CN101461772A - Method for preparing stem cell secretion factor for beauty treatment and skin-protection - Google Patents
Method for preparing stem cell secretion factor for beauty treatment and skin-protection Download PDFInfo
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Abstract
The invention relates to a preparation method for stem cell secretion factors used for beauty culture and skin protection. The preparation method comprises the following steps: cell separation, stem cell amplification, the acquisition and concentration of a stem cell factor system, and the acquisition of the system containing the stem cell active factors through storage. The system comprises the active factors secreted by various types of currently verified stem cells. The obtained stem cell active factor system neither contains foreign culture solution components nor contains dead cell decomposition components; in addition, the stem cell active factor system can store and maintain the tissue and cell repairing activity for a long period, thereby truly turning the ideal of stem cell beauty culture into a reality, and improving the traditional beauty culture concepts at the biomedical depth. Simultaneously, the preparation method is simple and feasible and is suitable for mass production, thereby providing a shortcut for the stem cells to serve the beauty culture cause.
Description
Technical field
The present invention relates to a kind ofly to can be used for improving looks, the preparation method of the stem cell secretion factor of skin protection.The composite factor of this stem cell secretion can be applied to cosmetics, health product, skin care item.
Background technology
Stem cell is a kind of cell with multidirectional differentiation potential, can be divided into embryonic stem cell and adult stem cell according to the difference of originating.Embryonic stem cell can be divided into any cell of health, so also be myeloid-lymphoid stem cell.Adult stem cell is meant that those have tissue or organ specific stem cell, can be divided into the cell line suitable with its location in adapt circumstance, is called pluripotent stem cell again.Stem cell can directed differentiation in particular environment, thereby reaches the effect of repair tissue and cell injury.The present inventor studies show that (Bc1-2 engineered MSCs inhibited apoptosis andimproved heart function ', Stem Cells, 2007 Aug; 25 (8): 2118-27..) stem cell can come repair tissue by being divided into specific cell, and paracrine action has been brought into play bigger effect in the reparation of tissue.That is to say that in stem-cell therapy tissue injury, the cytokine of stem cell secretion has been brought into play very important effect.This system comprises kind surplus stem cell factor (SCF), endothelial cell growth factor (ECGF) (VEGF), epithelical cell growth factor (EGF), tumor necrosis factor (TNF) hepatocyte growth factor (HGF), nerve growth factor (NGF), fiber mother cell growth factor (FGF), Insulin-Like growth factor (IGF), granulocyte colony-stimulating factor (G-CSF), the interleukin (IL) etc. 20.Wherein (Stem Cell Factor, it is needed SCF) to be not only the activity of earning a bare living, and the work of body is had and repairs in the opposing ionizing radiation and all be absolutely necessary in damaging due to the radiation for stem cell factor.In addition, also contain each collagen type (as I, II, III, IV collagen type) and various lysozyme etc. in the stem cell secretion factor.
Epidermal stem cells is the seed cell of skin renewal.All have every day a large amount of epidermis cell death to come off, the regenerative cell that also has respective numbers is replenished.Along with the increase at people's age, skin spots, wrinkle also increases gradually, epidermis has lost brilliance in the past, everything all comes from going down of epidermal stem cells regeneration capacity, how to make epidermal stem cells keep vigorous metabolism ability, will be the key point that keeps the skin health beauty.
Based on the demand of people to beauty, the exploitation of stem cell beautifying technique and Related product thereof is subjected to the attention of industry day by day.Occur a lot of so-called stem cell products in the market, do not had the feasibility of enforcement from technical standpoint.Chinese patent CN1817339A, N1872025A and CN1817339A patent relate to the content of product and have used active stem cell and Chinese medicinal components, at first how keep the activity of stem cell in the skin protection kind? secondly how to avoid producing the infringement of toxin after the stem cell necrosis to human body? these all are to be worth inquiring into and very insoluble problem.CN1872025A adopts cell breaking technology, and the cell culture technology under the normal condition obtains the stem cell active component.This method of obtaining the stem cell active component lacks foundation.Can't you obtain Fructus Mali pumilae just to cut apple tree broken as you? pulverize cell and just obtained the cell segment.Routine is under the normal growth state, the cell culture supernatant that is obtained, comprising in the supernatant more is metabolic waste, adopts α-MEN (wherein being added with hormone such as dexamethasone) culture medium simultaneously, how effectively removing hormone wherein, all is the problem that it faced.
The somatomedin of interpolation is also arranged in the market as bFGF and EGF in the beauty and skin care product, and the renewal of cell is the process of a system, though single somatomedin be added in a way can irritation cell metabolism, also cause dependency easily.
In summary it can be seen, have broad prospects based on the beauty curing of stem cell, but how science, effectively utilize the special performance of stem cell, develop stem cell beauty and skin care product truly, still allowing is a brand-new problem.
Summary of the invention
The object of the present invention is to provide a kind of preparation method that is used for the stem cell secretion factor of beauty and skin care, stem cell secretion factor can be used for the product of beauty and skin care.
The step that the preparation method that is used for the stem cell secretion factor of beauty and skin care provided by the invention comprises:
1) adopts conventional cell isolation method, from people or mammal respective organization, isolate stem cell.
2) amplification of stem cell, the conventional cultural method with corresponding stem cell comprises the a-MEM culture medium, adds 10% hyclone, 5%CO2 cultivates under 37 ℃ of conditions, regularly changes liquid, goes down to posterity, and then obtains the stem cell of requirement.
3) obtaining of stem cell factor system: in stem cell division the most vigorous period, discard normal culture medium, clean 1-3 time with PBS buffer (phosphate buffer is about pH=7.4), add PBS or normal saline then, cultivation is 4-24 hour in the cell culture incubator.Collect supernatant, can obtain being rich in the solution of stem cell factor system.
4) concentrate, store: the gained centrifugal, remove the cell that is scattered, concentrate, by 1 * 10
6The concentration of individual cell/m1 concentrates, and gets product, in-20 ℃ of long preservation, in order to using.
The secreted stem cell factor system of stem cell that derives from of the present invention, this system comprises stem cell factor (SCF), endothelial cell growth factor (ECGF) (VEGF), epithelical cell growth factor (EGF), tumor necrosis factor (TNF) hepatocyte growth factor (HGF), nerve growth factor (NGF), fiber mother cell growth factor (FGF), Insulin-Like growth factor (IGF), granulocyte colony-stimulating factor (G-CSF), interleukin (IL) etc.In addition, also contain each collagen type (as I, II, III, IV collagen type) and various lysozyme etc. in the stem cell secretion factor.
Stem cell factor system of the present invention is meant: derive from people or mammalian embryonic stem cell, navel blood stem cell, bone marrow stem cell, epidermal stem cells, endothelial stem cell, peripheral hematopoietic stem cells, fat stem cell etc., also can make a general reference all stem cell.
The product of gained of the present invention is equipped with the product that the lysate of medically accepting constitutes compositions as active component, is directly used in the purposes of beauty treatment, skin protection aspect, comprises reduce wrinkle, beauty treatment, speckle dispelling, reparation cicatrix etc.
The product of gained of the present invention can add in the beauty and skin care product of various preparations as active component.Be used to improve looks, the purposes of skin protection aspect, comprise reduce wrinkle, beauty treatment, speckle dispelling, reparation cicatrix etc.
Products obtained therefrom of the present invention can be used for improving looks, the various preparations in injection, facial cream, emulsion, water liquid or the cosmetics commonly used of skin-protection product, and, include but not limited to cleansing cream, cleaning breast, cleansing soap, facial film, astringent, massage cream, emulsion, nourishing cream, cosmetic cream, foundation cream or muffin etc. according to the various product of the skin protection and the beauty treatment of purpose classification.
Do not contain external culture fluid composition in the stem cell active factors system of gained of the present invention, do not contain dead cell cracking composition yet.And can long preservation and keep tissue and cytothesis activity.The present invention utilizes the multidirectional differentiation of stem cell and powerful the of self-replication capacity, external a large amount of expanding stem cells.Have the epidermal stem cells of promotion propagation, promote skin metabolism according to stem cell composite factor system, strengthen the vigor of Skin Cell, promote the effect of cellular material composition exchange, strengthen the absorption of skin other nutrient substance, and the discharging of body intracellular metabolite refuse.Adopt the secretion effect of stem cell under serum-free condition, do not contained the stem cell factor compound system of cell culture medium and cellular component.Because the present invention does not contain external culture medium and cellular component, thereby the injury of having avoided external additive and lysis toxin that skin is caused.Make the stem cell beauty treatment really move towards reality from ideal.In the biomedical degree of depth traditional Aesthetic idea is promoted.It is simple to be somebody's turn to do invention simultaneously, is suitable for large-scale production, serves beautiful cause for the stem cell cell shortcut is provided.
The specific embodiment
Embodiment 1
Mouse embryo stem cell: derive from the biological center of Nankai University
The cultivation of ES cell and amplification: (embryonic stem cell, ES), cell concentration is about 1 * 10 to get the 1ml embryonic stem cell
6Individual cell/ml, syringe or suction pipe piping and druming are for several times, with the assurance is single cell suspension, cell is laid in the culture bottle of former generation mouse embryo fibroblasts (MEF), ES keeps culture medium (Tianjin couple stars Bioisystech Co., Ltd) 10ml and cultivates, and changes culture fluid after the incubated overnight, after 2-3 days, digest, go down to posterity, cultivate again.Above repeatedly operation is to obtain the ES of sufficient amount.
The preparation of stem cell active factors: when the growth of ES cell is the most vigorous (covering with culture bottle 70-80% area approximately), discard culture medium, clean 2-3 time, add 10mlPBS then, continue to cultivate 24 hours with PBS (or normal saline).Get supernatant, centrifugal, must be rich in the stem cell factor system.
Cell dissociation, counting: wash in the culture bottle cell gently 1-2 times with phosphate buffer solution (PBS), add 3ml Digestive system (025% trypsin, 002%RDTA), 37 ℃ are incubated 2-3 minute, and the microscopically observation of cell separates, add and contain serum DMEM culture medium, dispel cell, 1500 rev/mins centrifugal 5 minutes, abandon supernatant, add 5mlPBS, fully dispel cell, carry out cell counting, press 1ml supernatant/1 * 10
6The concentration concentrated supernatant of individual cell.
The preservation of the stem cell composite reactive factor:, put-20 ℃ of refrigerators, but long preservation is standby with the supernatant (one or many merging) after above the concentrating.
Embodiment 2
This embodiment provides the preparation process of the stem cell factor system of derived from bone marrow, and is specific as follows:
1. obtaining of former generation bone marrow stem cell: get healthy people's bone marrow 10ml, PBS is diluted to 20ml, obtain myelomonocyte by Ficoll density gradient centrifugation, have the characteristic of tending to stick on the polystyrene surface, MSCs is separated with hematopoietic cell according to MSCs.Cultivate and began to occur little cell colony in 3 days later on.Cell reaches capacity after about 7-9 days, and go down to posterity this moment.The cell that goes down to posterity behind the 3h is promptly adherent fully, and after 2 days, cell presents typical fibroblast sample form, is evenly distributed in the culture bottle.Cell density promptly reaches 80-90% after 3-4 days.
2. the amplification of cell: when cell density reaches 80-90%, wash in the culture bottle cell gently 1-2 times with phosphate buffer solution (PBS), add 3ml Digestive system (025% trypsin, 002%RDTA), 37 ℃ are incubated 2-3 minute, the microscopically observation of cell separates, add and contain serum α-MEM culture medium, dispel cell, ratio in 3 bottles of 1 bottle of passages goes down to posterity, more joyous culture fluid after 2 days, when cell density reached 80-90% again, above repeatedly operation was to obtain the bone marrow stem cell of sufficient amount.
3. the preparation of stem cell active factors: (cover with culture bottle 70-80% area approximately) when growth of marrow mesenchyme stem cell is the most vigorous, discards culture medium, PBS or normal saline clean 2-3 time, add 10mlPBS then, continue to cultivate 24 hours.Get supernatant, centrifugal, must be rich in the stem cell factor system.Peptic cell carries out cell counting, presses 1ml supernatant/1 * 10
6The concentration concentrated supernatant of individual cell.
4. the preservation of the stem cell composite reactive factor:, put-20 ℃ of refrigerators, but long preservation is standby with the supernatant (one or many merging) after above the concentrating.
Embodiment 3
Present embodiment provides the preparation process of epidermal stem cells active factors system, and is specific as follows:
Cell source: the skin graft (or neonate foreskin) that derives from healthy people.
The isolated culture method of cell: cultivate according to a conventional method, specifically can be with reference to (referring to Zhang Zhuoran chief editor's " cultured cell is learned and cell culture technology ").
The preparation of stem cell active factors: when the epidermal stem cells growth is the most vigorous (covering with culture bottle 70-80% area approximately), discard culture medium, PBS or normal saline clean 2-3 time, add 10mlPBS then, continue to cultivate 24 hours.Get supernatant, centrifugal, must be rich in the supernatant of stem cell factor.Peptic cell carries out cell counting, presses 1ml supernatant/1 * 10
6The concentration concentrated supernatant of individual cell.
The preservation of the stem cell composite reactive factor:, put-20 ℃ of refrigerators, but long preservation is standby with the supernatant (one or many merging) after above the concentrating.
Embodiment 4
The application in cosmetics of embodiment 4, stem cell secretion factor, with stem cell factor system concentrated solution, (the stem cell factor system: ratio cosmetics stock solution) joins in the prescription of cosmetics, and homogenizing is handled, and makes the cosmetics that are rich in the stem cell factor system with 1-10:100.Such cosmetics that contain the stem cell factor system have beauty treatment, defying age, repair cell damage, strengthen the function of skin elasticity, make skin tenderer, beautiful, the more high resilience that becomes.
Claims (6)
1, a kind of preparation method that is used for the stem cell secretion factor of beauty and skin care is characterized in that the step that comprises:
1) separate stem cells: from people or mammal respective organization, isolate stem cell;
2) amplification of stem cell, the conventional cultural method with corresponding stem cell comprises the a-MEM culture medium, adds 10% hyclone, 5%CO2 cultivates under 37 ℃ of conditions, regularly changes liquid, goes down to posterity, and then obtains the stem cell of requirement;
3) obtaining of stem cell factor system: in stem cell division the most vigorous period, discard normal culture medium, with PBS buffer (phosphate buffer, about pH=7.4) clean 1-3 time, add PBS or normal saline then, cultivated 4-24 hour in the cell culture incubator, collect supernatant, can obtain being rich in the solution of stem cell factor system;
4) concentrate, store: the gained centrifugal, remove the cell that is scattered, concentrate, by 1 * 10
6The concentration of individual cells/ml concentrates, and gets product, can be in-20 ℃ of long preservation, in order to using.
2, method according to claim 1, it is characterized in that the described secreted stem cell factor system of stem cell that derives from, this system comprises that this system comprises stem cell factor, endothelial cell growth factor (ECGF), epithelical cell growth factor, tumor necrosis factor hepatocyte growth factor, nerve growth factor, fiber mother cell growth factor, Insulin-Like growth factor, granulocyte colony-stimulating factor, interleukin; In addition, also contain each collagen type in the stem cell secretion factor, comprise I, II, III, IV collagen type, and various lysozyme.
3, method according to claim 1, it is characterized in that described stem cell factor system is meant: derive from people or mammalian embryonic stem cell, navel blood stem cell, bone marrow stem cell, epidermal stem cells, endothelial stem cell, peripheral hematopoietic stem cells, fat stem cell, also can make a general reference all stem cell.
4, method according to claim 1, the product that it is characterized in that gained is equipped with the product that the lysate of medically accepting constitutes compositions as active component, is directly used in beauty treatment, skin protection, comprises, beauty treatment, speckle dispelling, reparation cicatrix.
5, method according to claim 1 is characterized in that the product of gained can add in the beauty and skin care product of various preparations as active component, comprises reduce wrinkle, beauty treatment, speckle dispelling, reparation cicatrix.
6, method according to claim 1, the product that it is characterized in that gained is used for improving looks, the injection of skin-protection product, facial cream, emulsion, liquid preparation, comprises cleansing cream, cleaning breast, cleansing soap, facial film, astringent, massage cream, emulsion, nourishing cream, cosmetic cream, foundation cream or muffin.
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| CN109097391A (en) * | 2018-07-11 | 2018-12-28 | 个体化细胞治疗技术国家地方联合工程实验室（深圳） | A kind of method and its application that decidua mescenchymal stem cell active factors are mass produced |
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| CN109536441A (en) * | 2018-11-30 | 2019-03-29 | 佛山科学技术学院 | A kind of culture medium and preparation method thereof of fatty stem cell secretion object |
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| CN113699202A (en) * | 2020-05-21 | 2021-11-26 | 华子昂 | Method for preparing collagen by using mixed cell and artificial cell culture nest |
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