CN112402357A - Preparation method of striae gravidarum repair liquid based on umbilical cord mesenchymal stem cell exosomes - Google Patents
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
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- A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
- A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
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- A61K8/00—Cosmetics or similar toiletry preparations
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention provides a preparation method of striae gravidarum repair liquid based on umbilical cord mesenchymal stem cell exosomes; the method comprises the following steps: step 1, culturing umbilical cord mesenchymal stem cells; step 2, preparing a stock solution rich in the exosomes derived from the umbilical cord mesenchymal stem cells, marking as a solution B, and storing at low temperature for later use; step 3, preparing a base solution, and marking as solution C; and 4, using the solution B and the solution C according to a certain proportion. The method concentrates the supernatant rich in the stem cell exosomes through centrifugation and nano-membrane filtration, reserves nutritional ingredients and active factors for promoting cell growth in the stem cell culture supernatant to the maximum extent, removes macromolecular proteins, cell debris and other components difficult to absorb, and adds centella asiatica extract, hyaluronic acid and the like with the effects of diminishing inflammation and preserving moisture, so that the method has multiple effects and promotes striae gravidarum repair.
Description
Technical Field
The invention belongs to the technical field of skin care; in particular to a preparation method of striae gravidarum repair liquid based on umbilical cord mesenchymal stem cell exosomes.
Background
The umbilical cord mesenchymal stem cells are derived from umbilical cord Wharton jelly tissues, and have the advantages of convenient material acquisition, high cell purity, low immunogenicity and the like compared with stem cells derived from bone marrow, fat, dental pulp and the like; more and more researches suggest that the umbilical cord mesenchymal stem cells can secrete a large amount of cytokines to act on damaged parts through a paracrine mechanism so as to promote tissue repair, have good clinical curative effects on various diseases including diabetes, nervous system diseases, liver diseases, cardiovascular diseases, autoimmune diseases and the like, and are also used for repairing skin damage and beautifying and protecting skin.
Exosomes (exosomes) are tiny vesicles secreted by a variety of cells under normal or pathological conditions, with diameters of 30-150nm, with lipid bilayer membrane structures. The function of the exosome depends on the cell type of the exosome, plays an important role in the aspects of body immune response, tumor growth and migration, cell differentiation, cell communication, tissue homeostasis and the like, and has good clinical application prospect in the aspect of tissue regeneration.
Research shows that the exosome derived from the stem cell and the target cell transfer the bioactive substances to the target cell after membrane fusion or endocytosis, mediate intercellular communication and influence the function of the target cell, and also has the functions of inhibiting inflammatory reaction, regulating immunity, generating blood vessels and participating in tissue repair. Compared with stem cells, the stem cell exosome is more stable and safer, is more convenient to store and better controls the quality, and is expected to become a new treatment method.
The striae gravidarum is mainly formed due to the influence of hormones in pregnancy, and the abdominal distension causes elastic fibers and collagen fibers of the skin to be damaged or broken, so that the abdominal skin becomes thin and thin, and pink or purple wavy patterns with different widths and lengths appear. After delivery, these lines gradually disappear, leaving white or silvery-white shiny scar lines, i.e. stretch marks. Stretch marks appear mainly on the abdominal wall, and may also appear on the inside and outside of the thighs, buttocks, chest, back waist, arms, and the like. The most obvious is the first lying-in woman. Once the striae gravidarum appears, the striae gravidarum cannot disappear, and the striae gravidarum, the breast drop and the abdominal fat accumulation are accompanied, so that the postpartum physical state and the physical and mental health of women are seriously influenced.
It can be seen that striae gravidarum is mainly the breakdown of fibrin in the dermis, resulting in the generation of striae gravidarum and pigmentation, and the promotion of regeneration of cells in the dermis is the key for repair. The traditional chemical products mainly have the functions of lubrication, moisture retention and moistening without cell repair; products containing the repair factors can not achieve the expected effect due to the defects of low content, poor activity, large molecular weight, difficult absorption and the like of the repair factors.
Disclosure of Invention
The invention aims to provide a preparation method of a striae gravidarum repair liquid based on umbilical cord mesenchymal stem cell exosomes. The method prepares the striae gravidarum repair stock solution rich in stem cell exosomes by extracting umbilical cord stem cells, and prepares the repair solution for postpartum striae gravidarum repair by adding the substrate solution with moisturizing and anti-inflammatory effects.
The invention is realized by the following technical scheme:
the invention relates to a preparation method of striae gravidarum repair liquid based on umbilical cord mesenchymal stem cell exosomes, which comprises the following steps:
step 1, culturing umbilical cord mesenchymal stem cells;
step 2, preparing a stock solution rich in the exosomes derived from the umbilical cord mesenchymal stem cells, marking as a solution B, and storing at low temperature for later use;
step 3, preparing a base solution, and marking as solution C;
and 4, using the solution B and the solution C according to the volume ratio of 9: 1.
Preferably, in step 1, the umbilical cord mesenchymal stem cells are cultured by the following specific steps: collecting umbilical cord of newborn delivered by full-term caesarean section, cutting into 2-3cm segments, cleaning, stripping off HUATONG gum, and cutting to 1mm2Spreading small pieces in culture flask uniformly, and placing at 37 deg.C and 5% CO2Culturing in an incubator with the concentration, adding 15ml of complete culture medium to cover the tissue block within 8-24h, culturing until the liquid is changed for half the fifth day, changing the liquid every 2-3 days, sucking the tissue block and adding the tissue block into a new culture bottle when the cell fusion degree reaches 70-80%, supplementing the culture medium to 15ml, observing new cells to climb out from the periphery of the tissue block after 1-2 days, carrying out passage when the cell fusion degree reaches about 70-80%, marking as P1, and continuing the passage operation to obtain the umbilical cord mesenchymal stem cells from the P2 generation to the P5 generation of the secondary adherent cells.
Preferably, in step 2, the preparation of the stock solution rich in the umbilical cord mesenchymal stem cell-derived exosomes comprises the following specific steps: inoculating a cell culture bottle of umbilical cord mesenchymal stem cells with good growth state, wherein the cell culture bottle is 5X106-175 square centimeters, and collecting culture supernatant when the fusion degree of the cells reaches 80-90% by using a serum-free commercial stem cell culture medium (manufactured by LONZA company); centrifuging the supernatant at 4 deg.C for 8-10min to remove intact cells and dead cell debris to obtain supernatant A; filtering the supernatant A with 0.45 μm and 0.22 μm filters respectively to obtain a stock solution B rich in exosomes; the solution B is rich in active factors and needs to be stored at a low temperature of 4 ℃;
preferably, in step 3, the base liquid comprises the following components in percentage by volume;
the components have the following functions in the invention:
the effect of the 100% glycerol is: moisturizing and lubricating;
the hyaluronic acid has the following functions: moisturizing and wrinkle removing;
the centella asiatica extract has the following effects: antibacterial and anti-inflammatory effects;
the olive oil has the following functions: moistening and lubricating;
the raw liquid B rich in exosome is rich in active factors and has the effects of promoting the growth of cells in a dermis layer, repairing the damage of the cells in the dermis layer and the like; the moisture retention and moistening effects of the solution C can promote the uniform distribution of the solution B on the surface layer of the skin, and is beneficial to the absorption and utilization of the skin.
Preferably, in step 4, the method used specifically is:
1) taking out the liquid B stored at low temperature, extruding 2-3 ml of the liquid B to the palm of the hand, and tapping the liquid B to the position of the abdominal stretch mark;
2) 2-3 ml of C liquid is squeezed out and is gently massaged on the abdomen to be completely absorbed.
In another use method, the device can also be used together with a physical low-temperature introduction device; mixing the solutions B and C at the palm, dipping with a low temperature probe, and gently massaging the abdomen until the solution is completely absorbed.
The invention has the following advantages:
(1) the method concentrates the supernatant rich in the stem cell exosomes through centrifugation and nano-membrane filtration, reserves nutritional ingredients and active factors for promoting cell growth in the stem cell culture supernatant to the maximum extent, removes macromolecular proteins, cell debris and other components difficult to absorb, and adds centella asiatica extract, hyaluronic acid and the like with the effects of diminishing inflammation and preserving moisture, so that the method has multiple effects and promotes striae gravidarum repair.
(2) The product related by the invention is to remove macromolecules which are difficult to absorb by enriching active factors, RNA (ribonucleic acid), enzyme and other active substances secreted to cell culture supernatant in the growth process of stem cells and filtering through a nano membrane, and achieves double effects of repairing and moistening by matching with components with inflammation diminishing, moisture preserving and lubricating functions in base fluid, thereby effectively improving striae gravidarum.
Drawings
FIG. 1 is a graph showing the effects of the use of example 1 of the present invention;
FIG. 2 is a graph showing the effect of the present invention in example 2.
Detailed Description
The present invention will be described in detail with reference to specific examples. It should be noted that the following examples are only illustrative of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The embodiment relates to a preparation method of striae gravidarum repair liquid based on umbilical cord mesenchymal stem cell exosomes, which comprises the following steps:
step 1, culturing umbilical cord mesenchymal stem cells;
step 2, preparing a stock solution rich in the exosomes derived from the umbilical cord mesenchymal stem cells, marking as a solution B, and storing at low temperature for later use;
step 3, preparing a base solution, and marking as solution C;
and 4, using the solution B and the solution C according to the volume ratio of 9: 1.
In the step 1, the specific steps of culturing the umbilical cord mesenchymal stem cells are as follows: collecting umbilical cord of newborn delivered by full-term caesarean section, cutting into 2-3cm segments, cleaning, stripping off HUATONG gum, and cutting to 1mm2Spreading small pieces in culture flask uniformly, and placing at 37 deg.C and 5% CO2Culturing in an incubator with the concentration, adding 15ml of complete culture medium to cover the tissue block within 8-24h, culturing till half liquid change in the fifth day, changing liquid every 2-3 days, sucking out the tissue block and adding the tissue block into a new culture bottle when the cell fusion degree reaches 70-80%, supplementing the culture medium to 15ml, observing new cells to climb out from the periphery of the tissue block after 1-2 days, carrying out passage when the cell fusion degree reaches about 70-80%, marking as P1, continuing passage operation, and obtaining P2 generation to P5 generation of secondary adherent cellsThe umbilical cord mesenchymal stem cells are used for experiments.
In the step 2, the preparation of the stock solution rich in the exosome derived from the umbilical cord mesenchymal stem cell comprises the following specific steps: inoculating a cell culture bottle of umbilical cord mesenchymal stem cells with good growth state, wherein the cell culture bottle is 5X106-175 square centimeters, and collecting culture supernatant when the fusion degree of the cells reaches 80-90% by using a serum-free commercial stem cell culture medium (manufactured by LONZA company); centrifuging the supernatant at 4 deg.C for 8-10min to remove intact cells and dead cell debris to obtain supernatant A; filtering the supernatant A with 0.45 μm and 0.22 μm filters respectively to obtain a stock solution B rich in exosomes; the solution B is rich in active factors and needs to be stored at a low temperature of 4 ℃;
in step 3, the base liquid comprises the following components in percentage by volume;
in step 4, the method used specifically comprises:
1) taking out the liquid B stored at low temperature, extruding 2-3 ml of the liquid B to the palm of the hand, and tapping the liquid B to the position of the abdominal stretch mark;
2) 2-3 ml of C liquid is squeezed out and is gently massaged on the abdomen to be completely absorbed;
3) the product is used for 1 time every day, and after the product is continuously used for 1 month, the dark purple striae gravidarum is lightened in color, the torn lines are lightened, and the abdomen is tight; the effect is shown in figure 1.
Example 2
The embodiment of the invention provides a preparation method of a striae gravidarum repair liquid based on umbilical cord mesenchymal stem cell exosomes, which comprises the following steps:
step 1, culturing umbilical cord mesenchymal stem cells;
step 2, preparing a stock solution rich in the exosomes derived from the umbilical cord mesenchymal stem cells, marking as a solution B, and storing at low temperature for later use;
step 3, preparing a base solution, and marking as solution C;
and 4, using the solution B and the solution C according to a certain proportion.
In the step 1, the specific steps of culturing the umbilical cord mesenchymal stem cells are as follows: collecting umbilical cord of newborn delivered by full-term caesarean section, cutting into 2-3cm segments, cleaning, stripping off HUATONG gum, and cutting to 1mm2Spreading small pieces in culture flask uniformly, and placing at 37 deg.C and 5% CO2Culturing in an incubator with the concentration, adding 15ml of complete culture medium to cover the tissue block within 8-24h, culturing until the liquid is changed for half the fifth day, changing the liquid every 2-3 days, sucking the tissue block and adding the tissue block into a new culture bottle when the cell fusion degree reaches 70-80%, supplementing the culture medium to 15ml, observing new cells to climb out from the periphery of the tissue block after 1-2 days, carrying out passage when the cell fusion degree reaches about 70-80%, marking as P1, and continuing the passage operation to obtain the umbilical cord mesenchymal stem cells from the P2 generation to the P5 generation of the secondary adherent cells.
In the step 2, the preparation of the stock solution rich in the exosome derived from the umbilical cord mesenchymal stem cell comprises the following specific steps: inoculating a cell culture bottle of umbilical cord mesenchymal stem cells with good growth state, wherein the cell culture bottle is 5X106-175 square centimeters, and collecting culture supernatant when the fusion degree of the cells reaches 80-90% by using a serum-free commercial stem cell culture medium (manufactured by LONZA company); centrifuging the supernatant at 4 deg.C for 8-10min to remove intact cells and dead cell debris to obtain supernatant A; filtering the supernatant A with 0.45 μm and 0.22 μm filters respectively to obtain a stock solution B rich in exosomes; the solution B is rich in active factors and needs to be stored at a low temperature of 4 ℃;
in step 3, the base liquid comprises the following components in percentage by volume;
in step 4, the method used specifically comprises:
the device is matched with a physical low-temperature lead-in device for use; mixing the solutions B and C at the palm, dipping with a low temperature probe, and gently massaging the abdomen until the solution is completely absorbed.
Comparative experiment:
wherein, the 1 side is introduced (the dosage of the solution B and the solution C is 1: 1) for 1 time per week and used for one month;
introducing solution C only into the other 1 side, and using for 1 month;
the experimental results are as follows: when the side 1 of the liquid B and the liquid C is used, the abdomen is firmer, the lines and the color of the striae gravidarum are lighter, and the effect is shown in figure 2.
The invention has the following advantages:
(1) the method concentrates the supernatant rich in the stem cell exosomes through centrifugation and nano-membrane filtration, reserves nutritional ingredients and active factors for promoting cell growth in the stem cell culture supernatant to the maximum extent, removes macromolecular proteins, cell debris and other components difficult to absorb, and adds centella asiatica extract, hyaluronic acid and the like with the effects of diminishing inflammation and preserving moisture, so that the method has multiple effects and promotes striae gravidarum repair.
(2) The product related by the invention is to remove macromolecules which are difficult to absorb by enriching active factors, RNA (ribonucleic acid), enzyme and other active substances secreted to cell culture supernatant in the growth process of stem cells and filtering through a nano membrane, and achieves double effects of repairing and moistening by matching with components with inflammation diminishing, moisture preserving and lubricating functions in base fluid, thereby effectively improving striae gravidarum.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (5)
1. A preparation method of striae gravidarum repair liquid based on umbilical cord mesenchymal stem cell exosomes is characterized by comprising the following steps:
step 1, culturing umbilical cord mesenchymal stem cells;
step 2, preparing a stock solution rich in the exosomes derived from the umbilical cord mesenchymal stem cells, marking as a solution B, and storing at low temperature for later use;
step 3, preparing a base solution, and marking as solution C;
and 4, using the solution B and the solution C according to the volume ratio of 9: 1.
2. The preparation method of the striae gravidarum repair solution based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein in the step 1, the specific steps of culturing umbilical cord mesenchymal stem cells are as follows: collecting umbilical cord of newborn delivered by full-term caesarean section, cutting into 2-3cm segments, cleaning, stripping off HUATONG gum, and cutting to 1mm2Spreading small pieces in culture flask uniformly, and placing at 37 deg.C and 5% CO2Culturing in an incubator with the concentration, adding 15ml of complete culture medium to cover the tissue block within 8-24h, culturing until the liquid is changed for half the fifth day, changing the liquid every 2-3 days, sucking the tissue block and adding the tissue block into a new culture bottle when the cell fusion degree reaches 70-80%, supplementing the culture medium to 15ml, observing new cells to climb out from the periphery of the tissue block after 1-2 days, carrying out passage when the cell fusion degree reaches about 70-80%, marking as P1, and continuing the passage operation to obtain the umbilical cord mesenchymal stem cells from the P2 generation to the P5 generation of the secondary adherent cells.
3. The method for preparing a striae gravidarum repair solution based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein in the step 2, the preparation of the stock solution rich in umbilical cord mesenchymal stem cell-derived exosomes comprises the following specific steps: inoculating a cell culture bottle with good growth state of umbilical cord mesenchymal stem cells of 5X106-175 square centimeters, using a serum-free commercial stem cell culture medium, and collecting culture supernatant when the fusion degree of the cells reaches 80-90%;
centrifuging the supernatant at 4 deg.C for 8-10min to remove intact cells and dead cell debris to obtain supernatant A;
the supernatant A was filtered through 0.45 μm and 0.22 μm filters, respectively, to obtain an exosome-rich stock B.
4. The method for preparing a striae gravidarum repair solution based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein in step 3, the base solution comprises the following components in percentage by volume;
5. the preparation method of the striae gravidarum repair solution based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein in the step 4, the method used specifically comprises the following steps:
1) taking out the liquid B stored at low temperature, extruding 2-3 ml of the liquid B to the palm of the hand, and tapping the liquid B to the position of the abdominal stretch mark;
2) 2-3 ml of C liquid is squeezed out and is gently massaged on the abdomen to be completely absorbed.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023045117A1 (en) * | 2021-09-24 | 2023-03-30 | 周桂英 | Exosomes of umbilical cord mesenchymal stem cells, and use thereof |
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| CN111184679A (en) * | 2020-01-18 | 2020-05-22 | 西安九州再生医学集团有限公司 | Striae gravidarum repair preparation containing stem cell exosomes and preparation method and application thereof |
| JP2020182415A (en) * | 2019-05-08 | 2020-11-12 | 日本メナード化粧品株式会社 | Agent for promoting differentiation of dermal stem cell |
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| JP2020182415A (en) * | 2019-05-08 | 2020-11-12 | 日本メナード化粧品株式会社 | Agent for promoting differentiation of dermal stem cell |
| CN111184679A (en) * | 2020-01-18 | 2020-05-22 | 西安九州再生医学集团有限公司 | Striae gravidarum repair preparation containing stem cell exosomes and preparation method and application thereof |
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| WO2023045117A1 (en) * | 2021-09-24 | 2023-03-30 | 周桂英 | Exosomes of umbilical cord mesenchymal stem cells, and use thereof |
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Application publication date: 20210226 |