CN117106705A - Placenta-umbilical cord mesenchymal stem cell culture composition and application thereof - Google Patents
Placenta-umbilical cord mesenchymal stem cell culture composition and application thereof Download PDFInfo
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Abstract
The application discloses a placenta-umbilical cord mesenchymal stem cell culture composition and application thereof, wherein the culture composition is culture supernatant obtained by culturing mesenchymal stem cells in an induction culture medium; the induction culture medium comprises seabuckthorn flavone, palmitoyl tripeptide-1, transferrin, linoleic acid and basic culture medium. The culture composition of the application promotes the generation or secretion of certain active cytokines with the effects of resisting aging and inhibiting the activity of the neuraminidase in stem cells by culturing the induction culture medium containing the seabuckthorn flavone and the palmitoyl tripeptide-1 with the mesenchymal stem cells, and the active cytokines and the seabuckthorn flavone and the palmitoyl tripeptide-1 cooperate together, thereby having good synergistic effects on reducing the activity of tyrosinase, improving the activity of the aged human skin fibroblasts, reducing the active oxygen fluorescence intensity of the aged human skin fibroblasts and promoting the aged human skin fibroblasts to generate collagen, and having the potential of developing cosmetics with the effects of resisting aging, whitening or brightening skin.
Description
Technical Field
The application relates to the technical field of stem cells, in particular to a placenta-umbilical cord mesenchymal stem cell culture composition and application thereof.
Background
Mesenchymal Stem Cells (MSCs) are derived from mesodermal tissues, belong to multipotent stem cells, and are characterized by wide sources, low immunogenicity, certain differentiation potential, self-renewal capacity and immunity regulation capacity, and are mainly classified into human umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, placenta mesenchymal stem cells and the like according to the sources thereof. In contrast, umbilical cord and placenta mesenchymal stem cells derived from fetal tissues have the advantages of innocuity collection, convenience in extraction, large acquisition amount and the like, and are favored by people.
These bioactive molecules secreted by MSCs cultured in vitro, which have tissue damage repair function, are released into the culture supernatant, which is called MSC conditioned medium after collection. The conditioned medium of MSCs refers to a cell culture supernatant collected after MSCs are cultured in vitro for a period of time, wherein the conditioned medium contains MSCs which secrete a large amount of active cytokines through a paracrine action mode in the growth process, so that the conditioned medium of MSCs has an anti-tissue aging effect, is expected to be applied to cosmetics for skin aging, but has a limited overall skin aging effect, and few reports that MSCs secrete active cytokines with tyrosinase activity reduction effects through the paracrine action mode in the growth process.
Based on the reasons, the application provides a conditional medium, and the placenta or umbilical cord mesenchymal stem cells are cultured by designing an induction medium, so that the active cytokines secreted by the conditional medium and the induction medium have synergistic effect, have better anti-aging effect and have remarkable whitening effect.
Disclosure of Invention
The application aims to overcome the defects of the prior art and provide a placenta-umbilical cord mesenchymal stem cell culture composition and application thereof, so as to solve the problems in the technical background.
In order to achieve the above object, the present application is realized by the following technical scheme:
in a first aspect, the present application provides a placenta-umbilical cord mesenchymal stem cell culture composition, which is a culture supernatant obtained after mesenchymal stem cells are cultured by an induction medium;
the induction culture medium comprises seabuckthorn flavone, palmitoyl tripeptide-1, transferrin, linoleic acid and a basal culture medium.
Further, the induction culture medium contains 0.1-5 g/L of seabuckthorn flavone, 0.5-10 mg/L of palmitoyl tripeptide-1, 15-25 ug/L of transferrin and 10-40 ug/L of linoleic acid.
Further, the induction medium contains 1.5g/L of seabuckthorn flavone, 5mg/L of palmitoyl tripeptide-1, 20ug/L of transferrin and 25ug/L of linoleic acid.
Further, the basal medium is DMEM/F12, DMEM or alpha-MEM.
Further, the mesenchymal stem cells are derived from human placenta or umbilical cord.
Further, the preparation method of the mesenchymal stem cell culture composition comprises the following steps: the preparation method of the mesenchymal stem cell culture composition comprises the following steps: mesenchymal stem cells were taken and prepared at 1X 10 6 Inoculating the culture medium into a culture dish at a density of one mL, changing the culture medium into an induction culture medium when the cell fusion degree reaches about 80%, continuously culturing for 48 hours, collecting cell culture medium supernatant, centrifugally filtering the supernatant to obtain the culture composition, and preserving the culture composition at 4 ℃ for later use.
In a second aspect, the application also provides application of the placenta-umbilical cord mesenchymal stem cell culture composition in preparing a skin care product, wherein the skin care product has anti-aging and whitening effects.
Compared with the prior art, the application has the beneficial effects that:
the culture composition obtained by culturing the mesenchymal stem cells by the induction culture medium containing the seabuckthorn flavone and the palmitoyl tripeptide-1 has better tyrosinase activity inhibition capability, and the stronger tyrosinase inhibition rate indicates that the composition possibly has stronger effect of inhibiting in-vivo melanin deposition, and has the potential of developing cosmetics with whitening or skin brightening effects; proved by performance tests, the mesenchymal stem cells are cultured by the induction culture medium containing the seabuckthorn flavone and the palmitoyl tripeptide-1, and the culture medium has good synergistic effect in reducing the activity of tyrosinase and reducing the synthesis of tyrosinase, because the seabuckthorn flavone, the palmitoyl tripeptide-1 and the mesenchymal stem cells are cultured, the production or secretion of certain active cytokines in the mesenchymal stem cells can be promoted, and the active cytokines can greatly inhibit the tyrosinase; the culture composition obtained by culturing the stem cells by using the induction culture medium of the seabuckthorn flavone or the palmitoyl tripeptide-1 alone or by using the induction culture medium of the seabuckthorn flavone and the palmitoyl tripeptide-1 in combination has far less inhibition of the aminopeptidase than the culture composition obtained by culturing the mesenchymal stem cells by using the seabuckthorn flavone and the palmitoyl tripeptide-1 in combination.
According to the application, cell experiments show that the culture composition obtained by culturing the mesenchymal stem cells through the induction culture medium containing the seabuckthorn flavone and the palmitoyl tripeptide-1 promotes the generation or secretion of certain active cytokines (with anti-aging effect) in the mesenchymal stem cells through the culture of the induction culture medium containing the seabuckthorn flavone and the palmitoyl tripeptide-1 and the mesenchymal stem cells, and the active cytokines and the seabuckthorn flavone and the palmitoyl tripeptide-1 cooperate together, so that the activity of the aged human skin fibroblasts can be greatly improved, the active oxygen fluorescence intensity of the aged human skin fibroblasts can be reduced, the collagen production of the aged human skin fibroblasts can be promoted, and the potential of the cosmetics with the anti-aging effect can be developed.
Drawings
FIG. 1 is a bar graph of the effect of the culture composition of the application on tyrosinase inhibition rate;
FIG. 2 is a bar graph of the effect of the culture composition of the application on cell viability of aged human skin fibroblasts;
FIG. 3 is a bar graph showing the effect of the culture composition of the present application on the fluorescence intensity of active oxygen of aged human skin fibroblasts;
FIG. 4 is a bar graph showing the effect of the culture composition of the present application on the amount of collagen type I secreted by fibroblasts of aged human skin.
Detailed Description
Other advantages and effects of the present application will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present application with reference to specific examples. The application may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present application. It should be noted that the following embodiments and features in the embodiments may be combined with each other without conflict.
The components and reagents involved in the following examples are all conventional commercial products. Such as DMEM/F12 medium (liquid, cat No. 21041025), DMEM medium (liquid, low sugar, cat No. 11885092/11054001), α -MEM medium (liquid, cat No. 41061037) available from Gibco; hippophae rhamnoides flavone, 95% gauge, purchased from Shaanxi North Biotechnology Co., ltd; palmitoyl tripeptide-1, cas:147732-56-7, 98% pure, from Chengdu Corp.
In the embodiment of the application, human placenta mesenchymal stem cells (hPMSCs) (product number CP-H204) and human umbilical cord mesenchymal stem cells (hUCMSCs) (product number CP-CL 11) are purchased from the Living technologies of Wuhanplauosai; or separately culturing from placenta and umbilical cord. Wherein, human placenta mesenchymal stem cells (hPMSCs) are isolated and cultured: the placenta is subjected to chorion, amniotic membrane and decidua tissue removal, and PBS buffer solution is repeatedly washed to clean residual blood. Cutting placenta tissue, adding 0.1% type IV collagenase, digesting at 37deg.C for 30min, grinding with 100 mesh sieve, collecting cells, centrifuging at 1500rpm for 10min, washing with PBS buffer for 2 times, adding complete culture medium (low sugar DMEM culture medium, 10% fetal bovine serum, 1% double antibody), re-suspending cells, counting, inoculating to 6-well plate, inoculating to 5% CO at 37deg.C 2 Culturing under the condition. 3d half-volume liquid exchange is carried out to remove non-adherent suspension cells. The culture medium is replaced every 3-4 days, and the culture medium is passaged for 1 time and 3 times after 7-8 daysCells were used for the experiment.
Isolated culture of human umbilical cord mesenchymal stem cells (hUCMSCs): taking umbilical cord tissue, washing residual blood with D-Hank's solution, cutting umbilical cord adventitia in longitudinal direction, peeling off blood vessel, taking Wharton's jelly tissue between blood vessels and between blood vessel and adventitia, and cutting umbilical cord Wharton's jelly tissue into pieces of about 1mm after washing with D-Hank's solution 3 Is immersed in a 0.075% type II collagenase solution, digested at 37℃for 18h, and centrifuged at 2500rpm to discard the supernatant. Digesting the rest liquid with 0.25% pancreatin at 37deg.C for 30min, centrifuging at 2500rpm, removing supernatant, adding complete culture medium (low sugar DMEM medium, 10% foetal calf serum, 1% double antibody), and adjusting cell density to 1×10 5 Per mL, 25cm into the vessel 2 Placing in culture flask at 37deg.C and 5% CO 2 Culturing in an incubator for 48 hours. Half of the liquid was changed to remove non-adherent cells, 1 time every 3 days. After that, the culture medium is changed every 3-4 d, when the cells grow to 80% and fuse, 0.125% pancreatin is used for digestion, and the cells are subcultured according to the proportion of 1:3, and the cells after 3 generations are used for experiments.
Example 1
A placenta mesenchymal stem cell culture composition, which is a culture supernatant obtained by culturing human placenta mesenchymal stem cells in an induction medium;
the induction culture medium comprises seabuckthorn flavone, palmitoyl tripeptide-1, transferrin, linoleic acid and a basal culture medium, wherein the basal culture medium is DMEM/F12. The induction medium takes DMEM/F12 liquid medium as a solvent to dissolve the following components: 0.1g/L seabuckthorn flavone, 10mg/L palmitoyl tripeptide-1, 25ug/L transferrin and 10ug/L linoleic acid.
Example 2
A placenta mesenchymal stem cell culture composition, which is a culture supernatant obtained by culturing human placenta mesenchymal stem cells in an induction medium;
the induction culture medium comprises seabuckthorn flavone, palmitoyl tripeptide-1, transferrin, linoleic acid and a basal culture medium, wherein the basal culture medium is DMEM. The induction medium takes DMEM liquid medium as a solvent to dissolve the following components: 1.5g/L seabuckthorn flavone, 5mg/L palmitoyl tripeptide-1, 20ug/L transferrin and 25ug/L linoleic acid.
Example 3
A placenta mesenchymal stem cell culture composition, which is a culture supernatant obtained by culturing human placenta mesenchymal stem cells in an induction medium;
the induction culture medium comprises seabuckthorn flavone, palmitoyl tripeptide-1, transferrin, linoleic acid and a basal culture medium, wherein the basal culture medium is alpha-MEM. The induction medium takes alpha-MEM liquid medium as a solvent to dissolve substances with the following components: 5g/L seabuckthorn flavone, 0.5mg/L palmitoyl tripeptide-1, 15ug/L transferrin and 40ug/L linoleic acid.
Example 4
An umbilical cord mesenchymal stem cell culture composition, which is a culture supernatant obtained by culturing human umbilical cord mesenchymal stem cells in an induction medium;
the induction culture medium comprises seabuckthorn flavone, palmitoyl tripeptide-1, transferrin, linoleic acid and a basal culture medium, wherein the basal culture medium is DMEM. The induction medium takes DMEM liquid medium as a solvent to dissolve the following components: 1.5g/L seabuckthorn flavone, 5mg/L palmitoyl tripeptide-1, 20ug/mL transferrin, 25ug/mL linoleic acid.
Comparative example 1
This example is similar to example 2, except that the induction medium does not contain seabuckthorn flavonoids; the method comprises the following steps: a placenta mesenchymal stem cell culture composition, which is a culture supernatant obtained by culturing human placenta mesenchymal stem cells in an induction medium;
the induction medium comprises palmitoyl tripeptide-1, transferrin, linoleic acid and a basal medium, wherein the basal medium is DMEM. The induction medium takes DMEM liquid medium as a solvent to dissolve the following components: 5mg/L palmitoyl tripeptide-1, 20ug/L transferrin, 25ug/L linoleic acid.
Comparative example 2
This example is similar to example 2 except that the induction medium does not contain palmitoyl tripeptide-1; the method comprises the following steps: a placenta mesenchymal stem cell culture composition, which is a culture supernatant obtained by culturing human placenta mesenchymal stem cells in an induction medium;
the induction culture medium comprises seabuckthorn flavone, transferrin, linoleic acid and a basal culture medium, wherein the basal culture medium is DMEM. The induction medium takes DMEM liquid medium as a solvent to dissolve the following components: 1.5g/L seabuckthorn flavone, 20ug/L transferrin and 25ug/L linoleic acid.
Comparative example 3
This example is similar to example 2 except that the induction medium does not contain seabuckthorn flavone and palmitoyl tripeptide-1; the method comprises the following steps: a placenta mesenchymal stem cell culture composition, which is a culture supernatant obtained by culturing human placenta mesenchymal stem cells in an induction medium;
the induction medium comprises transferrin, linoleic acid and a basal medium, and the basal medium is DMEM. The induction medium takes DMEM liquid medium as a solvent to dissolve the following components: 20ug/L transferrin, 25ug/L linoleic acid.
Comparative example 4
An induction culture medium comprises seabuckthorn flavone, palmitoyl tripeptide-1, transferrin, linoleic acid and a basal culture medium, wherein the basal culture medium is DMEM. The induction medium takes DMEM liquid medium as a solvent to dissolve the following components: 1.5g/L seabuckthorn flavone, 5mg/L palmitoyl tripeptide-1, 20ug/L transferrin and 25ug/L linoleic acid.
Comparative example 5
An induction medium comprising transferrin, linoleic acid, a basal medium, the basal medium being DMEM. The induction medium takes DMEM liquid medium as a solvent to dissolve the following components: 20ug/L transferrin, 25ug/L linoleic acid.
A mesenchymal stem cell culture composition or an induction medium was prepared according to the formulations of examples 1 to 4 and comparative examples 1 to 5 (see table 1), wherein the mesenchymal stem cell culture composition was prepared by: taking 3 rd generation human mesenchymal stem cells (more than 3 generations may be adopted) in subculture, and mixing the cells according to 1×10 6 The cells were seeded at a density of individual/mL in a petri dish and when fusedChanging the culture medium to an induction culture medium when the degree reaches about 80%, continuously culturing for 48 hours, collecting cell culture medium supernatant, centrifuging the supernatant at 4000rpm for 20 minutes, filtering the supernatant by a 0.22 mu m filter to obtain a conditioned medium, namely the mesenchymal stem cell culture composition, and preserving the conditioned medium at 4 ℃ for later use.
TABLE 1
1. Biochemical experiments
1. Inhibition tyrosinase assay
Tyrosinase is the most critical in the formation process of melanin in skin, and inhibition of tyrosinase activity can play a role in whitening. Thus, tyrosinase catalytic reaction systems were constructed to evaluate the inhibition effect of tyrosinase by the culture compositions or induction media provided in examples 1 to 4 and comparative examples 1 to 5, respectively.
Sample to be tested and grouping: groups 11 were set, wherein groups 1-9 correspond to the culture compositions or induction media provided in examples 1-4 and comparative examples 1-5, respectively, and groups 10-11 were 1.5g/L seabuckthorn flavone (dissolved in PBS buffer at pH=6.80), 5mg/L palmitoyl tripeptide-1 (dissolved in PBS buffer at pH=6.80).
The test method comprises the following steps: each group is tested according to a tyrosinase catalytic reaction system, and each group is repeated 4 times; the tyrosinase catalytic reaction system is divided into four groups A1, A2, A3 and A4 by referring to Table 2, and after the sample addition is finished, the reaction is carried out for 10min at a constant temperature in a water bath at 37 ℃, and the absorbance values of the groups A1, A2, A3 and A4 are measured at 475 nm.
Tyrosinase inhibition rates (%) = [ (A2-A1) - (A4-A3) ]/(A2-A1) ×100% were calculated for each experimental group, and the test results are shown in table 3 and fig. 1.
TABLE 2
| Reaction system | A1 | A2 | A3 | A4 |
| Sample to be measured (ul) | 0 | 0 | 200 | 200 |
| PBS (ul) of 0.2M, pH =6.80 | 1600 | 1100 | 1400 | 900 |
| 100U/mL tyrosinase solution (ul) | 0 | 500 | 0 | 500 |
| 0.5wt% tyrosine solution (ul) | 400 | 400 | 400 | 400 |
TABLE 3 Table 3
| Group of | Tyrosinase inhibition rate (%) |
| Example 1 | 88.94±0.89 a |
| Example 2 | 92.56±0.56 a |
| Example 3 | 90.49±1.68 a |
| Example 4 | 90.19±1.25 a |
| Comparative example 1 | 41.13±1.61 ab |
| Comparative example 2 | 44.20±0.96 ab |
| Comparative example 3 | 39.66±1.20 ab |
| Comparative example 4 | 36.24±0.62 ab |
| Comparative example 5 | 34.76±0.93 ab |
| Hippophae rhamnoides flavone | 16.79±0.54 b |
| Palmitoyl tripeptide-1 | 15.63±0.58 b |
As can be seen from table 3 and fig. 1, the tyrosinase inhibition rates of the culture compositions provided in examples 1-4 are significantly higher than those of the culture compositions or the induction media provided in comparative examples 1-5, seabuckthorn flavone and palmitoyl tripeptide-1, which indicates that the culture compositions provided in examples 1-4 of the present application have better antioxidant ability, and the stronger tyrosinase inhibition rate suggests that the culture compositions may have stronger effect of inhibiting melanin deposition in vivo, and have the potential of developing cosmetics with whitening or skin brightening effects; and comparative examples 1-3 do not have significant differences compared with comparative examples 4-5, while examples 1-4 have significantly enhanced ability of the culture composition provided in examples 1-4 to inhibit tyrosinase activity compared with comparative examples 1-3, demonstrating that the induction medium performs mesenchymal stem cell culture by containing seabuckthorn flavone and palmitoyl tripeptide-1, which has good synergistic effect in reducing tyrosinase activity and reducing tyrosinase synthesis, because the production or secretion of certain active cytokines in the mesenchymal stem cells can be promoted when the seabuckthorn flavone, palmitoyl tripeptide-1 and the mesenchymal stem cells are cultured, and the active cytokines can greatly inhibit the neuraminidase; the effect of inhibiting the activity of the complex ammonification enzyme is far less than that of the culture composition obtained by culturing the mesenchymal stem cells by using the induction culture medium of the seabuckthorn flavone or the palmitoyl tripeptide-1 alone or by using the induction culture medium of the seabuckthorn flavone and the palmitoyl tripeptide-1 in combination.
2. Cell experiment
Culture of human skin fibroblasts: human dermal fibroblasts, cat No. YX-HC099, available from all-biological technologies (Shanghai); the culture medium consisted of 1% diabody, 10% fetal bovine serum by volume fraction, 89% dmem/F12, which was passaged to naturally senescent cells and subjected to the following experiments.
1. Cell proliferation activity assay by CCK-8 method
2.1 samples tested and groupings: 6 test groups and 1 blank group were set, and the test groups correspond to the culture compositions of example 2, the culture compositions of comparative examples 1 to 3, and the induction media of comparative examples 4 to 5, respectively, according to the present application. The blank group uses DMEM medium.
2.2 test method: taking human skin fibroblast cells passaged to natural aging, preparing the cells into suspension, inoculating 100ul of cells/well into 96-well plate, and repeating each treatment for 4 times at 37deg.C and 5% CO 2 After cell attachment, the culture medium was discarded, PBS was washed, the culture composition of example 2 containing 10% fetal bovine serum, the culture compositions of comparative examples 1-3, and the induction medium of comparative examples 4-5 were added, respectively (i.e., the culture composition of example 2 containing 10% fetal bovine serum, the culture composition of comparative example 1 containing 10% fetal bovine serum, the culture composition of comparative example 2 containing 10% fetal bovine serum, the culture composition of comparative example 3 containing 10% fetal bovine serum, the induction medium of comparative example 4 containing 10% fetal bovine serum, the induction medium of comparative example 5 containing 10% fetal bovine serum, were added as a negative control group, the medium in the wells was discarded when culturing was performed for 36 hours, 10. Mu.L of CCK-8 and 90. Mu.L of serum-free DMEM were added to each well, and only 10. Mu.L of CCK-8 and 90. Mu.L serum free DMEM were added as a blank in 96 well plates; after incubation for 2 hours in a cell incubator, the absorbance values of the test group, the negative control group and the blank control group at the wavelength of 450nm are detected by an enzyme-labeled instrument, the cell proliferation rate is calculated, and the cell proliferation rate of the negative control group is counted as 100%, wherein the cell proliferation rate is shown in table 4 and fig. 2;
test group: absorbance values of test group cells, DMEM and CCK-8 solution
Negative control group: absorbance values of control group cells, DMEM and CCK-8 solution
Blank control group: absorbance values of DMEM and CCK-8 solutions
Cell proliferation rate (cell viability) = (test group-blank control group)/(negative control group-blank control group) ×100%, table 4
| Group of | Cell proliferation Rate (%) |
| Example 2 | 216.41±5.24 a |
| Comparative example 1 | 169.70±4.32 ab |
| Comparative example 2 | 164.09±4.74 ab |
| Comparative example 3 | 157.01±3.51 ab |
| Comparative example 4 | 128.67±3.73 abc |
| Comparative example 5 | 123.15±2.34 abc |
| Negative control group | 100.00 |
As can be seen from table 4 and fig. 2, the cell activity of the aged human skin fibroblasts cultured by the culture composition provided in example 2 is significantly higher than that of comparative examples 1 to 5, indicating that the culture composition provided in example 2 can improve the activity of the aged human skin fibroblasts, and has the potential of developing cosmetics with anti-aging effect. The activity of the culture composition provided in the embodiment 2 for improving the activity of the aged human skin fibroblasts is obviously higher than that of the comparative examples 4-5, and the activity of the culture composition provided in the embodiment 2 for improving the aged human skin fibroblasts is obviously higher than that of the comparative examples 1-3, which shows that the induction culture medium contains seabuckthorn flavone and palmitoyl tripeptide-1 for carrying out mesenchymal stem cell culture has good synergistic effect on improving the activity of the aged human skin fibroblasts, because the generation or secretion of certain active cytokines in the mesenchymal stem cells are promoted when the seabuckthorn flavone, palmitoyl tripeptide-1 and the mesenchymal stem cells are cultured, and the active cytokines and the seabuckthorn flavone and palmitoyl tripeptide-1 cooperate together, so that the activity of the aged human skin fibroblasts can be greatly improved.
2. Intracellular active oxygen levels
2.1 samples tested and groupings: 6 test groups and 1 blank group were set, and the test groups correspond to the culture compositions of example 2, the culture compositions of comparative examples 1 to 3, and the induction media of comparative examples 4 to 5, respectively, according to the present application. The blank group uses DMEM medium.
2.2 test method: collecting fibroblast cells of naturally aging human skin, and mixing the cells at a ratio of 1×10 5 Each well was seeded in 6-well plates, 4 replicates per treatment, at 37 ℃, 5% co 2 Culturing under the condition, removing culture medium after cell adhesion, washing with PBS, adding culture composition of example 2 containing 10% fetal bovine serum by volume fraction, culture compositions of comparative examples 1-3, and induction culture medium of comparative examples 4-5, respectively, adding DMEM culture medium containing 10% fetal bovine serum by volume fraction as negative control group, and detecting intracellular active oxygen level with active oxygen detection kit (Biyunshan organism) when culturing for 72 hr. Cells were digested with pancreatin, centrifuged, the supernatant was discarded, washed with PBS, centrifuged to discard the supernatant, 1000. Mu.L of 5. Mu.M DCFH-DA solution was added to each tube, incubated at 37℃for 20min, centrifuged at 1200rpm for 5min, and the supernatant was removedCells were washed twice and three times with serum-free DMEM, resuspended in PBS and detected by a fluorescent microplate reader (excitation wavelength 488nm, emission wavelength 525 nm), and the results are expressed as fluorescence intensity values as shown in table 5 and fig. 3.
TABLE 5
As can be seen from Table 5 and FIG. 3, the fluorescent intensity of cellular active oxygen of the culture composition provided in example 2 is significantly lower than that of comparative examples 1 to 5, demonstrating that the culture composition provided in example 2 has the best effect of reducing the fluorescent intensity of cellular active oxygen of the skin fibroblasts of aged people, and has the potential to develop cosmetics with anti-aging effect. The fluorescence intensity of active oxygen of cells in the comparative examples 1-3 is obviously lower than that in the comparative examples 4-5, and the fluorescence intensity of active oxygen of cells in the culture composition provided in the example 2 is obviously lower than that in the comparative examples 1-3, which shows that the induction culture medium contains seabuckthorn flavone and palmitoyl tripeptide-1 for carrying out mesenchymal stem cell culture has good synergistic effect on the effect of reducing the active oxygen of skin fibroblasts of aged people, because the generation or secretion of certain active cytokines in the mesenchymal stem cells are promoted when the seabuckthorn flavone, palmitoyl tripeptide-1 and the mesenchymal stem cells are cultured, and the active cytokines and the seabuckthorn flavone and palmitoyl tripeptide-1 cooperate together, so that the active oxygen fluorescence intensity of the skin fibroblasts of aged people can be greatly reduced.
3. Cell secretion collagen I content
3.1 samples tested and groupings: 6 test groups and 1 blank group were set, and the test groups correspond to the culture compositions of example 2, the culture compositions of comparative examples 1 to 3, and the induction media of comparative examples 4 to 5, respectively, according to the present application. The blank group uses serum-free DMEM medium.
3.2 test method: taking human skin fibroblast cells passaged to natural aging, inoculating the cells at a ratio of 1000 cells/well and 100ul per well to 96-well plate, 4 replicates of each treatment, 5% CO at 37deg.C 2 Culturing under the condition for 24 hours, discarding the culture medium, washing with PBS, respectively adding the culture composition of the example 2, the culture compositions of the comparative examples 1-3 and the induction culture medium of the comparative examples 4-5, which contain 10% of fetal bovine serum, adding the DMEM culture medium, which contains 10% of fetal bovine serum, as a blank control group, incubating the blank control group in a 37 ℃ incubator for 36 hours, taking a cell supernatant sample, measuring the content of collagen by using a type I collagen (COL-I) ELISA kit (Shanghai Sitang Biotech Co., ltd.) and evaluating the type I collagen synthesis promotion effect on human fibroblasts. The results of the type I collagen content test are shown in table 6 and fig. 4.
TABLE 6
As can be seen from Table 6 and FIG. 4, the secretion level of the type I collagen promoted by the cells of the culture composition provided in example 2 is significantly higher than that of comparative examples 1 to 5, demonstrating the potential of the culture composition provided in example 2 to promote collagen production by the fibroblasts of the skin of an aging human and to develop cosmetics with anti-aging effects. The secretion content of the type I collagen of the comparative examples 1-3 is obviously higher than that of the comparative examples 4-5, and the secretion content of the type I collagen of the culture composition provided in the example 2 is obviously higher than that of the comparative examples 1-3, which shows that the induction culture medium contains seabuckthorn flavone and palmitoyl tripeptide-1 for carrying out mesenchymal stem cell culture has good synergistic effect on promoting the secretion of the type I collagen by the skin fibroblast of the aged people, because the generation or secretion of certain active cytokines in the mesenchymal stem cells are promoted when the seabuckthorn flavone, palmitoyl tripeptide-1 and the mesenchymal stem cells are cultured, the active cytokines and the seabuckthorn flavone and palmitoyl tripeptide-1 cooperate together, and the activity of the skin fibroblast of the aged can be greatly increased and the regeneration of the collagen can be promoted.
The placenta-umbilical cord mesenchymal stem cell culture composition provided by the application can be applied to the preparation of skin care products, wherein the skin care products have the effects of resisting aging and whitening.
The foregoing examples merely illustrate specific embodiments of the application, which are described in greater detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application.
Claims (7)
1. The placenta-umbilical cord mesenchymal stem cell culture composition is characterized by being a culture supernatant obtained by culturing mesenchymal stem cells in an induction culture medium;
the induction culture medium comprises seabuckthorn flavone, palmitoyl tripeptide-1, transferrin, linoleic acid and a basal culture medium.
2. The composition of claim 1, wherein the induction medium comprises 0.1-5 g/L seabuckthorn flavone, 0.5-10 mg/L palmitoyl tripeptide-1, 15-25 ug/L transferrin, and 10-40 ug/L linoleic acid.
3. The placenta-umbilical cord mesenchymal stem cell culture composition of claim 2, wherein the induction medium comprises 1.5g/L seabuckthorn flavone, 5mg/L palmitoyl tripeptide-1, 20ug/L transferrin, 25ug/L linoleic acid.
4. The placenta-umbilical cord mesenchymal stem cell culture composition of claim 1, wherein the basal medium is DMEM/F12, DMEM or alpha-MEM.
5. The placenta-umbilical cord mesenchymal stem cell culture composition of claim 1, wherein the mesenchymal stem cells are derived from a human placenta or umbilical cord.
6. The placenta-umbilical cord mesenchymal stem cell culture composition of claim 1, wherein,the preparation method of the mesenchymal stem cell culture composition comprises the following steps: mesenchymal stem cells were taken and prepared at 1X 10 6 Inoculating the culture medium into a culture dish at a density of one mL, changing the culture medium into an induction culture medium when the cell fusion degree reaches about 80%, continuously culturing for 48 hours, collecting cell culture medium supernatant, centrifugally filtering the supernatant to obtain the culture composition, and preserving the culture composition at 4 ℃ for later use.
7. The use of the placenta-umbilical cord mesenchymal stem cell culture composition of any one of claims 1-6 for preparing a skin care product, wherein the skin care product has anti-aging and whitening effects.
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