Umbilical cord mesenchymal stem cell culture composition and application thereof
By combining short peptide A and short peptide B with umbilical cord mesenchymal stem cell culture supernatant, an umbilical cord mesenchymal stem cell culture composition is solved, and the problem of insufficient antioxidant and collagen production in the prior art is solved. , achieved significant antioxidant and collagen production effects, and had significant anti-skin aging effects.
Patent Information
- Application Number
- CN202411660488.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-02-25
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to effectively delay skin aging, especially in terms of antioxidant and collagen production.
By combining short peptide A and short peptide B with umbilical cord mesenchymal stem cell culture supernatant, an umbilical cord mesenchymal stem cell culture composition is formed to promote the secretion of collagen by fibroblasts and have antioxidant effects.
This composition significantly enhances the antioxidant damage effect and promotes the production of collagen, with significant anti-skin aging effect.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to an umbilical cord mesenchymal stem cell culture composition, in particular to an umbilical cord mesenchymal stem cell culture composition comprising a combination of umbilical cord mesenchymal stem cell culture supernatant and active short peptides. The present invention also relates to the application of the umbilical cord mesenchymal stem cell culture composition in skin anti-oxidation and aging. Background Art
[0002] Skin is the outermost barrier of the human body, which can prevent physical, chemical and microbial damage, protect the body's water, electrolytes and nutrients from loss, and maintain the stability of the body's internal environment. Skin aging is caused by many factors, including internal factors, such as the increase in the production of reactive oxygen species (ROS) and the weakening of the activity of antioxidant-related enzymes (SOD) with cell aging, as well as external factors, such as cells being exposed to ultraviolet radiation (UVB) for a long time. The manifestations of skin aging include sagging skin and wrinkles, and the important reason is that collagen is damaged or the production is reduced. Although aging is an inevitable law in the human life process, with the improvement of living standards, people are eager to delay skin aging and hope to have more effective anti-aging skin care products. Summary of the invention
[0003] In one aspect, the present invention provides an umbilical cord mesenchymal stem cell culture composition, comprising: 1) Umbilical cord mesenchymal stem cell culture supernatant I supplemented with short peptide A and short peptide B; or 2) Umbilical cord mesenchymal stem cell culture supernatant II, The umbilical cord mesenchymal stem cell culture supernatant I is collected from a cell culture of umbilical cord mesenchymal stem cells cultured in a serum-free medium, and the umbilical cord mesenchymal stem cell culture supernatant II is collected from a cell culture of umbilical cord mesenchymal stem cells cultured in a serum-free medium supplemented with the short peptide A and the short peptide B. The sequence of short peptide A is shown in SEQ ID NO: 1, and the sequence of short peptide B is shown in SEQ ID NO: 2.
[0004] In some embodiments, for situation 1), the amount of the short peptide A and the short peptide B added to the umbilical cord mesenchymal stem cell culture supernatant I is no less than 6.0 μg / mL.
[0005] In some embodiments, for case 2), the amounts of the short peptide A and the short peptide B added to the serum-free medium are both no less than 0.6 μg / mL.
[0006] In some embodiments, for case 2), the amounts of the short peptide A and the short peptide B added to the serum-free medium are both no less than 6.0 μg / mL.
[0007] In some embodiments, the serum-free medium is a serum-free complete medium.
[0008] In some embodiments, the serum-free culture medium is StemPro™ MSC SFM.
[0009] In some embodiments, the umbilical cord mesenchymal stem cell culture supernatants I and II are collected from a cell culture of umbilical cord mesenchymal stem cells grown to a confluence of 70-80%.
[0010] On the other hand, the present invention provides a use of the umbilical cord mesenchymal stem cell culture composition in preparing cosmetics for anti-skin aging.
[0011] The umbilical cord mesenchymal stem cell culture composition provided herein has excellent antioxidant effect, can promote fibroblasts to secrete collagen, has anti-skin aging effect, and can be used in various skin care products. DETAILED DESCRIPTION
[0012] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
[0013] When we studied the whitening effect of oligopeptides in tilapia skin hydrolysate, we found that the combination of two short peptides and umbilical cord mesenchymal stem cell culture supernatant had a good antioxidant effect. Further research found that when these two short peptides were added to the umbilical cord mesenchymal stem cell culture medium, the collected culture supernatant had a better antioxidant effect and also promoted the secretion of collagen by fibroblasts, which had an anti-skin aging effect.
[0014] The present invention is further illustrated by the following specific examples.
[0015] Example 1 Discovery and preparation of short peptides We used tilapia skin as raw material, subjected it to multiple protease enzymolysis, and separated the various short peptides produced by enzymolysis by capillary electrophoresis, trying to find one or more short peptide combinations with whitening function. The separated short peptides were sequenced and synthesized by liquid phase synthesis to study their tyrosinase inhibitory ability. Although no single short peptide or combination of two short peptides was found to have a better tyrosinase inhibitory effect, it was found that the combination of short peptides numbered #035 and #086 had a certain antioxidant effect. The amino acid sequence of the short peptide numbered #035 is: DGRSGPPGP (SEQ ID NO: 1), and the amino acid sequence of the short peptide numbered #086 is: TPGDEGNI (SEQ ID NO: 2).
[0016] Example 2 Antioxidant effect of short peptides Hacat cells (human immortalized keratinocytes) in logarithmic growth phase were cultured at 10 4 Each well was inoculated in a 96-well plate, with 100 μL (DMEM+10%FBS+1%PS) per well, and cultured for 24 h. Different concentrations of #035 short peptide, different concentrations of #086 short peptide or their combination (dissolved in PBS) were added to the experimental group, and the positive control group was added with an equal volume (20 μL) of PBS, and cultured for another 24 h, and then treated with 150 μmol / L H2O2 for 4 h. After replacing the fresh culture medium (DMEM+10%FBS+1%PS) and culturing for 24 h, the cell activity was detected using the CCK-8 kit. The negative control group was operated in the same way, but was not treated with short peptides and H2O2. The blank control group was not inoculated with Hacat cells, and only culture medium and CCK-8 reagent were added. Five replicate wells were set up for each group. The cell proliferation rate (cell activity) was calculated by the following formula: Cell proliferation rate = (OD 450 Value-blank control group OD 450 value) / (OD of negative control group 450 Value-blank control group OD 450 value)×100%.
[0017] The results are listed in Table 1 (cell proliferation rate is the average of 5 wells).
[0018] Table 1 Cell proliferation rate (%) test results
[0019] It means that the concentration of short peptide #035 and short peptide #086 is 0.01 μg / mL, and the same is true for 0.1 μg / mL and 1.0 μg / mL.
[0020] # There is a significant difference compared with the positive control group. p < 0.05.
[0021] From the results in Table 1, it can be seen that the single short peptide #035 or short peptide #086 did not show obvious antioxidant damage effect, but when the two were used in combination, at a higher concentration (1.0 μg / mL), they showed antioxidant protection effect on cells.
[0022] Example 3 Antioxidant damage effect of mesenchymal stem cell culture supernatant supplemented with short peptides We then combined these two short peptides with other substances with antioxidant effects in order to further enhance the antioxidant effect. The results showed that the antioxidant effect was significantly enhanced when they were used in combination with the culture supernatant of umbilical cord mesenchymal stem cells.
[0023] The experimental process is as follows.
[0024] 3.1 Preparation of umbilical cord mesenchymal stem cell culture supernatant Umbilical cord mesenchymal stem cells (ScienCell) were used at 10 4 Pieces / cm 2 The cells were inoculated at a density of 100 μg / mL in a T75 cell culture flask, and the medium (serum-free medium, StemPro™ MSC SFM) was used in an amount of 15 mL. The cells were cultured at 37°C and 5% CO2 for about 2-3 days, and the culture supernatant was collected when the cell confluence was about 70-80%. The supernatant was centrifuged at 3000 g for 10 minutes at 4°C, filtered through a 0.22 μm filter membrane, and stored at -80°C for later use.
[0025] 3.2 Antioxidant damage test The antioxidant effects of the following various test substances (each added to the Hacat cell culture medium in an amount of 20 μL) were detected according to the method described in Example 2.
[0026] Test substance A: umbilical cord mesenchymal stem cell culture supernatant.
[0027] Test substance B: #035 short peptide + #086 short peptide, dissolved in PBS, the concentration of the short peptides was 6.0 μg / mL (so that the final concentration of the short peptides in the Hacat cell culture medium was 1.0 μg / mL).
[0028] Test substance C: umbilical cord mesenchymal stem cell culture supernatant to which short peptide #035 and short peptide #086 were added, with the short peptide concentrations of both being 6.0 μg / mL.
[0029] The results are listed in Table 2.
[0030] Table 2 Cell proliferation rate (%) test results
[0031] Different lowercase letters indicate significant differences among the groups ( p <0.05, the same lowercase letters indicate no significant difference between the groups ( p >0.05) From the results in Table 2, it can be seen that the supernatant of umbilical cord mesenchymal stem cell culture supplemented with short peptide #035 and short peptide #086 is superior to the supernatant of umbilical cord mesenchymal stem cell culture or the combination of short peptide #035 and short peptide #086 in terms of anti-oxidative damage effect, with significant difference ( p < 0.05).
[0032] Example 4 Enhancement of the Antioxidant Effect of Mesenchymal Stem Cell Culture Supernatant Further studies found that adding short peptide #035 and short peptide #086 to the mesenchymal stem cell culture medium had better effects than directly adding them to the prepared mesenchymal stem cell culture supernatant.
[0033] Umbilical cord mesenchymal stem cells (ScienCell) were used at 10 4 Pieces / cm 2 The cells were inoculated at a density of 15 mL in a T75 cell culture flask, and the culture medium (serum-free medium, StemPro™ MSC SFM) was used. The cells were cultured at 37°C and 5% CO2 for about 2-3 days, and the culture supernatant was collected when the cell confluence was about 70-80%. The supernatant was centrifuged at 3000g for 10 minutes at 4°C, filtered through a 0.22μm filter membrane, and stored at -80°C for later use. The serum-free culture medium used was supplemented with 0.6 μg / mL of #035 short peptide and #086 short peptide, 6.0 μg / mL of #035 short peptide and #086 short peptide, or without these two short peptides. The culture supernatants prepared were recorded as supernatant I, supernatant II, and supernatant III, respectively.
[0034] The antioxidant effects of various culture supernatants were detected according to the method described in Example 2, and the amount of culture supernatant added was 20 μL.
[0035] Table 3 Cell proliferation rate (%) test results
[0036] Different lowercase letters indicate significant differences among the groups ( p <0.05) Compared with the test substance group C in Table 2, under the condition of basically the same short peptide dosage (6.0 μg / mL), the supernatant II has a better antioxidant damage effect, and the cell proliferation rate is increased by about 12%. It is speculated that these two short peptides (#035 short peptide and #086 short peptide) can promote the secretion of certain antioxidant substances or other types of antioxidant substances by mesenchymal stem cells in addition to their own antioxidant effects.
[0037] Example 5 Anti-skin aging effect Type I collagen (COL-1) is an important component of the skin's extracellular matrix and plays an important role in the migration and adhesion of skin cells. The reduction of its content is an important cause of skin aging and wrinkles.
[0038] After culturing human dermal fibroblasts (HDF) in a 6-well culture plate for 24 h, 1 / 3 volume of the above test substance C, supernatant I, II, III or serum-free medium StemPro™ MSCSFM was added to the culture medium (DMEM + 10% FBS + 1% PS), and the culture was continued for 48 h. The content of COL-1 in the culture supernatant was detected using a collagen kit. The results are listed in Table 4.
[0039] Table 4 COL-1 content test results (μg / mL)
[0040] Different lowercase letters indicate significant differences among the groups ( p <0.05, the same lowercase letters indicate no significant difference between the groups ( p >0.05) The results showed that both culture supernatants I and II prepared in Example 4 could significantly promote the secretion of collagen I ( p <0.01, compared with supernatant III and serum-free medium), and the effect was enhanced as the content of the two short peptides in the mesenchymal stem cell culture medium increased during the preparation of the supernatant ( p <0.01, compared with supernatants I and II). The effect of test substance C was similar to that of supernatant I, but the amount of short peptide used was 10 times that of the latter.
[0041] The above embodiments describe preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be regarded as equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. An umbilical cord mesenchymal stem cell culture composition comprising 1) Umbilical cord mesenchymal stem cell culture supernatant I supplemented with short peptide A and short peptide B; or 2) Umbilical cord mesenchymal stem cell culture supernatant II, The umbilical cord mesenchymal stem cell culture supernatant I is collected from a cell culture of umbilical cord mesenchymal stem cells cultured in a serum-free medium, and the umbilical cord mesenchymal stem cell culture supernatant II is collected from a cell culture of umbilical cord mesenchymal stem cells cultured in a serum-free medium supplemented with the short peptide A and the short peptide B. The sequence of short peptide A is shown in SEQ ID NO: 1, and the sequence of short peptide B is shown in SEQ ID NO:
2.
2. The umbilical cord mesenchymal stem cell culture composition according to claim 1, wherein for situation 1), the amount of the short peptide A and the short peptide B added to the umbilical cord mesenchymal stem cell culture supernatant I is not less than 6.0 μg / mL.
3. The umbilical cord mesenchymal stem cell culture composition according to claim 1, wherein for situation 2), the amount of the short peptide A and the short peptide B added to the serum-free culture medium is not less than 0.6 μg / mL. 4 . The umbilical cord mesenchymal stem cell culture composition according to claim 1 , wherein for situation 2), the amount of the short peptide A and the short peptide B added to the serum-free medium is not less than 6.0 μg / mL. The umbilical cord mesenchymal stem cell culture composition according to claim 1 , wherein the serum-free medium is a serum-free complete medium.
6. The umbilical cord mesenchymal stem cell culture composition of claim 1, wherein the serum-free culture medium is the culture medium StemPro™ MSC SFM. 7 . The umbilical cord mesenchymal stem cell culture composition according to claim 1 , wherein the umbilical cord mesenchymal stem cell culture supernatants I and II are collected from cell cultures of umbilical cord mesenchymal stem cells grown to a confluence of 70-80%.
8. Use of the umbilical cord mesenchymal stem cell culture composition according to any one of claims 1 to 7 in the preparation of cosmetics for anti-skin aging.