CN106420390A - Stem cell preparation for skin beauty and preparation method thereof - Google Patents
Stem cell preparation for skin beauty and preparation method thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/10—General cosmetic use
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
Abstract
The invention provides a stem cell preparation for skin beauty and a preparation method thereof, wherein the method comprises: a) providing stem cells acquired from a mammal animal; b) subjecting the stem cells to primary culture; c) subjecting the stem cells to subculture in a stem cell medium with vitamin A; d) changing with phenol-red-free culture liquid with astragaloside, and continuously culturing under hypoxic condition; e) subjecting the stem cells and the culture liquid to ultrasonic disruption and centrifuging, collecting supernate, and filtering to remove bacteria; f) detecting total protein concentration of the supernate, and adjusting the total protein concentration of the supernate. By using the preparation method of the stem cell preparation for skin beauty, the key problems of conventional culture methods, such as low in-vitro culture cell quantity and low factor content are solved.
Description
Technical field
The present invention relates to beauty treatment, skin care industry are and in particular to a kind of stem cell medicine for beauty and skin care and its preparation
Method.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC) is that a class has self renewal and Multidirectional Differentiation
The seed cell of potential, can not only be differentiated to form bone, cartilage, fat, muscle etc. mesoblastic under different inductive conditions
Cell is moreover it is possible to form ectodermic neuron, neurogliocyte, Skin Cell and endoblastic Ink vessel transfusing across differentiation of germinal layers
The Various Tissues cell such as chrotoplast.In addition to having multi-lineage potential, MSC also can secrete cytokine profiles, and MSC passes through other point
Secrete form secretion EGF, KGF, VEGF, angiogenesis hormone -1 (angiopoietin-1, Ang-1), bFGF, IGF-1,
Wnt1/3A/5A, HGF, PDGF-BB and EPO etc. are it is known that angiogenesis, the synthesis of promotion organization cell biological and energy can be promoted
The cell active substance of amount metabolism, it also can strengthen wound healing.In addition, these factors can be to regulation epidermis and epithelial cell life
The aspects such as length, differentiation and proliferation, promotion angiogenesis, improvement growth microenvironment, holding cytoactive play an important role[1].
Document report, EGF, FGF of denier can strongly facilitate division and the growth of Skin Cell, thus reach promoting application on human skin
Metabolism, promotion Regeneration and Repair, slowing down skin aging, make skin again recover elasticity and gloss[2-3].
Mostly presently commercially available cytokine product is single component cosmetics and skincare product, also has and is become for activity with EGF with bFGF
Divide and be added in cosmetics and skincare product, one or two single cytokines can not meet the demand of skin repair, cell turnover,
Multiple agents applications could promote epidermal renewal, and multiple-factor product also will become the inexorable trend of cosmetic industry development.
Chinese patent CN1872025A and CN101461772A all refers to the cytokine of stem cell secretion, all in air
Carry out culture amplification, document report under oxygen concentration (about 21%)[4], O in microenvironment2Concentration is very important factor, between pair
The impact of mesenchymal stem cells is many aspects, and the cytokine amount of the mescenchymal stem cell secretion of culture under air oxygen concentration is relatively
Few, through hypoxia condition (O2Concentration is 4%) the various factor amounts of the mescenchymal stem cell cultivated secretion all have different degrees of increasing
Plus, in addition, document report[5], O in human normal tissue and interstice2, in 3%-9%, human chondrocytes are (no for concentration
Vascular tissue) the oxysome fraction of growing environment is 0-7%, bone marrow intracavity concentration, in 1%-7% scope, illustrate that mesenchyme is done carefully
Born of the same parents are in low-oxygen environment in vivo, and it is that secrete cytokines play various functions with this understanding.
Pertinent literature has been reported can increase its content to the respective cells factor to a certain extent using hypoxia culture, but
Need long-time low oxygen process, condition of culture is had high demands, and Nostoc commune Vanch method, stem cell occurs breaking up after repeatedly passing on
Phenomenon, just no longer possesses the ability of secretion stem cell correlation factor, and noble cellss can not continue Secondary Culture once differentiation,
Problem above is unsuitable for producing in enormous quantities.
The feature of cytokine profiles can be secreted based on stem cell, the present invention develops human stem cell beauty skin care product, and
The active substance that its cosmetics and skincare product adds, selection comes from the mankind, solves part population because producing using chemical reagent product
Allergy, this make people psychology, be physiologically easier to accept the skin quality that this kind of product improves skin sensitive people.Solution
The problem that certainly traditional physical chemistry beauty treatment cann't be solved, inherently improves skin.
The present invention passes through specific culture technique, adds vitamin A and makes stem cell pass on multiple increase, when being passaged to for 20 generation
Still there is the function of stem cell secretion cytokine, adopt astragaloside to stimulate culture to cultivate combined process with hypoxia simultaneously, increase
Plus stem cell factor secretion, develop the system that a kind of cultural method is simple, cytokine secretion function is strong, cytokine amount to obtain is high
Preparation Method, realizes batch with a small amount of raw material and obtains, be a kind of preparation method being suitable for industrialization, is that cytokine is applied to protect
Skin beauty industry, tissue injury repair field and lay the foundation.
Content of the invention
One aspect of the present invention provides a kind of method preparing stem cell medicine, and it includes：
A) provide the stem cell obtaining from mammal；
B) in stem cell media, original cuiture is carried out to stem cell；
C) in being added with 0.8-1.2 μM of the stem cell media of vitamin A, stem cell is carried out with Secondary Culture to
15 to 20 generations；
D) it is replaced by the no phenol red culture fluid being added with 0.8-1.2 μM of astragaloside, in 1% to 5%O2Under conditions of continue
Continuous culture 24 hours；
E) stem cell after cultivating step d) carries out ultrasonication together with culture fluid, centrifugation, collects supernatant, and mistake
Filter bacterium；
F) total protein concentration of the supernatant that detecting step e) obtains, supernatant total protein concentration is adjusted to 9-11 μ g/ μ
L.
In a specific embodiment, described stem cell media is the α-MEM culture containing 2% serum substitute
Liquid.
In a specific embodiment, described no phenol red culture fluid is DMEM culture fluid or DMEM/F12 culture fluid.
In an optional embodiment, described stem cell is selected from navel blood stem cell, umbilical cord mesenchymal stem cells, epidermis
Stem cell, mesenchymal stem cells MSCs, fat mesenchymal stem cell.
In a preferred embodiment, described mammal is people.
In a specific embodiment, wherein in c), passed on when cell confluency reaches 75%-85%.
In an optional embodiment, said method is also included g)：Supernatant by adjusted total protein concentration in f)
Liquid lyophilization, obtains freeze-drying product.
In an optional embodiment, 0.8-1.2 μM of astragaloside can also be substituted using 1mg/mL astragalus polysaccharidess.
Wherein, the main active ingredient in astragalus polysaccharidess is polysaccharide and astragaloside.Astragaloside is divided into Astragalosid I, Astragaloside II, astragaloside
Ⅳ.Wherein most preferably astragaloside IV is astragaloside to biological activity.
In a specific embodiment, the method preparing above-mentioned stem cell medicine comprises the following steps：
1) provide the stem cell obtaining from mammal：Using corresponding cell separation technology, divide from the respective organization of people
Separate out stem cell；
2) in stem cell media, original cuiture is carried out to stem cell：Using the α-MEM containing 2% serum substitute
Culture fluid, in 37 DEG C, 5%CO2Under the conditions of carry out cultivating, expand using cell culture incubator；
3) Secondary Culture of stem cell：In being added with 1 μM of the stem cell media of vitamin A, stem cell is carried out
Secondary Culture, the growth conditions of visual cell carry out changing liquid, pass on, and every other day pass on once, when Secondary Culture is to 20 generation, and
When fusion rate reaches 75%-85%, abandoning supernatant, change the no phenol red culture fluid DMEM containing 1 μM of astragaloside；
4) cytokine enrichment：By step 3) cell that obtains put into 1% to 5% O2Continue culture in hypoxia incubator
24h, makes cytokine secretion amount increase；
5) preparation of stem cell medicine：By step 4) cell that processed carry out together with no phenol red culture fluid DMEM ultrasonic broken
Broken；Cytokine is completely discharged in cultivating system；Again 15min is centrifuged with 4000r/min, collects supernatant, discard precipitation, on
Clear liquid is with 0.22 μm of membrane filtration so as to aseptic；
6) total protein concentration mensure is carried out using BCA method, adjustment protein concentration is 10 μ g/ μ L；Alternatively protein concentration is surveyed
Determine method such as Lowry method, sulfosalisylic acid precipitation method, Bradford method；
7) preparation of the freeze-drying product of stem cell medicine：By 6) in multiple-factor be combined stem cell medicine according to 2-3ml/
Bottle fill, in cillin bottle, takes Freeze Drying Technique to obtain the freeze-drying product of stem cell medicine.
Alternatively, in step 4) and step 5) between can also carry out cytobiology coherent detection：Divided using ELISA method
The secretory volume of several factors in analysis culture supernatant, including EGF, bFGF, VEGF, KGF, Ang-I, PDGF, IGF-1, HGF.
Another aspect of the present invention provides a kind of stem cell medicine, and it is prepared by above-mentioned method.
In a specific embodiment, described stem cell medicine include stem cell secretion cytokine include but not
Be confined to epithelical cell growth factor (EGF), fibroblast growth factor (bFGF), VEGF (VEGF),
Human angiogenin I (Ang-I), erythropoietin (EPO), hepatocyte growth factor (HGF), transforming growth factor-β
(TGF-β), interleukin 6 (IL-6), IL-10 (IL-10), PGE2 (PGE2), insulin like growth factor (IGF-
1), platelet-derived growth factor (PDGF).
In a specific embodiment, described stem cell medicine also including collagen, laminin, fine even egg
In vain, hyaluronic acid.
Another aspect of the invention, there is provided the stem cell medicine of above-mentioned stem cell medicine or said method preparation is in system
Purposes in standby beauty skin care product and/or cosmetics.
In a specific embodiment, described stem cell medicine is liquid form.In another specific embodiment party
In case, described stem cell medicine is the freeze-drying product of the solid form being obtained by lyophilization.
In particular embodiments, stem cell medicine (the i.e. stem cell medicine of liquid form that the present invention is obtained
Stock solution) can be directly appended in beauty skin care product, the such as various dosage forms such as beauty treatment aqueous, frost, cream, dry powder, injection, Emulsion.
Including but not limited to facial cream, emulsion, astringent, toner/smoothing toner, cleanser, cleansing soap, facial film, massage cream, vanishing cream.Can also
The freeze-drying product of the stem cell medicine of the solid form that the present invention is obtained is added in the middle of beauty skin care product, this freezing
It is non-degradable that dry product can preserve its activity for a long time, meet long-term storage, have the advantages such as factor concentration scalable.In addition,
This freeze-drying product can be applied directly to skin surface after being dissolved with certain dissolving medium, or imports this beautifying liquid by apparatus
Subcutaneous play a role so as to fully absorb.
In particular embodiments, the described stem cell medicine of the described stem cell medicine of liquid form or solid form
Freeze-drying product coordinate certain solvent medium, include adding Moisture factor, rush absorption factor, whitening factor isoreactivity material straight
Connect for improving looks, skin protection, reach repair tissue, promote skin metabolism effect.
In particular embodiments, the described stem cell medicine of the described stem cell medicine of liquid form and solid form
Freeze-drying product can be added in beauty skin care product as a kind of cosmetic active agent, such as beauty treatment aqueous, frost, cream, dry
The various dosage form such as powder, injection, Emulsion.Including but not limited to facial cream, emulsion, astringent, toner/smoothing toner, cleanser, cleansing soap, face
Film, massage cream, vanishing cream.
Another aspect of the invention, there is provided the stem cell medicine of above-mentioned stem cell medicine or said method preparation is in system
It is ready for use on the purposes in the medicine for the treatment of skin injury.
Specifically, described dermal tissue insult includes but is not limited to skin burn, scald, ultraviolet injury, wound, sugar
Urinate dermal tissue insult, the difficulty that foot disease disease, operative incision, chronic wound (as chronic granulo wound surface, ulcer, decubital ulcer etc.) etc. cause
Wounds.
Stimulated altogether in vitro with reference to astragaloside using vitamin A from the above as can be seen that present method be advantageous in that
The stem cell of culture, and hypoxia culture process, pass on multiple using the mescenchymal stem cell that vitamin A makes In vitro culture bright
Aobvious increase, amplification quantity dramatically increases, and astragaloside stimulates mescenchymal stem cell so that its cytokine amount secreted substantially is increased,
Solve conventional culture methods cultured cell in vitro number few, the low critical problem of factor content.It is dry thin that this method obtains
Born of the same parents' preparation effect is remarkably reinforced, and its freeze-drying product is not only easy to use, achievable industrialization, and is easy to protect for a long time
Deposit, greatly reduce the requirement of preservation condition, solve holding time this bottleneck problem short.The stem cell system of present invention preparation
The freeze-drying product of agent can coordinate certain dissolving medium to be applied directly on skin surface, or imports subcutaneous；Can also be used as one
Kind of beauty and skin care composition is added to beauty treatment, in skin-protection product, is such as added to the products such as facial cream, astringent, Emulsion, muffin.Also may be used
To be added in the middle of the repair in trauma series of products with cytokine as raw material.
Brief description
Fig. 1 is the freeze-drying product pictorial diagram of the stem cell medicine of the present invention.
Fig. 2A to Fig. 2 D is the impact to human umbilical cord mesenchymal stem cells growing multiplication for the stem cell medicine of the present invention.Its
In, Fig. 2A is the growth result of the human umbilical cord mesenchymal stem cells of culture in blank control group；Fig. 2 B is to add embodiment 1 to prepare
Stem cell medicine freeze-drying product culture human umbilical cord mesenchymal stem cells growth result；Fig. 2 C is to add embodiment 2
The growth result of the human umbilical cord mesenchymal stem cells of stem cell medicine freeze-drying product culture of preparation；Fig. 2 D is to add embodiment
The freeze-drying product of the stem cell medicine of 3 preparations cultivates the growth result of human umbilical cord mesenchymal stem cells.
Fig. 3 A to Fig. 3 C is the rear example of stem cell medicine treatment ultraviolet injury animal model of the present invention.Wherein, scheme
3A is ultraviolet injury model；Fig. 3 B is purple using the stock solution treatment of the mesenchymal stem cells MSCs preparation of preparation in embodiment 2
Outside line damage results；Fig. 3 C is the stock solution treatment ultraviolet injury result using fat mesenchymal stem cell preparation in embodiment 3.
Specific embodiment
Below in conjunction with accompanying drawing, the present invention is further illustrated by embodiment, but not as limitation of the present invention.Hereinafter carry
Supply concrete material and its source used in embodiment of the present invention.It is to be understood that these are only example
Property it is not intended to limit the present invention, same or similar with the type of following reagent and instrument, model, quality, property or function
Material may be incorporated for implement the present invention.Experimental technique used in following embodiments if no special instructions, is routine
Method.Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
Embodiment 1：Prepare stem cell medicine and its freeze-drying product by umbilical cord mesenchymal stem cells
1st, the acquisition of primary umbilical cord mesenchymal stem cells and culture
Multipara's informed consent, hepatitis B virus antigen, hepatitis C surface antibody, HIV (human immunodeficiency virus), treponemal
Body antibody etc. is detected as feminine gender, obtains the neonatal umbilical cord through aseptic process.
The neonatal umbilical cord of aseptic collection is put in normal saline, soaks 2min with 75% ethanol, then soaked with PBS clear
Wash, remove the remaining blood of Ink vessel transfusing, umbilical cord scissories are broken into lmm3The piece of tissue of size, by evenly laid out for piece of tissue in culture dish
In (corning, article No. 430167), it is inverted culture 48h, behind tissue patch jail, add 5ml stem cell media (containing 2% serum
Substitute (Pall；Article No. 15950-017)-MEM culture fluid (Hyclone；Article No. SH30265.01)), in 37 DEG C, 5%
CO2(v/v) cultivate in incubator, cultivate 3-5d, the cell growing is primary umbilical cord mesenchymal stem cells.
2nd, the amplification of umbilical cord mesenchymal stem cells and Secondary Culture
Primary umbilical cord mesenchymal stem cells fusion rate reaches 80% about, carefully discards culture fluid, adds appropriate physiology salt
Water cleans cell surface, to remove remaining culture liq.Outwell washing liquid, add 0.125g/L trypsin SCIENTIFC
RESEAROH SPECIAL；Article No. 27250018) 2ml, when Microscopic observation most cells are become round by fusiform, gently pats
Culture dish is so as to float.- MEM the culture fluid (stem cell media) that 2ml contains 2% serum substitute is added to terminate pancreatin to thin
The Digestion of born of the same parents.Clean culture dish with normal saline, the cell suspension that digestion is got off pours 50ml centrifuge tube, 1500r/ into
Min is centrifuged 5min, and centrifugation takes out centrifuge tube after terminating, and abandons supernatant.30ml is added to contain-MEM the culture fluid of 2% serum substitute,
Mix homogeneously, takes a small amount of cell suspension to count, with 5 × 106Individual/bottle cell density is inoculated in T182 bottle (BIOFIL；Article No.
TCF011600, in), in every bottle of-MEM culture fluid adding 25ml to contain 2% serum substitute, tie up according to final concentration of 1 μM simultaneously
Raw element A (Shanghai Yuan Ye bio tech ltd；Article No. S13006-5) be added in culture fluid, put into incubator (37 DEG C,
5%CO2(v/v)) cultivate.When cell confluency reaches 75%-85%, according to above-mentioned steps Secondary Culture, during every culture, do
All add 1 μM of vitamin A in cell culture medium, expanded for the 20th generation.
3rd, the cytokine enrichment strategy of umbilical cord mesenchymal stem cells secretion
When expanding for 20 generation, the mescenchymal stem cell fusion rate of above-mentioned culture is 75%-85% (when about 80%), will
Original fluid changes into containing 1 μM of astragaloside (Shanghai Yuan Ye bio tech ltd；Article No. S31401-500) no phenol red
Culture fluid (DMEM culture fluid (ThermoFisher, article No. 11054020) or DMEM/F12 (ThermoFisher, article No.
21041025), 18ml/T182 bottle, puts into 3%O2(v/v) continue culture 24h in hypoxia incubator, make cytokine secretion amount
Increase.
4th, the preparation of the stem cell medicine of umbilical cord mesenchymal stem cells
By the umbilical cord mesenchymal stem cells supernatant collection through above-mentioned culture in centrifuge tube, add the washing of 10ml physiology salt
Wash once, add 0.125g/L trypsin 5ml and digest adherent cell, Microscopic observation most cells are become round by fusiform
When, gently pat culture bottle so as to floating, add-MEM the culture fluid that 5ml contains 2% serum substitute to terminate pancreatin to cell
Digestion.Clean culture bottle with normal saline, the cell suspension that digestion is got off is put in 200ml centrifuge tube, 1500r/
Min is centrifuged 10min, and centrifugation takes out centrifuge tube after terminating, and abandons supernatant, adds above-mentioned cell supernatant in cell precipitation, mixes,
Carry out ultrasonication, make cell cracking, 15min is centrifuged with 4000r/min, collect supernatant, discard precipitation, supernatant uses 0.22
μm membrane filtration so as to aseptic, carries out protein concentration survey using BCA protein concentration detection kit (the green skies, article No. P0009)
Fixed, adjustment concentration is that (with sterilized water for injection adjustment, if concentration is higher than 10 μ g/ μ L, dilute with water, if dense for 10 μ g/ μ L
Degree is less than 10 μ g/ μ L, then mix the sample high with detectable concentration according to volume ratio it is ensured that concentration is 10 μ g/ μ L after mixing),
Obtain multiple cytokine activity liquid, it comprises the cell growth factor of umbilical cord mesenchymal stem cells secretion, and fills between umbilical cord
Matter Stem Cell Activity albumen, i.e. stem cell medicine.
5th, the preparation of the freeze-drying product of umbilical cord mesenchymal stem cells preparation
By multiple cytokine activity liquid achieved above with 2ml/ bottle, fill, in cillin bottle, covers lyophilization special
With rubber stopper, put into freezer dryer (LGJ-10F vacuum freeze drier；Beijing development in science and technology company limited of Song Yuan Huaxing)
In, carry out lyophilization, -70 DEG C of freezing 12h by following procedure, then heated up with the speed of 5 DEG C/h, when temperature reaches -10 DEG C,
Keep 5h；When temperature reaches 20 DEG C, keep 3h；When temperature reaches 25 DEG C, keep 3h；Obtain the dry thin of umbilical cord mesenchymal stem cells
The freeze-drying product of born of the same parents' preparation.
Embodiment 2：Prepare stem cell medicine and its freeze-drying product by mesenchymal stem cells MSCs
1st, gather volunteer bone marrow 15ml under the acquisition of Primary bone marrow mescenchymal stem cell and culture aseptic condition, heparin resists
Solidifying, add 10ml normal saline, mix, be slowly added in 50ml centrifuge tube along tube wall using 5ml syringe, in centrifuge tube
It is previously added Percoll cell separation liquid (solarbio, article No. P8370) 15ml, density gradient centrifugation, 2000r/min, from
The heart, 30min, collects middle tunica albuginea confluent monolayer cells in sterile centrifugation tube, adds 30ml brine cell, with 1500r/
Min, centrifugation, 10min, abandoning supernatant, repeated washing 2 times, it is eventually adding-MEM the culture that 20ml contains 2% serum substitute
Liquid, in 37 DEG C, 5%CO2(v/v) cultivate in incubator, cultivate 3-5d, the cell growing is primary umbilical cord mesenchyma and does carefully
Born of the same parents.
2nd, mesenchymal stem cells MSCs amplification and Secondary Culture, cytokine enrichment, the step of lyophilizing preparation are with embodiment 1
In method.
Embodiment 3：Stem cell medicine and its freeze-drying product are prepared by fat mesenchymal stem cell
Through informed consent, adipose-derived in women abdominal part liposuction procedures person, 70ml fat adds appropriate saline to wash twice,
Remove part hemocyte, add equal-volume 1.5%I Collagenase Type (solarbio, article No. C8140), in 37 DEG C of waters bath with thermostatic control
Pot concussion digestion 40min, takes off layer mononuclearcell in 50ml centrifuge tube, addition 30ml brine cell 3 times,
20ml is added to contain-MEM the culture fluid of 2% serum substitute, in 37 DEG C, 5%CO afterwards2Cultivate in incubator, the growth of visual cell
State carries out changing liquid or Secondary Culture.Other preparation processes are with embodiment 1.
Embodiment 4：The impact of stem cell medicine cell growth propagation
1st, experiment material
6 well culture plates (corning, article No. 3516)；- MEM culture fluid (Hyclone；Article No. SH30265.01)；Dry thin
Born of the same parents' preparation (stem cell medicine of embodiment 1 preparation, i.e. umbilical cord mesenchymal stem cells multiple cytokine freeze-drying product)；When long
Between cell dynamic measuring instrument (Essen Bioscience)
2nd, experimental technique
Using-MEM culture fluid suspension the human umbilical cord mesenchymal stem cells of 2% serum substitute, then with 1 × 105Individual/hole
It is inoculated in 2 piece of 6 well culture plate, be placed in incubator culture 6h so as to fully adherent, be divided into A, B, C, D group, wherein A group is 3
Individual culture hole changes-MEM culture fluid matched group into, and B group changes the freeze-drying product of 10% embodiment 1 preparation into for 3 culture hole
(i.e. the stock solution of 2ml umbilical cord mesenchymal stem cells preparation is prepared into one bottle of freeze-drying product to-MEM culture fluid, then takes this bottle of freezing
The dry product solution obtaining after 2ml sterilizing water dissolution, this solution accounts for the 10% of the volume of-MEM culture fluid), C group is 3 and trains
Foster hole changes freeze-drying product (the freeze-drying product addition 2ml sterilizing of the stock solution preparation of 2ml volume of 10% embodiment 2 preparation into
Water dissolution)-MEM culture fluid, D group for 3 culture hole change into 10% embodiment 3 preparation freeze-drying product (2ml volume
The freeze-drying product of stock solution preparation adds 2ml to sterilize water dissolution)-MEM culture fluid, change dynamic using long-time cell after liquid
Detector is observed, and takes pictures once every 4h, every other day changes liquid once.
3rd, result and discussion
Compare with control wells, during In vitro culture 2d, add stem cell medicine and the growth of human umbilical cord mesenchymal stem cells is increased
Grow and there is obvious facilitation, result is shown in Fig. 2A to Fig. 2 D, this also illustrates the stem cell system of the method preparation in embodiment 1-3
Agent all can promote cell division to breed, and has plerosis function to tissue injury.
Embodiment 5：The repair to ultraviolet injury animal model for the stem cell medicine
1st, experiment material
(embodiment 2 prepares the stock solution of mesenchymal stem cells MSCs preparation to stem cell medicine；Embodiment 3 is prepared and is filled between fat
The stock solution of matter stem cell medicine)；SD male rat is (purchased from the long-living Bioisystech Co., Ltd in Liaoning；Quality certification number SCXK (distant)
2015-0001)(180-220g)；Uviol lamp UVB；Shears；Razor；10% chloral hydrate (solarbio, article No. T8590)；Aluminum
Foil paper；Micropin roller (540 pins, the long 1.0mm of pin)
2nd, experimental technique
2.1 animal feeding
Every 5 of rat puts into a cage raising, and under identical natural environment, (day alternates with night 12 hours) raises, conventional feeding
And drinking-water, no undesirable element impact is so as to proceed by experiment after adapting to laboratory environment.
2.2 depilation
Mark pre-irradiation scope with marker pen, first cut off the coarse wool of Mus back with shears, with shaver by remaining floss
Hair shaves clean, depilation area about 9cm2, so that skin of back is fully exposed.
2.3 models are set up and experiment process method
Animal adaptability is fed 1 week, and second week starts, by SD rat 36, to be randomly divided into 4 groups each 9：Normal group, model
Matched group, model experiment group 1, model experiment group 2.UVB Burdick lamp preheats 15min, stabilizing UV intensity.Medium wave ultraviolet
UVB irradiation in rats prepares that skin pipe is aging, ultraviolet injury model, after skin condition is stable, using 10% chloral hydrate fiber crops
Liquor-saturated rat, is carried out according to 0.5ml/100g dosage, and model control group and model experiment group rat are carried out ultra-vioket radiation, and distance is shone
Penetrate irradiation medium wave ultraviolet 1h/ time at the 40cm of position, irradiate twice daily, Continuous irradiation one week, non-irradiated position is hidden with aluminium-foil paper
Lid, to avoid ultra-vioket radiation to damage.
Daily to micropin roller at experimental group rat baring skin once, then uniform application 0.5ml stem cell medicine hereafter,
Stock solution, model group 1 smears the stock solution of embodiment 2 preparation, and model group 2 smears the stock solution of embodiment 3 preparation, all smears two daily
Secondary, all continuously smear 15 days, after observing ultraviolet injury, skin appearance improves situation.
3rd, result and discussion
Normal group laboratory animal skin smooth high resilience, water are tender；Compare with normal group, model control group skin occurs red
Swollen, scurf increases, coarse plumpness, erythema, deep wrinkle, drying, the aging, damage characteristic such as follow the string.Model experiment group skin
Outward appearance is all obviously improved compared with model control group, and skin has the coarse plump sense of elasticity, anoderm, also no deep wrinkle, skin is relatively
Water is tender.This explanation stem cell medicine can effectively facilitate generation, the secretion of hyaluronic acid and the glycoprotein of rat skin neoblast
Generate, damaged skin structure can be adjusted, promote damaged skin quickly to repair, make skin smoothness, water tender and high resilience, to skin
Skin photoaging has repair.Result is shown in Fig. 3 A, Fig. 3 B and Fig. 3 C.
Embodiment 6：Cytokine content analysis in stem cell medicine
Using ELISA kit (producer arigo), according to EGF in test kit operating procedure analysis stem cell medicine,
bFGF、VEGF、KGF、Ang-I、PDGF-BB、IGF-1、HGF.This lists above-mentioned factor content in 3 batches of stem cell medicines, tool
Body the results are shown in Table 1.
Cytokine content in table 1 stem cell medicine
It can be seen that, the stem cell medicine that the present invention provides and its freeze-drying product can be used directly, also can be as biological activity
Material is added in beauty skin care product.The stem cell medicine of the present invention directly acts on skin target cell, in active cell
Metabolic process, accelerates its metabolism, is recovering cytoactive, is promoting the aspect prospects such as propagation renewal, reparation damaged tissues wide
Wealthy.Cytokine is applied to improve looks, and has broken away from traditional physics, the constraint of chemistry beauty treatment, beautiful from the inherent realization of human body
Beautiful, young state.At present, domestic also very deficient in the theoretical research of beauty treatment fields and practice with regard to cytokine, deep further
Enter to study the essence that cytokine plays a role, being applied to beauty treatment for more multiple cytokine provides more fully theoretical basiss,
Tradition beauty treatment will be replaced with cytokine by the biological beauty of representative[6].
List of references
[1] Tang Yanhong, royal power is auspicious, Tang Ming. the protective effect [J] to skin for the hEGF. Chinese medical is beautiful
Beauty treatment magazine, 2004,10 (1):53-55.
[2] pay dogface, Sun Xiaoqing, Sun Tongzhu, etc. epithelical cell growth factor treats the stem cell island phenomenon that wound surface occurs
[J]. Chinese Medical Journal, 2001,81 (12):733-736.
[3] pay dogface, Sun Xiaoqing, Sun Tongzhu, etc. epithelical cell growth factor passes through the differentiation of induced skin stem cell and accelerates
It is wound the research [J] of promoting epidermization. Chinese Reconstructive surgery magazine, 2002,16 (1):31-35.
[4] Song Xi takes. the impact [D] to human umbilical cord mesenchymal stem cells biological characteristicses and propagation for the hypoxia. coordinate in Beijing
Medical college graduate school.
[5] Wang Liwei, Huang Wen, Zhao Yu. the impact [J] to mesenchymal stem cell biological for the hypoxia culture. China group weaver
Journey research, 2012,16 (23):4329-4334.
[6] Zhang Zhenmin etc. the new opplication [J] of biological beauty-cytokine. medical research and education .2011.3 (28).
Claims (10)
1. a kind of method preparing stem cell medicine, it includes：
A) provide the stem cell obtaining from mammal；
B) in stem cell media, original cuiture is carried out to stem cell；
C) in being added with 0.8-1.2 μM of the stem cell media of vitamin A, stem cell is carried out Secondary Culture to the 15th to
20 generations；
D) it is replaced by the no phenol red culture fluid being added with 0.8-1.2 μM of astragaloside, in 1% to 5%O2Under conditions of continue culture
24 hours；
E) stem cell after d) middle culture is being carried out ultrasonication together with no phenol red culture fluid, centrifugation, collect supernatant, and mistake
Filter bacterium；
F) obtain the total protein concentration of supernatant in detection e), supernatant total protein concentration is adjusted to 9-11 μ g/ μ L.
2. the method preparing stem cell medicine according to claim 1, described stem cell media is containing 2% blood serum substituting
α-MEM the culture fluid of thing.
3. the method preparing stem cell medicine according to claim 1 and 2, wherein said no phenol red culture fluid is DMEM training
Nutrient solution or DMEM/F12 culture fluid.
4. the method preparing stem cell medicine according to any one of claim 1 to 3, wherein said stem cell is selected from umbilicuss
Hemocytoblast, umbilical cord mesenchymal stem cells, epidermal stem cells, mesenchymal stem cells MSCs, fat mesenchymal stem cell.
5. the method preparing stem cell medicine according to any one of claim 1 to 4, wherein said mammal is
People.
6. the method preparing stem cell medicine according to any one of claim 1 to 5, wherein in c), when cell melts
Conjunction rate reaches and is passed on during 75%-85%.
7. the method preparing stem cell medicine according to any one of claim 1 to 6, it is also included g)：By warp in f)
The supernatant lyophilization of adjustment total protein concentration, obtains freeze-drying product.
8. a kind of stem cell medicine, it passes through the method preparation preparing stem cell medicine any one of claim 1 to 7
Obtain.
9. purposes in preparation beauty skin care product and/or cosmetics for the stem cell medicine according to claim 8.
10. stem cell medicine according to claim 8 is used for the purposes in the medicine treating dermal tissue insult in preparation；
Alternatively, described dermal tissue insult include skin burn, scald, ultraviolet injury, wound, diabetic foot, operative incision,
Dermal tissue insult that chronic wound causes, refractory wound surface.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107126556A (en) * | 2017-05-17 | 2017-09-05 | 何伟 | A kind of stem cell extract and preparation method thereof and the application in skin wound preparation for repairing is prepared |
| CN107488626A (en) * | 2017-08-10 | 2017-12-19 | 河南省银丰生物工程技术有限公司 | A kind of umbilical cord mesenchymal stem cells extract and its application in terms of beauty |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503672A (en) * | 2008-02-05 | 2009-08-12 | 湖南惠霖生命科技有限公司 | Composition of cytokine and use thereof |
| CN103550117A (en) * | 2013-10-18 | 2014-02-05 | 上海润生生物技术有限公司 | Beauty and skin care product capable of promoting skin repairation and regeneration as well as preparation method and application thereof |
| CN104013644A (en) * | 2014-05-30 | 2014-09-03 | 成都清科生物科技有限公司 | Conditioned medium preparation of mesenchymal stem cells used for skin aging restoration |
| CN105543313A (en) * | 2015-12-29 | 2016-05-04 | 四川新生命干细胞科技股份有限公司 | Human-derived mesenchymal stem cell factor, and preparation method and application thereof |
-
2016
- 2016-11-25 CN CN201611050918.2A patent/CN106420390A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503672A (en) * | 2008-02-05 | 2009-08-12 | 湖南惠霖生命科技有限公司 | Composition of cytokine and use thereof |
| CN103550117A (en) * | 2013-10-18 | 2014-02-05 | 上海润生生物技术有限公司 | Beauty and skin care product capable of promoting skin repairation and regeneration as well as preparation method and application thereof |
| CN104013644A (en) * | 2014-05-30 | 2014-09-03 | 成都清科生物科技有限公司 | Conditioned medium preparation of mesenchymal stem cells used for skin aging restoration |
| CN105543313A (en) * | 2015-12-29 | 2016-05-04 | 四川新生命干细胞科技股份有限公司 | Human-derived mesenchymal stem cell factor, and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 刘丹等: "维生素A对体外培养人脐带间充质干细胞生长的影响", 《中国细胞生物学学报》 * |
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