CN109200011A - A kind of skin care item and preparation method thereof - Google Patents
A kind of skin care item and preparation method thereof Download PDFInfo
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- CN109200011A CN109200011A CN201811409317.5A CN201811409317A CN109200011A CN 109200011 A CN109200011 A CN 109200011A CN 201811409317 A CN201811409317 A CN 201811409317A CN 109200011 A CN109200011 A CN 109200011A
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- A—HUMAN NECESSITIES
- 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
- A61K8/65—Collagen; Gelatin; Keratin; 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
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
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- A—HUMAN NECESSITIES
- 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
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/84—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions otherwise than those involving only carbon-carbon unsaturated bonds
- A61K8/85—Polyesters
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- A—HUMAN NECESSITIES
- 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
- A61K8/96—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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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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
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/59—Mixtures
- A61K2800/592—Mixtures of compounds complementing their respective functions
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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
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
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Abstract
The present invention relates to biological skin care fields, in particular to a kind of skin care item and preparation method thereof.A kind of skin care item, the parts by weight of each component are as follows: component A: 3 ± 0.2 parts of polyglycol distearate, 11 ± 0.5 parts of triple pressed stearic acid, 2 ± 0.2 parts of mineral oil, 1.3 ± 0.2 parts of lecithin, 3.7 ± 0.5 parts of Tween-60,0.1 ± 0.05 part of Span-60,2 ± 0.2 parts of propylben, 2 ± 0.2 parts of hyaluronic acid, 2 ± 0.2 parts of silk-fibroin；Component B: 18 ± 1 parts of glycerol, 0.2 ± 0.05 part of methylparaben, 54-55 parts of pure water；Component C: excretion body freeze-dried powder 1 × 10‑9~7 × 10‑8Part.Skin care item provided by the invention, each component coordinated, obtained skin care item performance are stablized, and nutrition composition therein cooperates, and have and preferably remove wrinkle, increase the effect of skin elasticity.
Description
Technical field
The present invention relates to biological skin care fields, in particular to a kind of skin care item and preparation method thereof.
Background technique
Skin is stacked by the fine and close multilayer tissue of construction with cell, is broadly divided into epidermis, corium and subcutaneous tissue.Skin
The aging of skin is that physiologic factor and external factor are coefficient as a result, physiologic factor such as age, endocrine and immune function etc.,
External factor such as ultraviolet light, wind etc..The main reason for aging of skin corium is skin aging, skin corium is directly and containing transparent
The matrix of many kinds of substance such as matter acid, compound protein sugar is connected, these matrix are the metabolic conversions such as various water-soluble substances, electrolyte
Place, have most direct relationship with skin aging.The aging for delaying skin corium matrix delays the reduction of matrix capilary amount,
Aging course can be effectively relieved.The prolonged and repeated irradiation of solar UV is the influence most important factor of skin aging in environment,
It can result in pachylosis, wrinkle intensification, pigmentation, blood vessel dilatation, the denaturation of corium elastomer etc..
In the development course of skin care item, the first generation is the grease type cosmetics of simple physical protection, such as sheep oil, second
Dai Ze is the aliphatic compound of synthesis, such as odium stearate, and the main component of the third generation is the extract of natural plants.4th
For biotechnology skin care item, is extracted using biotechnology and manufacture biological fine similar with human body self structure and with high-affinity
Magnificent substance, umbilical cord mesenchymal stem cells excretion body are exactly one type, and the various factors entrained by excretion body can protect skin
Reason turns to cellular level, fundamentally realizes the rejuvenation of skin condition.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of skin care item, each component cooperation, with better skin effect.
The second object of the present invention is to provide the preparation method of the skin care item, and entire temperature is lower, operates conveniently,
Obtained skin care item each component dispersion is uniform, has better skin effect.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The parts by weight of a kind of skin care item, each component are as follows:
Component A: 3 ± 0.2 parts of polyglycol distearate, 11 ± 0.5 parts of triple pressed stearic acid, 2 ± 0.2 parts of mineral oil,
1.3 ± 0.2 parts of lecithin, 3.7 ± 0.5 parts of Tween-60,0.1 ± 0.05 part of Span-60,2 ± 0.2 parts of propylben,
2 ± 0.2 parts of hyaluronic acid, 2 ± 0.2 parts of silk-fibroin；
Component B: 18 ± 1 parts of glycerol, 0.2 ± 0.05 part of methylparaben, 54-55 parts of pure water；
Component C: excretion body freeze-dried powder 1 × 10-9~7 × 10-8Part.
Skin care item provided by the invention, each component coordinated, obtained skin care item performance is stablized, and nutrition therein
Component cooperation, has and preferably removes wrinkle, increase the effect of skin elasticity.
Further, the excretion body freeze-dried powder is prepared using following methods:
Mescenchymal stem cell excretion body is resuspended with PBS, and xanthan gum mixing is added, then obtains institute through vacuum freeze-drying method
State mescenchymal stem cell excretion body freeze-dried powder；
The additive amount of the xanthan gum is the 15%-20% of the mescenchymal stem cell excretion body weight.
The present invention prepares excretion soma powder using xanthan gum, and compared to trehalose, xanthan gum has preferably protection excretion
The active effect of effective component in body reaches the longer skin care time, after skin care item provided by the invention, when skin care
Between persistently, 15 more than hour do not have the impression of skin hair shaft.
Mescenchymal stem cell (mesenchymal stem cells, MSC) be mesoderma origin, have height self more
The multipotential stem cell of new ability and multi-lineage potential.In fact, MSC is widely present in whole body Various Tissues, almost source
In all histoorgans of body, such as marrow, periosteum, adipose tissue, dental pulp, synovial membrane, umbilical cord, placenta, amniotic fluid and fetal tissue
Deng.The mescenchymal stem cell of separate sources has similar form, expresses identical surface markers, has similar biological characteristics
Property aspect, can such as cultivate amplification in vitro, and can be divided under given conditions thin including nerve cell, osteoblast, cartilage
The cell of more organization systems including born of the same parents, muscle cell, fat cell.MSC in addition to multidirectional histocyte differentiation capability it
Outside, important regulative also is played in hematopoiesis, immunization inflammatory reaction, angiogenesis et al. body critical function.
Umbilical cord mesenchymal stem cells (UC-MSC), which refer to, is present in one of neonatal umbilical cord tissue versatile stem cell.
Umbilical cord mesenchyma is a kind of low immunogenicity cell, and has very strong immunoloregulation function.Umbilical cord mesenchymal stem cells possess
The complete characteristic of MSC, and rich content are easily isolated culture.
Compared with the MSC in other sources, UC-MSC is more original, is dry between embryonic stem cell and adult stem cell
Cell, proliferation is lower than embryonic stem cell with differentiation capability, but is apparently higher than adult stem cell.
Umbilical cord mesenchymal stem cells have more differentiation potentials, and under specific inductive condition, UC-MSC can not only be divided into
The mesoblastemas such as bone, cartilage, fat, tendon, and can inwardly embryonic tissue cell (such as cardiac muscle cell, liver cell) and outside
Embryonic tissue cell (such as nerve cell) differentiation.UC-MSC does not have oncogenicity, can different inductive condition and it is suitable in vivo
It grows in microenvironment, safely directed differentiation is different tissue lines, has the ability for repairing various tissues and organ.
Excretion body (exosomes) is a kind of vesicles of lipid molecular film package.Electric cup-shaped under the microscope, diameter is about
For 40-100nm, density is about 1.13-1.19g/mL.Excretion body contains the cytoplasm and lipid envelope ingredient of derived cell, excretion
Internal portion includes mRNA abundant, microRNA and protein component.
The formation of excretion body is the cellular compartment of more vesica endosomes (MVE) to being formed by way of endogenous budding, intracellular vesicle
It is interior to contain protein, microRNA and mRNA.As MVE and cell membrane fusion, intracellular vesicle is just released to outside extracellular become
Body is secreted, the excretion body released can run to the behavior and change physiologically that cell is influenced apart from farther away tissue.
Because excretion body carries the RNA of derived cell, microRNA and protein ingredient, participates in cell and intercellular information passes
It passs, therefore during stem-cell therapy, the excretion body of source of human stem cell plays critically important effect on tissue reconstruction, especially
Albumen or RNA ingredient and damaged cell that it, which is in excretion body, is included carry out the function of information interchange, can to target cell into
The regulation of row biological function achievees the purpose that reparation, and also therefore excretion body has obtained extensive experiment in terms of regenerative medicine
Research.
Due to excretion body and its small and similar with cell culture constituents, therefore, it is very difficult to separate.Existing separation
Method is supercentrifugation, filter centrifugation method, affine in immunity or molecular exclusion chromatography, the poly precipitation method and micro-fluidic skill respectively
Art.Supercentrifugation is the goldstandard of excretion body separation, can be isolated outside in laboratory by the way that ultracentrifuge is convenient and efficient
Secrete body.Other methods are required to buy the consumptive materials such as specific instrument and splitter, excretion at high cost, complicated for operation and isolated
Body purity is without supercentrifugation height.
In addition, the acquisition limited source of body secretion, limits its application.
Further, the mescenchymal stem cell excretion body is prepared by the following method:
Umbilical cord mesenchymal stem cells secondary culture 8-14h, siphons away culture solution and not adherent cell, and cleaned, so
The dedicated complete culture solution of excretion body is added afterwards to be cultivated；
Culture solution is collected after 72-96h into centrifuge tube, obtains the culture solution rich in mescenchymal stem cell excretion body；
The culture solution rich in mescenchymal stem cell excretion body is centrifuged 5-10min at 4 ± 2 DEG C with 2000-2200g,
Dead cell and fragment are removed, then filtering removal vesica；
Obtained liquid is centrifuged 4-8h in 100000-120000g, abandons supernatant, and the liquid on centrifuge container tube wall is gone
It removes, obtains mescenchymal stem cell excretion body；
The dedicated complete medium of excretion body adds following components: without excretion body on the basis of without phenol red α-MEM
FBS percentage by volume be 10% ± 2%, 200mM glutamine percentage by volume be 1% ± 0.05%, bFGF concentration be 20 ±
2ng/mL, EGF concentration are 20 ± 2ng/mL.
The present invention is using the dedicated complete culture solution of excretion body to the umbilical cord mesenchymal stem cells for cultivating a period of time after passage
It is cultivated, effectively promotes the content of excretion body, promote 25% or more；It is dry thin that umbilical cord mesenchyma is handled by specific method
Born of the same parents obtain the culture solution rich in mescenchymal stem cell excretion body, then separate to it, obtain that concentration is higher, purity is higher
Mescenchymal stem cell excretion body.
Further, the no excretion body FBS using following methods prepare: FBS in 100000g-120000g revolving speed from
Heart 8-14h；
Draw upper layer clarify serum, and use 0.22 μm of membrane filtration, filtrate as without excretion body FBS.
The preparation method of no excretion body FBS provided by the invention, is centrifuged, then again using certain revolving speed and time
It is filtered, filtrate is without excretion body FBS.This method is easy, it is easily operated obtain it is outer without being not detected in excretion body FBS
Secrete body.
Further, the filtrate obtained ensures germ-free condition, sealing, spare.
Further, culture P3 is passed on when cell length to 85% ± 10% fusion for umbilical cord mesenchymal stem cells.
In the step, P3 is as follows for the component of umbilical cord mesenchymal stem cells culture and passage culture medium used: with α-
On the basis of MEM, add following components: FBS percentage by volume is that 10% ± 2%, 200mM glutamine percentage by volume is 1%
± 0.05%, bFGF concentration are 20 ± 2ng/mL, and EGF concentration is 20 ± 2ng/mL.
Further, it is described passage according to spread cultivation 3-5 times ratio progress.That is, one bottle of cell passes on to obtain 3-5
Bottle cell.
Further, it is carefully cleaned 2-3 times with PBS.Cleaning be in order to further remove the raffinate not removed and other
Fragment.
Further, the dedicated complete culture solution of excretion body of 4%-6% volume is added in each culture bottle.
In T75 Tissue Culture Flask (250mL), the dedicated complete culture solution of excretion body of 10-15mL is added, such as T75 cell
It can be 10mL, 12mL, 15mL etc. in culture bottle (250mL).
Further, the aperture of strainer used in filtering removal vesica is 0.22 μm.By filtering, larger vesica is removed.
In separation process, low-temperature centrifugation is to be centrifuged 5-10min at 4 ± 2 DEG C with 2000-2200g.By the rate carry out from
The heart removes dead cell and fragment.By ultracentrifugation, excretion body precipitates.Liquid on centrifuge container tube wall is removed, from
The substance of heart container bottom is mescenchymal stem cell excretion body.By removing liquid, purer mescenchymal stem cell is obtained
Excretion body.
Centrifuge container is generally centrifuge tube.
Further, further comprising the steps of: obtained mescenchymal stem cell excretion body is resuspended with PBS, is saved.Such as may be used
It is saved at -80 DEG C.
The mescenchymal stem cell excretion body of the container bottom obtained is added PBS and is resuspended, and saves.
The isolated mescenchymal stem cell excretion body of the present invention is detected, as under Electronic Speculum and surface markers albumen
Detection, meet the characteristic of mescenchymal stem cell excretion body, illustrate the isolated mescenchymal stem cell excretion body of the present invention
It is reliable and stable.
Further, the components also include essence.
Further, by weight, essence 0.00005-0.00015 parts.
The present invention also provides the preparation methods of the skin care item, comprising the following steps:
Lecithin is dissolved in 60 ± 5 DEG C of mineral oil, remaining component of component A is added under stiring, is heated to 70 ± 5
℃；
Component B, emulsifying is added, stirring is cooled to 40 ± 5 DEG C；
It is gradually added component C, is cooled to room temperature and forms soft frost；
If being added together with component C containing essence.
The preparation method of skin care item provided by the invention, whole operation temperature is lower, operates safer simplicity, shield obtained
Skin product effect stability, reliably.
Compared with prior art, the invention has the benefit that
(1) skin care item provided by the invention, each component coordinated, obtained skin care item performance are stablized, and therein
Nutrition composition cooperation, has and preferably removes wrinkle, increase the effect of skin elasticity.
(2) the dedicated complete medium of excretion body provided by the invention, effectively improves umbilical cord mesenchymal stem cells incubation
25% or more the content of middle excretion body.
(3) preparation method of no excretion body FBS provided by the invention, method is simple, is easy to industrialization production.
(4) separation method of mescenchymal stem cell excretion body provided by the invention, method is simple, between isolated
Mesenchymal stem cells excretion body is reliable and stable.
(5) preparation method of skin care item provided by the invention, whole operation temperature is lower, operates safer simplicity, is made
Skin care item effect stability, reliably.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is to observe figure to the Electronic Speculum of mescenchymal stem cell excretion body in experimental example 1 of the present invention；
Fig. 2 is in experimental example 1 of the present invention to the detection figure of the surface markers PROTEIN C D9 of mescenchymal stem cell excretion body；
Fig. 3 is in experimental example 1 of the present invention to the detection figure of the surface markers PROTEIN C D63 of mescenchymal stem cell excretion body；
The comparative diagram that Fig. 4 treats for skin care item provided by the invention in experimental example 2 of the present invention；
Another comparative diagram that Fig. 5 treats for skin care item provided by the invention in experimental example 2 of the present invention；
Another comparative diagram that Fig. 6 treats for skin care item provided by the invention in experimental example 2 of the present invention.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
1.1 umbilical cord mesenchymal stem cells excretion body separation methods
1) prepared without excretion body FBS: under aseptic condition by FBS pour into it is dedicated surpass from centrifuge tube, every pipe 38.6mL, 4 pipes,
It after confirming trim, is put into ultracentrifuge, 120000g, is centrifuged 12h.
2) second day, careful upper layer clarification serum of drawing was into new sterile centrifugation tube, and 0.22 μm of membrane filtration is to another
In centrifuge tube, it is ensured that the germ-free condition of serum, sealing are spare.
1.2 umbilical cord mesenchymal stem cells culture mediums
1) common complete medium
On the basis of α-MEM, add following components: FBS percentage by volume is 10%, 200mM glutamine volume basis
Number is that 1%, bFGF concentration is 20ng/mL, and EGF concentration is 20ng/mL.
2) the dedicated complete medium of excretion body
On the basis of without phenol red α-MEM, add following components: no excretion body FBS percentage by volume is 10%, 200mM
Glutamine percentage by volume is that 1%, bFGF concentration is 20ng/mL, and EGF20 concentration is 20ng/mL.
1.3 umbilical cord mesenchymal stem cells excretion body separation methods, steps are as follows:
T75 Tissue Culture Flask, common complete medium culture P3 melt for umbilical cord mesenchymal stem cells to cell length to 95%
When conjunction, passed on according to 1:5.
Second day, the dedicated complete medium of excretion body was prepared with no excretion body FBS.Carefully siphon away common complete medium and
Not adherent cell is carefully cleaned 3 times, every bottle of dedicated complete medium of addition 10mL excretion body with PBS.
Collection culture medium arrives the culture medium rich in mescenchymal stem cell excretion body into centrifuge tube after 72h.
General low temperature centrifuge and ultracentrifuge are opened in advance, are cooled to 4 DEG C or so in advance.
2000g is centrifuged 7min, removes dead cell and fragment.
0.22 μm of membrane filtration removes larger vesica.
Supernatant is gone in ultracentrifugation pipe, and with PBS trim, 100000g is centrifuged 4h.
It discards supernatant, tube wall residual liquid is carefully siphoned away with aseptic filter paper, tube bottom is mescenchymal stem cell excretion body.
Each pipe excretion weight is hanged with 1mL PBS, is mixed, -80 DEG C of preservations.
Embodiment 2
1.1 umbilical cord mesenchymal stem cells excretion body separation methods
1) prepared without excretion body FBS: under aseptic condition by FBS pour into it is dedicated surpass from centrifuge tube, every pipe 38.6mL, 4 pipes,
It after confirming trim, is put into ultracentrifuge, 100000g, is centrifuged 14h.
2) second day, careful upper layer clarification serum of drawing was into new sterile centrifugation tube, and 0.22 μm of membrane filtration is to another
In centrifuge tube, it is ensured that the germ-free condition of serum, sealing are spare.
1.2 umbilical cord mesenchymal stem cells culture mediums
1) common complete medium
On the basis of α-MEM, add following components: FBS percentage by volume is 10%, 200mM glutamine volume basis
Number is that 1%, bFGF concentration is 20ng/mL, and EGF concentration is 20ng/mL.
2) the dedicated complete medium of excretion body
On the basis of without phenol red α-MEM, add following components: no excretion body FBS percentage by volume is 10%, 200mM
Glutamine percentage by volume is that 1%, bFGF concentration is 20ng/mL, and EGF concentration is 20ng/mL.
1.3 umbilical cord mesenchymal stem cells excretion body separation methods, steps are as follows:
T75 Tissue Culture Flask, common complete medium culture P3 melt for umbilical cord mesenchymal stem cells to cell length to 90%
When conjunction, passed on according to 1:4.
Second day, the dedicated complete medium of excretion body was prepared with no excretion body FBS.Carefully siphon away common complete medium and
Not adherent cell is carefully cleaned 3 times, every bottle of dedicated complete medium of addition 10mL excretion body with PBS.
Collection culture medium arrives the culture medium rich in mescenchymal stem cell excretion body into centrifuge tube after 80h.
General low temperature centrifuge and ultracentrifuge are opened in advance, are cooled to 4 DEG C in advance.
2000g is centrifuged 10min, removes dead cell and fragment.
0.22 μm of membrane filtration removes larger vesica.
Supernatant is gone in ultracentrifugation pipe, and with PBS trim, 120000g is centrifuged 5h.
It discards supernatant, tube wall residual liquid is carefully siphoned away with aseptic filter paper, tube bottom is mescenchymal stem cell excretion body.
Each pipe excretion weight is hanged with 1mLPBS, is mixed, is saved.
Embodiment 3
1.1 umbilical cord mesenchymal stem cells excretion body separation methods
1) prepared without excretion body FBS: under aseptic condition by FBS pour into it is dedicated surpass from centrifuge tube, every pipe 38.6mL, 4 pipes,
It after confirming trim, is put into ultracentrifuge, 120000g, is centrifuged 8h.
2) second day, careful upper layer clarification serum of drawing was into new sterile centrifugation tube, and 0.22 μm of membrane filtration is to another
In centrifuge tube, it is ensured that the germ-free condition of serum, sealing are spare.
1.2 umbilical cord mesenchymal stem cells culture mediums
1) common complete medium
On the basis of α-MEM, add following components: FBS percentage by volume is 10%, 200mM glutamine volume basis
Number is that 1%, bFGF concentration is 20ng/mL, and EGF concentration is 20ng/mL.
2) the dedicated complete medium of excretion body
On the basis of without phenol red α-MEM, add following components: no excretion body FBS percentage by volume is 10%, 200mM
Glutamine percentage by volume is that 1%, bFGF concentration is 20ng/mL, and EGF20 concentration is 20ng/mL.
1.3 umbilical cord mesenchymal stem cells excretion body separation methods, steps are as follows:
T75 Tissue Culture Flask, common complete medium culture P3 melt for umbilical cord mesenchymal stem cells to cell length to 85%
When conjunction, passed on according to 1:3.
Second day, the dedicated complete medium of excretion body was prepared with no excretion body FBS.Carefully siphon away common complete medium and
Not adherent cell is carefully cleaned 2 times, every bottle of dedicated complete medium of addition 15mL excretion body with PBS.
Collection culture medium arrives the culture medium rich in mescenchymal stem cell excretion body into centrifuge tube after 96h.
General low temperature centrifuge and ultracentrifuge are opened in advance, are cooled to 4 DEG C or so in advance.
2200g is centrifuged 5min, removes dead cell and fragment.
0.22 μm of membrane filtration removes larger vesica.
Supernatant is gone in ultracentrifugation pipe, and with PBS trim, 120000g is centrifuged 8h.
It discards supernatant, tube wall residual liquid is carefully siphoned away with aseptic filter paper, tube bottom is mescenchymal stem cell excretion body.
Each pipe excretion weight is hanged with 1mLPBS, is mixed, is saved.
Comparative example 1
As different from Example 2, the α-MEM in common complete medium replaces with DMEM, the dedicated complete training of excretion body
Replacing with without phenol red α-MEM without phenol red DMEM in base is supported, other are all the same.
Comparative example 2
As different from Example 2, the α-MEM in common complete medium replaces with RPMI1640, and excretion body is dedicated complete
Replacing with without phenol red α-MEM without phenol red RPMI 1640 in full culture medium, other are all the same.
Comparative example 3
As different from Example 2, the α-MEM in common complete medium replaces with DMEM/F12, and excretion body is dedicated complete
Replacing with without phenol red α-MEM without phenol red DMEM/F12 in full culture medium, other are all the same.
Comparative example 4
As different from Example 2, the dedicated complete medium of excretion body replaces with the culture medium of following components: without phenol red
α-MEM on the basis of, add following components: the concentration of PDGF-BB is 30ng/mL, and the concentration of EPO is 2U/mL.
It should be noted that unmentioned condition of culture in the present invention, it is accordingly to be regarded as at 37 DEG C, 5% CO2Saturated humidity item
It is cultivated under part.
Experimental example 1
It is measured using BCA determination of protein concentration kit:
1) it prepares protein standard solution: 0.8mL protein standard preparation liquid being added in 20mg BSA, dissolves it sufficiently
It is further diluted to final concentration of 0.5mg/mL afterwards.
2) configuration of BCA working solution: BCA reagent A: reagent B=50:1 prepares appropriate body according to sample number plus standard items number
Product BCA working solution.
3) 96 orifice plates are taken, it is respectively that 0,1,2,4,8,12,16,20 μ L protein standard substance (0.5mg/mL) and sample to be tested is suitable
Amount, complements to 20 μ L for each pore volume with standard dilutions PBS.
4) 200 μ L BCA working solutions, 37 DEG C of placement 20-30min are added in every hole more than.
5) wavelength 562nm is selected in microplate reader, measurement standard curve is simultaneously outer according to made from each group of standard curve calculating
Secrete bulk concentration.And calculate that the results are shown in Table 1 to calculate excretion bulk concentration by cell quantity.
Excretion bulk concentration obtained in the different groups of table 1
| Group | Excretion bulk concentration (g/1 × 10 μ7cells) |
| Embodiment 1 | 95.1±1.7 |
| Embodiment 2 | 96.3±1.2 |
| Embodiment 3 | 95.4±1.8 |
| Comparative example 1 | 76.3±1.7 |
| Comparative example 2 | 55.5±1.2 |
| Comparative example 3 | 75.2±1.3 |
| Comparative example 4 | 67.4±1.6 |
From the foregoing it can be that relative to comparative example, method provided by the invention, obtained mescenchymal stem cell excretion body
Content at least promotes 25% or more.
The extracted excretion bulk concentration of method provided by the invention is very high, has the yield being obviously improved.
In addition, detection excretion body purity, for different groups between 80%-90%, embodiment is not obvious with comparative example
Difference.
In addition, the mescenchymal stem cell excretion body isolated to the present invention detects, as under Electronic Speculum and surface is marked
The detection for remembering albumen, meets the characteristic of mescenchymal stem cell excretion body, wherein electron microscope is as shown in Figure 1, surface markers albumen
Detection figure is as shown in Figures 2 and 3.Illustrate that the isolated mescenchymal stem cell excretion body of the present invention is reliable and stable.
Embodiment 4
Excretion body freeze-dried powder:
Xanthan gum is added in excretion body made from embodiment 2, and it is dry thin then to obtain the mesenchyma through vacuum freeze-drying method
It is extracellular to secrete body freeze-dried powder；
The additive amount of xanthan gum is the 15%-20% of mescenchymal stem cell excretion body weight.
A kind of skin care item take each component spare by parts by weight shown in table 2:
2 each component of table
It prepares according to the following steps:
Lecithin is dissolved in 60 DEG C of mineral oil, remaining component of component A is added under stiring, is heated to 70 DEG C；
Component B, emulsifying is added, stirring is cooled to 40 DEG C or so；
It is gradually added component C, is cooled to room temperature and forms soft frost.
Comparative example 5
As different from Example 4, silk-fibroin, hyaluronic acid and lecithin are not added.
Comparative example 6
As different from Example 4, when prepared by excretion body freeze-dried powder, xanthan gum is replaced with trehalose.
Experimental example 2
Soft frost made from embodiment 1 and comparative example 5 and comparative example 6 is subjected to Skin Irritation Test, does not observe skin
Skin stimulate the reaction shows that these soft frosts have safety in utilization.
Recruit 20 Chinese women subjects, the age 40~60 years old.Every subject is apprised of entire research process, and
Sign informed consent form.Wherein, ointment made from embodiment 4 is used by 10 people, and comparative example 5 and 6 respectively has 5 people use, user
Method: smearing skin of face, smear sooner or later daily, do not use other cosmetics, and cleaning skin is all made of mild facial cleanser, system
One allots, and after 28d is used continuously, the same time cleans the smearing position of subject weekly, uses the U.S. by tester's detection
The Visia skinanalysis apparatus of Canfield company is tested, and the data of wrinkle and elasticity are obtained.As a result as shown in Table 3-5.
3 embodiment of table, 4 skin effect
4 comparative example of table, 5 skin effect
5 comparative example of table, 6 skin effect
Specifically, the part effect of skin care item treatment provided by the present application is as shown in Fig. 4-6, wherein A indicates figure before treatment
Piece, B indicate picture after treatment.
It was found that, for going wrinkle effect: the effective scope that skin care item provided by the invention remove wrinkle is
9%-36%, average rate of decrease 18.7%；And comparative example 5 is 4.9%-11% to the effective scope that wrinkle removes, it is average to drop
Low rate is 8.3%；Comparative example 6 is 4.9%-13.5%, average rate of decrease 8.4% to the effective scope that wrinkle removes.
For increasing elastic effect: skin care item provided by the invention are 5.7%-15.4% to the expanded reach of elasticity, are put down
Equal enhancing rate is 10.2%；5 pairs of elastic expanded reaches of comparative example are 3.3%-7.8%, and average enhancing rate is 5.5%；Comparison
6 pairs of elastic expanded reaches of example are 4.4%-8.2%, and average enhancing rate is 5.9%.
Above content is it is found that soft frost provided by the invention has more superior skin effect to skin of face, hence it is evident that is better than
Other groups.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of skin care item, which is characterized in that the parts by weight of each component are as follows:
Component A: 3 ± 0.2 parts of polyglycol distearate, 11 ± 0.5 parts of triple pressed stearic acid, 2 ± 0.2 parts of mineral oil, lecithin
It is 1.3 ± 0.2 parts of rouge, 3.7 ± 0.5 parts of Tween-60,0.1 ± 0.05 part of Span-60,2 ± 0.2 parts of propylben, transparent
2 ± 0.2 parts of matter acid, 2 ± 0.2 parts of silk-fibroin；
Component B: 18 ± 1 parts of glycerol, 0.2 ± 0.05 part of methylparaben, 54-55 parts of pure water；
Component C: excretion body freeze-dried powder 1 × 10-9~7 × 10-8Part.
2. skin care item according to claim 1, which is characterized in that the excretion body freeze-dried powder is prepared using following methods:
Mescenchymal stem cell excretion body is resuspended with PBS, and xanthan gum mixing is added, then through vacuum freeze-drying method obtain it is described between
Mesenchymal stem cells excretion body freeze-dried powder；
The additive amount of the xanthan gum is the 15%-20% of the mescenchymal stem cell excretion body weight.
3. skin care item according to claim 2, which is characterized in that the mescenchymal stem cell excretion body is by the following method
Preparation:
Umbilical cord mesenchymal stem cells secondary culture 8-14h, siphons away culture solution and not adherent cell, and cleaned, then plus
Enter the dedicated complete culture solution of excretion body to be cultivated；
Culture solution is collected after 72-96h into centrifuge tube, obtains the culture solution rich in mescenchymal stem cell excretion body；
The culture solution rich in mescenchymal stem cell excretion body is centrifuged 5-10min, removal at 4 ± 2 DEG C with 2000-2200g
Dead cell and fragment, then filtering removes vesica；
Obtained liquid is centrifuged 4-8h in 100000-120000g, abandons supernatant, and the liquid on centrifuge container tube wall is removed, obtains
To mescenchymal stem cell excretion body；
The dedicated complete medium of excretion body adds following components: without excretion body FBS body on the basis of without phenol red α-MEM
It is 1% ± 0.05%, bFGF concentration is 20 ± 2ng/ that product percentage, which is 10% ± 2%, 200mM glutamine percentage by volume,
ML, EGF concentration are 20 ± 2ng/mL.
4. skin care item according to claim 3, which is characterized in that the no excretion body FBS is prepared using following methods:
FBS is centrifuged 8-14h in the revolving speed of 100000g-120000g；
Draw upper layer clarify serum, and use 0.22 μm of membrane filtration, filtrate as without excretion body FBS.
5. skin care item according to claim 3, which is characterized in that P3 is for umbilical cord mesenchymal stem cells for culture, long to cell
It is passed on when being merged to 85% ± 10%；
Further, it is described passage according to spread cultivation 3-5 times ratio progress.
6. skin care item according to claim 3, which is characterized in that the cleaning is cleaned 2-3 times with PBS；
Further, the dedicated complete culture solution of excretion body of 4%-6% volume is added in each culture bottle.
7. skin care item according to claim 3, which is characterized in that the aperture of strainer used in filtering removal vesica is 0.22
μm。
8. skin care item according to claim 1-7, which is characterized in that the components also include essence.
9. skin care item according to claim 8, which is characterized in that by weight, 0.00005-0.00015 parts of essence.
10. the preparation method of the described in any item skin care item of claim 1-9, which comprises the following steps:
Lecithin is dissolved in 60 ± 5 DEG C of mineral oil, remaining component of component A is added under stiring, is heated to 70 ± 5 DEG C；
Component B, emulsifying is added, stirring is cooled to 40 ± 5 DEG C；
It is gradually added component C, is cooled to room temperature and forms soft frost；
If being added together with component C containing essence.
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| CN201811409317.5A CN109200011A (en) | 2018-11-23 | 2018-11-23 | A kind of skin care item and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110151678A (en) * | 2019-06-24 | 2019-08-23 | 广州远想生物科技有限公司 | It is a kind of for improving the composition and cosmetics comprising source of human stem cell excretion body of wrinkle |
| CN110151679A (en) * | 2019-06-24 | 2019-08-23 | 广州远想生物科技有限公司 | A kind of excretion body freeze-dried powder and the preparation method and application thereof |
| WO2022198771A1 (en) * | 2021-03-26 | 2022-09-29 | 广州赛莱拉生物基因工程有限公司 | Composition containing animal placental exosomes, preparation method therefor and use thereof |
-
2018
- 2018-11-23 CN CN201811409317.5A patent/CN109200011A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110151678A (en) * | 2019-06-24 | 2019-08-23 | 广州远想生物科技有限公司 | It is a kind of for improving the composition and cosmetics comprising source of human stem cell excretion body of wrinkle |
| CN110151679A (en) * | 2019-06-24 | 2019-08-23 | 广州远想生物科技有限公司 | A kind of excretion body freeze-dried powder and the preparation method and application thereof |
| WO2022198771A1 (en) * | 2021-03-26 | 2022-09-29 | 广州赛莱拉生物基因工程有限公司 | Composition containing animal placental exosomes, preparation method therefor and use thereof |
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