CN111700911A - Research method for skin tightening effect by adopting stem cell exosomes - Google Patents

Research method for skin tightening effect by adopting stem cell exosomes Download PDF

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CN111700911A
CN111700911A CN202010385535.0A CN202010385535A CN111700911A CN 111700911 A CN111700911 A CN 111700911A CN 202010385535 A CN202010385535 A CN 202010385535A CN 111700911 A CN111700911 A CN 111700911A
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skin tightening
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朱学义
韦丹
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Henan Tissue Cell Bank Co ltd
Henan Genetic Resources Cell Bank Co ltd
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Henan Genetic Resources Cell Bank Co ltd
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Abstract

The invention discloses a research method for skin tightening effect by using stem cell exosomes, which comprises the steps of extracting mesenchymal stem cells, culturing the mesenchymal stem cells, preparing stem cell exosome freeze-dried powder, performing skin tightening effect experiments, and performing data statistical analysis. When the mesenchymal stem cells are extracted, the liquid to be separated in the liquid storage tank is sucked out by the delivery pump through the inlet pipe, the required MSC liquid is left after being filtered by the filter screen and flows out to the culture medium, and the culture sleeve is provided with a plurality of cavities which can be used for accommodating the culture medium, so that accidents such as infection in the transportation process are avoided, and the problem of mutual infection is also avoided; the oxygen concentration of the mesenchymal stem cell culture is 2-3%, the temperature is 37C, the concentration of the seed and carbon dioxide is 5%, and under the condition of low oxygen concentration, the expansion of the adult stem cells is promoted, and the cell doubling period is shortened.

Description

Research method for skin tightening effect by adopting stem cell exosomes
Technical Field
The invention relates to a research method of a stem cell exosome, in particular to a research method of skin tightening effect by adopting the stem cell exosome, and belongs to the technical field of stem cell application.
Background
Exosomes are nanoscale outer lipid bilayer vesicles released outside cells by exocytosis, and can be present in various types of extracellular body fluids such as blood, urine, amniotic fluid, saliva, cerebrospinal fluid and even breast milk, and researches show that the exosomes contains cell-specific proteins, lipids and nucleic acids which can be used as signal molecules to be transmitted to other cells so as to change the functions of other cells, such as: exosomes are involved in the regulation of basic physiological processes such as neuronal communication, antigen presentation, immune response, organ development and reproduction, as well as in the regulation of pathological processes such as cancer progression, cardiovascular disease, inflammation, neurodegenerative disease, viral infection and prion transmission.
Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, and are non-hematopoietic cells of mesodermal origin. It is rich in source, and can be separated from placenta, deciduous teeth, fat, bone marrow, skeleton, periosteum, skeletal muscle, skin, peripheral blood, and umbilical cord. MSCs secrete a variety of growth factors (e.g., epidermal growth factor, vascular endothelial growth factor, collagen, fibronectin, etc.) and inflammatory factors (e.g., interferon gamma, interleukin 9, tumor necrosis factor alpha, etc.), and have significant anti-aging effects in inhibiting skin aging, tightening skin, assisting fat transplantation, promoting hair regeneration, etc.
In recent years, the application of stem cells in cosmetics for tightening skin is a hot point of research, however, most of the so-called stem cell injections in the cosmetic industry on the market have single active ingredients and are not good for keeping the skin tight, and many products for keeping the skin tight, such as jasmine essential oil, rosemary essential oil and the like, exist on the market at present, so that the market is wide, but the products can cause rebound when the products are not used for a long time.
Disclosure of Invention
The present invention is directed to solving the above problems by providing a method for studying skin tightening effects using stem cell exosomes.
The invention realizes the purpose through the following technical scheme: a research method for skin tightening effect by using stem cell exosomes comprises the following steps:
step one, extracting mesenchymal stem cells, and performing serum-free culture by using adult mesenchymal stem cells extracted from blood;
step two, culturing mesenchymal stem cells, putting the extracted mesenchymal stem cells into a serum-free culture medium for culture, and adding a serum promoiety, L-glutamine, deoxyribose nucleic acid, FGF and TGF-B into the culture medium;
wherein, the volume fraction of the serum pioneer in the serum-free medium is 15-20%, the concentration of L-glutamine euphoria is 280-300ng/mol, the concentration of deoxyribose nucleic acid is L0-20ng/mol, the concentration of fibroblast growth factor is 15-25ng/mol, the concentration of transforming growth factor is 15-25ng/mol, the concentration of vitamin B5 is 3-5ng/mol, and the concentration of vitamin B12 is 0.02 ng/mol.
Step three, preparing a dry cell exosome freeze-dried powder, collecting supernatant in a culture medium, mashing rosemary, cinnamon and non-photosensitive grapefruit, soaking the mashed rosemary, the cinnamon and the non-photosensitive grapefruit in sterile water soaked by jasmine, mixing liquid of the minced rosemary, the cinnamon and the grapefruit with the supernatant, vitamin C and vitamin E, filtering the collected mixed liquid by using a sterile filter of 0.2-0.22 md, and then concentrating, freeze-drying to prepare a dry cell exosome freeze-dried powder compact preparation;
step four, performing a skin tightening effect experiment, namely performing a mouse skin tightening effect experiment, and applying the exosome freeze-dried powder tightening preparation on an epidermal layer or introducing exosomes into an epidermal deep layer and a dermal layer by adopting any one of a micro-needle, a roller needle or a water light needle;
wherein, when the mouse skin tightening efficacy experiment is carried out, a blank experiment group is carried out at the same time.
And step five, data statistics and analysis, namely respectively counting the skin tightening change degree and time of the mice in the skin tightening efficacy experiment group and the blank experiment group, and carrying out comparative analysis.
As a still further scheme of the invention: in the first step, when extracting the mesenchymal stem cells, the transfer pump is utilized to suck out the to-be-separated liquid in the liquid storage tank through the introducing pipe, the required MSC liquid is left after filtering through the filter screen and flows out of the culture medium, and the culture sleeve is provided with a plurality of cavities which can be used for accommodating the culture medium.
As a still further scheme of the invention: in the second step, the oxygen concentration of the mesenchymal stem cell culture is 2-3%, and the temperature is 37C, and the concentration of the seed and the carbon dioxide is 5%.
As a still further scheme of the invention: in the second step, the serum-free medium is alpha-MEM medium as a basic medium, and serum substitute, L-glutamic acid amide, ribokernel-shake, deoxyribokernel-shake, fibroblast growth factor and transforming growth factor-8 are added.
As a still further scheme of the invention: in the third step, the rosemary, the cinnamon and the non-photosensitive grapefruit are smashed and fully contacted with the sterile water soaked by the jasmine flower and the vitamins, the temperature of the mixed liquid is 37-40 ℃, the soaking time is 12 hours under the high-pressure environment, the concentration of the vitamin C is 30-50ng/mol, and the concentration of the vitamin E is 0.3-0.5 ng/mol.
The invention has the beneficial effects that: the research method for the skin tightening effect by adopting the stem cell exosomes is reasonable in design:
1. when the mesenchymal stem cells are extracted, the liquid to be separated in the liquid storage tank is sucked out by the delivery pump through the inlet pipe, the required MSC liquid is left after being filtered by the filter screen and flows out to the culture medium, and the culture sleeve is provided with a plurality of cavities which can be used for accommodating the culture medium, so that accidents such as infection in the transportation process are avoided, and the problem of mutual infection is also avoided;
2. the oxygen concentration of the mesenchymal stem cell culture is 2-3%, the temperature is 37C, the concentration of the seed carbon dioxide is 5%, and under the condition of low oxygen concentration, the expansion of the adult stem cells is promoted, the cell doubling period is shortened, the gene expression of growth factors in the adult stem cells is stimulated, and the secretion of the growth factors is promoted;
3. the used serum-free culture medium takes an alpha-MEM culture medium as a basic culture medium, and serum substitutes, L-glutamine, ribokernel-shake, deoxyribokernel-shake, fibroblast growth factors and transforming growth factors-8 are added, so that the safety performance is high, the cells are not easily polluted in the cell culture process, the growth cycle of the adult stem cells can be greatly shortened, the proliferation speed of the adult stem cells is improved, the adult stem cells are promoted to secrete a large amount of growth factors, and the obtained lyophilized adult stem cell powder has high purity and high bioactivity and can effectively repair damaged cells;
4. the prepared adult stem cell freeze-dried powder contains polypeptide substances such as epidermal growth factors, fibroblast growth factors and the like, and can repair damaged cells, promote the growth and proliferation of skin cells, eliminate skin wrinkles, shrink pores and improve the state of skin.
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FIG. 1 is a schematic view of the flow structure of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, a method for studying skin tightening effect using stem cell exosomes includes the following steps:
step one, extracting mesenchymal stem cells, and performing serum-free culture by using adult mesenchymal stem cells extracted from blood;
step two, culturing mesenchymal stem cells, putting the extracted mesenchymal stem cells into a serum-free culture medium for culture, and adding a serum promoiety, L-glutamine, deoxyribose nucleic acid, FGF and TGF-B into the culture medium;
wherein, the volume fraction of the serum pioneer in the serum-free medium is 15-20%, the concentration of L-glutamine euphoria is 280-300ng/mol, the concentration of deoxyribose nucleic acid is L0-20ng/mol, the concentration of fibroblast growth factor is 15-25ng/mol, the concentration of transforming growth factor is 15-25ng/mol, the concentration of vitamin B5 is 3-5ng/mol, and the concentration of vitamin B12 is 0.02 ng/mol.
Step three, preparing a dry cell exosome freeze-dried powder, collecting supernatant in a culture medium, mashing rosemary, cinnamon and non-photosensitive grapefruit, soaking the mashed rosemary, the cinnamon and the non-photosensitive grapefruit in sterile water soaked by jasmine, mixing liquid of the minced rosemary, the cinnamon and the grapefruit with the supernatant, vitamin C and vitamin E, filtering the collected mixed liquid by using a sterile filter of 0.2-0.22 md, and then concentrating, freeze-drying to prepare a dry cell exosome freeze-dried powder compact preparation;
step four, performing a skin tightening effect experiment, namely performing a mouse skin tightening effect experiment, and applying the exosome freeze-dried powder tightening preparation on an epidermal layer or introducing exosomes into an epidermal deep layer and a dermal layer by adopting any one of a micro-needle, a roller needle or a water light needle;
wherein, when the mouse skin tightening efficacy experiment is carried out, a blank experiment group is carried out at the same time.
And step five, data statistics and analysis, namely respectively counting the skin tightening change degree and time of the mice in the skin tightening efficacy experiment group and the blank experiment group, and carrying out comparative analysis.
Further, in the embodiment of the present invention, in the first step, when the mesenchymal stem cells are extracted, the fluid to be separated in the fluid storage tank is sucked out through the inlet tube by using the delivery pump, the required MSC fluid is left after being filtered by the filter screen and flows out onto the culture medium, and the culture sleeve is provided with a plurality of cavities for accommodating the culture medium, so that accidents such as infection during transportation are avoided, and the problem of mutual infection is also avoided.
Further, in the second step of the present invention, the oxygen concentration of the mesenchymal stem cell culture is 2 to 3%, the temperature is 37C, and the concentration of the seed carbon dioxide is 5%, and under the low oxygen concentration, the expansion of the adult stem cells is promoted, the cell doubling cycle is shortened, the gene expression of the growth factor in the adult stem cells is stimulated, and the secretion of the growth factor is promoted.
Furthermore, in the embodiment of the present invention, in the second step, the serum-free medium used in the step two is an alpha-MEM medium as a basic medium, and serum substitutes, L-glutamine, ribokernel-shake, deoxyribokernel-shake, fibroblast growth factor and transforming growth factor-8 are added, so that the serum-free medium has high safety, is not easily contaminated during a cell culture process, can greatly shorten a growth cycle of adult stem cells, improve a proliferation rate of the adult stem cells, and promote the adult stem cells to secrete a large amount of growth factors, and the obtained lyophilized powder of the exosomes of the adult stem cells has high purity and bioactivity, and can effectively repair damaged cells.
Further, in the third step of the present invention, rosemary, cinnamon and non-photosensitive grapefruit are mashed and fully contacted with the jasmine flower soaked sterile water and the vitamins, the temperature of the mixed liquid is 37 ℃ to 40 ℃, the soaking time is 12 hours under a high pressure environment, the concentration of the vitamin C is 30 ng/mol to 50ng/mol, and the concentration of the vitamin E is 0.3 ng/mol to 0.5ng/mol, so that the factors in each material for keeping skin tight can be fully mixed with the solution, the preparation is convenient, and the good efficacy of the preparation is ensured.
The working principle is as follows: when the research method for the skin tightening effect of the stem cell exosomes is used, the stem cell lyophilized powder is prepared by extracting and culturing the mesenchymal stem cells, the mouse is subjected to a tightening effect experiment and a blank control group experiment simultaneously by using the stem cell exosome lyophilized powder, in the comparison experiment, hUCM-SCS is injected subcutaneously around the wound surface of a BALB/C mouse, HE staining and immunohistochemistry are carried out to observe the skin condition of the injection part of the mouse, and the result shows that the tightening effect of the experiment group is obviously higher than that of the blank control group at each time point, the protein expression of the vascular endothelial cell growth factor is also obviously increased, and the hUCM-SCS is prompted to be capable of differentiating and regenerating new cells and new blood vessels when the skin is loosened, so that the repair and reconstruction of skin damaged cells are promoted, and the condition of skin collapse is improved.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (5)

1. A research method for skin tightening effect by using stem cell exosomes is characterized in that: the method comprises the following steps:
step one, extracting mesenchymal stem cells, and performing serum-free culture by using adult mesenchymal stem cells extracted from blood;
step two, culturing mesenchymal stem cells, putting the extracted mesenchymal stem cells into a serum-free culture medium for culture, and adding a serum promoiety, L-glutamine, deoxyribose nucleic acid, FGF and TGF-B into the culture medium;
wherein the volume fraction of the serum pioneer in the serum-free medium is 15-20%, the concentration of L-glutamine euphoria is 280-300ng/mol, the concentration of deoxyribose nucleic acid is L0-20ng/mol, the concentration of fibroblast growth factor is 15-25ng/mol, the concentration of transforming growth factor is 15-25ng/mol, the concentration of vitamin B5 is 3-5ng/mol, and the concentration of vitamin B12 is 0.02 ng/mol;
step three, preparing a dry cell exosome freeze-dried powder, collecting supernatant in a culture medium, mashing rosemary, cinnamon and non-photosensitive grapefruit, soaking the mashed rosemary, the cinnamon and the non-photosensitive grapefruit in sterile water soaked by jasmine, mixing liquid of the minced rosemary, the cinnamon and the grapefruit with the supernatant, vitamin C and vitamin E, filtering the collected mixed liquid by using a sterile filter of 0.2-0.22 md, and then concentrating, freeze-drying to prepare a dry cell exosome freeze-dried powder compact preparation;
step four, performing a skin tightening effect experiment, namely performing a mouse skin tightening effect experiment, and applying the exosome freeze-dried powder tightening preparation on an epidermal layer or introducing exosomes into an epidermal deep layer and a dermal layer by adopting any one of a micro-needle, a roller needle or a water light needle;
wherein, when the mouse skin tightening effect experiment is carried out, a blank experiment group is carried out at the same time;
and step five, data statistics and analysis, namely respectively counting the skin tightening change degree and time of the mice in the skin tightening efficacy experiment group and the blank experiment group, and carrying out comparative analysis.
2. The method for studying the skin tightening effect of stem cell exosomes according to claim 1, wherein the stem cell exosomes are selected from the group consisting of: in the first step, when extracting the mesenchymal stem cells, the transfer pump is utilized to suck out the to-be-separated liquid in the liquid storage tank through the introducing pipe, the required MSC liquid is left after filtering through the filter screen and flows out of the culture medium, and the culture sleeve is provided with a plurality of cavities which can be used for accommodating the culture medium.
3. The method for studying the skin tightening effect of stem cell exosomes according to claim 1, wherein the stem cell exosomes are selected from the group consisting of: in the second step, the oxygen concentration of the mesenchymal stem cell culture is 2-3%, and the temperature is 37C, and the concentration of the seed and the carbon dioxide is 5%.
4. The method for studying the skin tightening effect of stem cell exosomes according to claim 1, wherein the stem cell exosomes are selected from the group consisting of: in the second step, the serum-free culture medium is alpha-MEM culture medium as a basic culture medium, and serum substitute, L-glutamic acid amide, deoxyribokernel-shake, fibroblast growth factor and transforming growth factor-8 are added.
5. The method for studying the skin tightening effect of stem cell exosomes according to claim 1, wherein the stem cell exosomes are selected from the group consisting of: in the third step, the rosemary, the cinnamon and the non-photosensitive grapefruit are smashed and fully contacted with the sterile water soaked by the jasmine flower and the vitamins, the temperature of the mixed liquid is 37-40 ℃, the soaking time is 12 hours under the high-pressure environment, the concentration of the vitamin C is 30-50ng/mol, and the concentration of the vitamin E is 0.3-0.5 ng/mol.
CN202010385535.0A 2020-05-09 2020-05-09 Research method for skin tightening effect by adopting stem cell exosomes Pending CN111700911A (en)

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Cited By (1)

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CN113559238A (en) * 2021-08-16 2021-10-29 北京戴域生物技术有限公司 A pharmaceutical or cosmetic containing active peptide and having antiaging effect

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