field. The results are shown in FIG. 3.

As can be seen from the results in FIG. 3, the decrease in Caspase-3 expression was significant (P <0.05) with increasing CpY-201 polypeptide concentration compared to the control group. At a concentration of 500. mu.g/mL, Caspase-3 had a mean absorbance of only 0.022. + -. 0.003.

Example 5 Effect of CpY-201 polypeptide inhibitors on UVB ultraviolet radiation on human dermal fibroblasts

The skin fibroblasts were added at 5X 10 per well ³ The individual cells were seeded in 96-well plates, and when the cells grew to 70% -80% confluency, the supernatant of the cells in each well was discarded, 80. mu.L of PBS was added, and then the concentration was 150mJ/cm ² UVB irradiation, PBS in each well was changed to 150. mu.L of medium immediately after irradiation, and placed at 37 ℃ 5% C0 ₂ Culturing in incubators for 12h, 24h, 36h and 48h respectively. And (3) adding 20 mu L of 5mg/mL MTT solution into each well 4h before the experiment is finished, incubating for 4h, absorbing and removing a supernatant, adding 100 mu L of LDMSO into each well, shaking for 15min at room temperature to dissolve granular substances at the bottom of each well, carrying out color comparison on a 490nm microplate reader, recording, and comparing OD values. Wherein the experimental group is added with polypeptide with a final concentration of 10mM or the positive control with the same concentration for pretreatment for 30min before irradiation, and the blank control group is not added with polypeptide. The results are shown in FIG. 4.

As can be seen from the results of FIG. 4, the human skin fibroblasts received 150mJ/cm ² After UVB irradiation, the cell activity gradually decreased at 12h, 24h, 36h and 48h4 different time points, and the cell activity was lowest at 48h after irradiation. However, after the positive control or the polypeptide pretreatment of the invention is added, the damage of UVB irradiation to cells can be obviously inhibited, particularly, the polypeptide of the invention can reverse and improve the cell viability at a faster speed, and the cell viability is improved at 48hAchieves (95.1 +/-2.0)%, and has remarkable effect improvement.

Example 6 bone marrow mesenchymal stem cell-promoted wound healing experiment

The bone marrow mesenchymal stem cells identified in example 2 were cultured in F12-DMEM medium containing 10% fetal bovine serum, and bone marrow mesenchymal stem cells in the logarithmic phase and in good growth condition were taken and subjected to conventional digestion to prepare a single cell suspension. Inoculating to 96-well plate, regulating cell number to 1 × 10 per well ⁴ The cells were cultured in F12-DMEM medium containing 10% fetal bovine serum and 20. mu.g/mL of CpY-201 polypeptide, and bone marrow mesenchymal stem cells were collected as an experimental group. The control group was 20. mu.g/mL of AC-DEVD-CHO as a positive control group.

Experimental grouping and preparation of cell models experimental grouping 2 groups in total: (1) experimental groups: and (3) directly co-culturing the hMSCs and the HEKa. The hMSCs were suspended in serum-free medium and then inoculated onto the surface of HEKa fused into a plate for 12h of co-culture. Wherein the mesenchymal stem cells adopt three forms of polypeptide treatment, AC-DEVD-CHO treatment and no treatment respectively; (2) control group: the HEKa-only culture group. Each group was cultured in a serum-free medium containing deoxythymidine. Epidermal wounds of 100 μm width were prepared for each group by growing confluent sheets of monolayer cells by scraping with a pipette tip under an inverted microscope along a marked line previously delineated at the bottom of the plate.

Detecting the healing speed of the epidermal wound surface: after each group of cells are cultured for 72h, the healing condition of the epidermal wound surface is observed under an inverted phase contrast microscope, and the wound surface healing rate is calculated by Sigma Scan Pro software, wherein the wound surface healing rate is the area occupied by the cells migrating to the epidermal wound surface/the total area of the epidermal wound surface multiplied by 100 percent. The results are shown in FIG. 5.

As can be seen from fig. 5, at 72h after epidermal wound, wound healing rate was calculated by using Sigma Scan Pro software, and the bone marrow mesenchymal stem cell co-culture group cultured with CpY-201 polypeptide and the positive control group were significantly higher than those of the bone marrow mesenchymal stem cell co-culture group not treated with polypeptide and the simple HEKa culture group, and the difference between the former three and the latter was significant (P < 0.05). The wound healing rate of the bone marrow mesenchymal stem cell co-culture group cultured by the CpY-201 polypeptide reaches 0.73 +/-0.05, and the wound healing rate has a better healing effect.

It should be understood that the above describes only some embodiments of the present invention and that various other changes and modifications may be affected therein by one of ordinary skill in the related art without departing from the scope or spirit of the invention.

Sequence listing

<110> Beijing Yue Pan Biotechnology Co., Ltd

Application of anti-aging active peptide and bone marrow mesenchymal stem cells in preparation of anti-aging medicine or cosmetic product

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ala Phe Gln Phe Ala Ala Gly Gly Met Ala His Tyr Phe Pro Gln Pro

1 5 10 15

Cys Pro Asn Arg Val Lys Pro Asp

20

Claims

1. The application of Caspase-3 protein inhibitory peptide CpY-201 in preparing a pharmaceutical composition for preventing human skin from being damaged by ultraviolet rays, wherein the sequence of the inhibitory peptide CpY-201 is shown as SEQ ID NO: 1 is shown.

2. The use according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

3. The use according to claim 2, wherein the pharmaceutical composition further comprises an antioxidant.

4. The use according to claim 3, wherein the antioxidant comprises one or more selected from glutathione, cysteine, cysteamine, ubiquinol, and ascorbic acid.