CN114796089A - Application of umbilical cord mesenchymal stem cell exosome and rhodiola rosea polypeptide in beauty treatment and face nourishing - Google Patents
Application of umbilical cord mesenchymal stem cell exosome and rhodiola rosea polypeptide in beauty treatment and face nourishing Download PDFInfo
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- CN114796089A CN114796089A CN202210585754.2A CN202210585754A CN114796089A CN 114796089 A CN114796089 A CN 114796089A CN 202210585754 A CN202210585754 A CN 202210585754A CN 114796089 A CN114796089 A CN 114796089A
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Abstract
The invention discloses an application of umbilical cord mesenchymal stem cell exosome in combination with rhodiola rosea polypeptide in beauty treatment and face nourishing; the human umbilical cord mesenchymal stem cell exosome and the rhodiola rosea polypeptide are used in a combined manner, so that the skin tissue function can be better activated, the physiological function of each tissue is improved, the proliferation of fibroblasts and the generation of collagen are effectively stimulated, and the effects of quickly repairing the skin, removing wrinkles and resisting aging are achieved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of umbilical cord mesenchymal stem cell exosome in combination with rhodiola rosea polypeptide in beauty treatment and face nourishing.
Background
With the increase of age, the continuous change of social environment and the increase of living pressure, various problems often occur to the skin of people, meanwhile, with the improvement of social progress and the improvement of living standard of people, the problems of the skin are more and more emphasized, and the skin plays a very important role in protecting individuals from the external influence, namely the so-called barrier function. The barrier function is a function of protecting against various external stimuli (chemicals, air pollutants, dry environment, ultraviolet rays, etc.) and preventing excessive dispersion of water in the body through the skin, and the function is maintained only when the stratum corneum consisting of keratinocytes has been normally formed.
Exosomes refer to small membrane vesicles (30-150nm) containing complex RNAs and proteins, which today refer specifically to discoid vesicles with diameters between 40-100 nm; exosomes may modulate the biological activity of recipient cells by the proteins, nucleic acids, lipids, etc. they carry.
At present, various cosmetics such as whitening, wrinkle removing, moisturizing and the like are available on the market aiming at various problems of skin, in addition, exosome has been applied to the existing cosmetics to achieve the effect of beautifying, face nourishing, however, the existing products have the problem of poor effect, and can not better meet the requirements of people.
Disclosure of Invention
The invention aims to provide an application of umbilical cord mesenchymal stem cell exosome and rhodiola rosea polypeptide in beauty treatment and beautification.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides an application of umbilical cord mesenchymal stem cell exosome in combination with rhodiola rosea polypeptide in beauty treatment and face nourishing.
Preferably, the mass ratio of the umbilical cord mesenchymal stem cell exosomes to the rhodiola polypeptides is (400-800): (1-5).
Preferably, the rhodiola polypeptides are prepared by the following method:
(a) pulverizing radix Rhodiolae, mixing with water, and steaming to obtain mixed solution;
(b) adding complex enzyme into the cooled mixed solution for enzymolysis, inactivating enzyme, centrifuging, and collecting supernatant;
(c) carrying out ultrafiltration on the supernatant to obtain a peptide fragment with the molecular weight of 1000-2000 Da, and freeze-drying to obtain a rhodiola polypeptide crude product;
(d) preparing the rhodiola polypeptide crude product into a solution and filtering the solution by adopting a 0.45 mu m microporous filter membrane to obtain a filtrate; eluting the filtrate with Sephadex G-75 gel column, collecting eluate at the third eluting peak, and freeze drying to obtain radix Rhodiolae polypeptide.
Preferably, the cooking temperature is 105-115 ℃, and the time is 10-20 min; the elution rate was 0.8 ml/min.
Preferably, the enzymolysis temperature is 35-38 ℃, the time is 6-12 h, and the pH value is 4-6.
Preferably, the complex enzyme consists of pectinase, cellulase and trypsin according to the ratio of 2: 2-3: 2-4; the addition amount of the compound enzyme is 0.8-2% of the mass of the rhodiola rosea.
Preferably, the umbilical cord mesenchymal stem cell exosome is prepared by the following method:
carrying out subculture on the primary umbilical cord mesenchymal stem cells, and collecting third generation culture supernatant of the umbilical cord mesenchymal stem cells; centrifuging the supernatant at 3000-4000 Xg to remove cells and cell debris;
adding an exosome precipitation reagent into the supernatant, fully mixing uniformly, standing, centrifuging for 20-40 min at the speed of (10000-50000) Xg, and collecting the precipitate;
and (2) resuspending and precipitating by using PBS, placing the suspension precipitate in a purification column, placing the purification column in a collecting pipe, centrifuging the collecting pipe at 4 ℃ by (3000-4000) Xg for 8-12 min, and freeze-drying the solution in the collecting pipe to obtain the umbilical cord mesenchymal stem cell exosome.
The second aspect of the invention provides an exosome beauty fluid for beautifying and nourishing skin, which comprises the umbilical cord mesenchymal stem cell exosome and rhodiola rosea polypeptide.
Preferably, the mass ratio of the umbilical cord mesenchymal stem cell exosomes to the rhodiola polypeptides is (400-800): (3-5).
Preferably, the content of the umbilical cord mesenchymal stem cell exosomes in the exosome beauty fluid is 40-80 mg/ml, and the content of the rhodiola polypeptides is 0.3-0.5 mg/ml.
Compared with the prior art, the invention has the beneficial effects that at least:
research shows that the human umbilical cord mesenchymal stem cell exosome and rhodiola polypeptide are combined for use, so that the skin tissue function can be better activated, the physiological function of each tissue is improved, the proliferation of fibroblasts and the generation of collagen are effectively stimulated, and the effects of quickly repairing and removing wrinkles of the skin and resisting aging are achieved.
Drawings
In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
FIG. 1 is a chromatogram of the crude rhodiola rosea polypeptide of example 2 of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
Example 1
The embodiment is a preparation method of human umbilical cord mesenchymal stem cell exosome, which comprises the following steps:
1. instruments and consumables, D-MEM/F12 culture medium, fetal calf serum, an inverted microscope, an ultra-clean workbench, a CO2 incubator, a centrifuge, cytokines, ITS, Pbs and the like;
2. reagent configuration
D-MEM/F12 Medium configuration: D-MEM/F12 medium containing 10% fetal bovine serum, one cytokine, ITS5 ml;
2. the cell factor contains 20ng/ml EGF and 5ng/ml bFGF;
3. preparation before experiment
5 scissors, 5 hemostats, 3 tissue forceps, 3 common forceps, 2 kidney-shaped discs, 3 beakers of 50ml, 2 spoons (about 20 cm) and 500ml pbs for autoclaving at 121 ℃ for 30 minutes;
4. experimental procedures
(1) Ultraviolet irradiation is carried out on the superclean bench for 30 minutes, and the table top is wiped;
(2) placing the sterilized experimental apparatus on a workbench, igniting an alcohol lamp, taking out a kidney-shaped disc, pouring half of 75% alcohol, placing the umbilical cord in the kidney-shaped disc, and sterilizing for 30 seconds;
(3) pouring a 50ml beaker into half of the sterilized pbs, putting the umbilical cord into the beaker, and repeatedly washing for 3 times;
(4) taking out the other kidney-shaped disc, putting the umbilical cord into the kidney-shaped disc, pouring a proper amount of pbs, taking the scissors with the right hand and taking the common forceps with the left hand, and shearing the umbilical cord into small sections of about 2 cm;
(5) holding the hemostatic forceps with the left hand and holding the tissue forceps with the right hand, and slowly removing the umbilical cord skin and the blood vessel;
(6) placing the rest tissues into a clean beaker, and cutting the tissues into about 3mm by using scissors;
(7) the tissue blocks are paved in bottles of T75 by using a medicine spoon, and all the tissue blocks are paved after every 100 bottles of the tissue blocks are paved;
(8) slowly adding 15ml of culture medium into one side of a T75 bottle; put in CO 2 Incubating in an incubator;
(9) changing the solution once within 48 hours, observing the climbing condition of the stem cells after one week, if the climbing condition is better, beating the tissue block and abandoning the tissue block, and adding a culture medium;
(10) after 48h, the cells were digested, digested by adding 1ml trypsin, collected by centrifugation (1200 rpm, 5 minutes), the supernatant discarded, the cells were resuspended by adding medium, and the number of cells was determined (1X 10) 6 ) Inoculating cells, namely P1 generation;
(11) digesting and centrifuging after 48 hours, counting, and carrying out passage to obtain P2 generation;
(12) after two days, the cells were counted in 4.5X 10 medium by taking the supernatant (for detection of sterility, mycoplasma, endotoxin) 6 Freezing and storing;
(13) recovering the cells of the P2 generation according to production needs, and transferring the cells to the P3 generation to take the supernatant;
(14) centrifuging the supernatant at 4000 Xg to remove cells and cell debris;
(15) adding an exosome precipitation reagent into the supernatant, fully mixing uniformly, standing, centrifuging for 30min at 10000 Xg, and collecting the precipitate;
(16) and placing the sediment in a purification column after adopting PBS to resuspend the sediment, placing the purification column in a collecting tube, centrifuging the collecting tube at the temperature of 4 ℃ at 4000 Xg for 10min, and freeze-drying the solution in the collecting tube to obtain the human umbilical cord mesenchymal stem cell exosome.
Example 2
The embodiment is a preparation method of rhodiola polypeptide, which comprises the following steps:
(a) pulverizing radix Rhodiolae, sieving with 100 mesh sieve, mixing undersize product with water, and steaming at 110 deg.C for 15min to obtain mixed solution, wherein the mass of water is 3 times of undersize product;
(b) adding a complex enzyme into the cooled mixed solution, adjusting the pH value to 4-6, performing enzymolysis for 10 hours at 35-38 ℃, performing high-temperature enzyme deactivation, centrifuging, and collecting supernatant, wherein the complex enzyme consists of pectinase, cellulase and trypsin according to a ratio of 2: 2.5: 3; the adding amount of the complex enzyme is 1.2 percent of the mass of the rhodiola rosea undersize;
(c) carrying out ultrafiltration on the supernatant to obtain a peptide fragment with the molecular weight of 1000-2000 Da, and freeze-drying to obtain a rhodiola polypeptide crude product;
(d) preparing the rhodiola polypeptide crude product into a solution and filtering the solution by adopting a 0.45 mu m microporous filter membrane to obtain a filtrate; eluting the filtrate with Sephadex G-75 gel column at 0.8ml/min with distilled water as eluent, with the elution result shown in FIG. 1; collecting eluates at different elution peaks according to the peak appearance sequence, and freeze drying the eluates to obtain rhodiola rosea polypeptide A1 (peak appearance time is about 16 min), rhodiola rosea polypeptide A2 (about 18min), and rhodiola rosea polypeptide A3 (about 20 min).
Example 3
The embodiment is an exosome beauty fluid for beauty treatment, which comprises the umbilical cord mesenchymal stem cell exosome of the embodiment 1 and the rhodiola rosea polypeptide at the third elution peak of the embodiment 2, wherein the mass ratio of the umbilical cord mesenchymal stem cell exosome to the rhodiola rosea polypeptide is 597: 3; the content of the umbilical cord mesenchymal stem cell exosomes in the exosome beauty liquid is 6%.
Example 4
This example is a study of the effect of different contents of exosomes (denoted as B) of example 1 and rhodiola polypeptides (a3) of example 2 on fibroblasts:
fibroblast NIH/3T3 was formulated as 1X 10 6 Inoculating 200 mul/ml cell suspension into a 96-well plate, and culturing cells at 37 ℃ and 5% carbon dioxide concentration for 2 h;
then, respectively adding A3 polypeptide to final concentration of 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 0/4mg/ml, 0.5 mg/ml; example 1 the final exosome concentrations were 10mg/ml, 20ml/mg/ml, 40ml/mg/ml, 60ml/mg/ml, 80 ml/mg/ml; finally, cell culture was carried out at 37 ℃ for 24h at 5% carbon dioxide concentration;
processing according to the specification of an MTT kit, detecting the OD value of each 570nm position of each hole by using an enzyme-labeling instrument, calculating the cell survival rate according to the OD value, adding polypeptide and exosome to form an experimental group, and enabling the cell survival rate to be the OD value of the experimental group/the OD value of a blank group multiplied by 100%; the calculation results are shown in table 1:
TABLE 1
As can be seen from Table 1:
the polypeptide has the best proliferation effect on cells when the content of the polypeptide is 0.3-0.5 mg/ml, and the content of the polypeptide increases firstly and then decreases; however, the cell survival rate gradually increases with increasing content of extracellular exosome B, but the increasing tendency is slow, and therefore, it is considered that the exosome content is not less than 40 mg/ml.
Example 5
This example is a study of the effect of the exosomes of example 1 and the rhodiola polypeptides of example 2 on fibroblasts:
fibroblast NIH/3T3 was formulated as 1X 10 6 Inoculating 200 mul/ml cell suspension into a 96-well plate, and culturing cells at 37 ℃ and 5% carbon dioxide concentration for 2 h;
then, A1, A2, A3, the exosome of example 1 (as B), A1+ the exosome of example 1 (as A1B), A2+ the exosome of example 1 (as A2B), A3+ the exosome of example 1 (as A3B) were added, and a blank control group was set for 3 replicates, wherein the final addition concentrations of A1, A2, and A3 were 0.3mg/ml, and the final addition concentration of the exosome was 59.7 mg/ml; finally, cell culture was carried out at 37 ℃ for 24h at 5% carbon dioxide concentration;
processing according to the specification of an MTT kit, detecting the OD value of each 570nm position of each hole by using an enzyme-labeling instrument, calculating the cell survival rate according to the OD value, adding polypeptide and exosome to form an experimental group, and enabling the cell survival rate to be the OD value of the experimental group/the OD value of a blank group multiplied by 100%; the calculation results are shown in table 2:
TABLE 2
As can be seen from Table 2:
compared with the A1-A3 polypeptide, the A3 polypeptide has better promotion of proliferation and survival of fibroblasts, and the survival rate of cells can be obviously improved when the A3 polypeptide and exosome are jointly used for proliferation of the fibroblasts according to the data of exosome and polypeptide combination.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
Claims (10)
1. The application of the umbilical cord mesenchymal stem cell exosome in combination with rhodiola rosea polypeptide in maintaining beauty and keeping young.
2. The use of claim 1, wherein the mass ratio of the umbilical cord mesenchymal stem cell exosomes to the rhodiola rosea polypeptide is (400-800) to (3-5).
3. The use of claim 1 or 2, wherein the rhodiola polypeptides are prepared by the following method:
(a) pulverizing radix Rhodiolae, mixing with water, and steaming to obtain mixed solution;
(b) adding complex enzyme into the cooled mixed solution for enzymolysis, inactivating enzyme, centrifuging, and collecting supernatant;
(c) carrying out ultrafiltration on the supernatant to obtain a peptide fragment with the molecular weight of 1000-2000 Da, and freeze-drying to obtain a rhodiola polypeptide crude product;
(d) preparing the rhodiola polypeptide crude product into a solution and filtering the solution by adopting a 0.45 mu m microporous filter membrane to obtain a filtrate; eluting the filtrate with Sephadex G-75 gel column to obtain an eluate of distilled water, collecting the eluate of the third elution peak in order of appearance, and freeze drying to obtain the radix Rhodiolae polypeptide.
4. The use according to claim 3, wherein the cooking temperature is 105-115 ℃ and the time is 10-20 min; the elution rate was 0.8 ml/min.
5. The application of claim 3, wherein the enzymolysis temperature is 35-38 ℃, the time is 6-12 h, and the pH value is 4-6.
6. The use of claim 3, wherein the complex enzyme is composed of pectinase, cellulase and trypsin according to the ratio of 2: 2-3: 2-4; the addition amount of the compound enzyme is 0.8-2% of the mass of the rhodiola rosea.
7. The use according to claim 2, wherein the umbilical cord mesenchymal stem cell exosome is prepared by the following method:
carrying out subculture on the primary umbilical cord mesenchymal stem cells, and collecting third generation culture supernatant of the umbilical cord mesenchymal stem cells; centrifuging the supernatant at 3000-4000 Xg to remove cells and cell debris;
adding an exosome precipitation reagent into the supernatant, fully mixing uniformly, standing, centrifuging for 20-40 min at the speed of (10000-50000) Xg, and collecting the precipitate;
and (2) resuspending and precipitating by using PBS, placing the suspension precipitate in a purification column, placing the purification column in a collecting pipe, centrifuging the collecting pipe at 4 ℃ by (3000-4000) Xg for 8-12 min, and freeze-drying the solution in the collecting pipe to obtain the umbilical cord mesenchymal stem cell exosome.
8. An exosome beauty fluid for beauty treatment, which comprises the umbilical cord mesenchymal stem cell exosome of claim 1 and rhodiola polypeptide.
9. The exosome beauty fluid according to claim 6, wherein the mass ratio of the umbilical cord mesenchymal stem cell exosome to the rhodiola rosea polypeptide is (400-800) to (3-5).
10. The exosome beauty fluid according to claim 6, wherein the content of the umbilical cord mesenchymal stem cell exosomes in the exosome beauty fluid is 40-80 mg/ml, and the content of the rhodiola polypeptide is 0.3-0.5 mg/ml.
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