CN109316373A - A kind of preparation method and application of umbilical cord mesenchymal stem cells factor coniplexes - Google Patents

A kind of preparation method and application of umbilical cord mesenchymal stem cells factor coniplexes Download PDF

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Publication number
CN109316373A
CN109316373A CN201811409240.1A CN201811409240A CN109316373A CN 109316373 A CN109316373 A CN 109316373A CN 201811409240 A CN201811409240 A CN 201811409240A CN 109316373 A CN109316373 A CN 109316373A
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umbilical cord
stem cells
mesenchymal stem
cell
cord mesenchymal
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张权
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Beijing Anyi Biotechnology Co Ltd
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Beijing Anyi Biotechnology Co Ltd
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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • 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
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • 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
    • A61Q19/08—Anti-ageing preparations

Abstract

The present invention relates to cell factor cosmetic fields, in particular to a kind of preparation method and application of umbilical cord mesenchymal stem cells factor coniplexes.A kind of preparation method of umbilical cord mesenchymal stem cells factor coniplexes, comprising the following steps: umbilical cord mesenchymal stem cells carry out adhere-wall culture；Liquid is changed, continues to cultivate, until cell is covered with to 85%-95%, culture solution is collected, obtains the culture solution for the factor secreted containing a large amount of umbilical cord mesenchymal stem cells；The culture solution filtering removal cell fragment, obtains the liquid containing the umbilical cord mesenchymal stem cells factor coniplexes.The preparation method of umbilical cord mesenchymal stem cells factor coniplexes provided by the invention, after culture appropriate, cytokine content is abundant, and method is easy.Obtained umbilical cord mesenchymal stem cells factor coniplexes, can activate Skin Cell from inside to outside, improve skin function from skin corium, and by this and face, improvement can more persistently.

Description

A kind of preparation method and application of umbilical cord mesenchymal stem cells factor coniplexes
Technical field
The present invention relates to cell factor cosmetic fields, multiple in particular to a kind of umbilical cord mesenchymal stem cells factor Close the preparation method and application of object.
Background technique
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.MSC is widely present in whole body Various Tissues, almost derives from body institute Some histoorgans, such as marrow, periosteum, adipose tissue, dental pulp, synovial membrane, umbilical cord, placenta, amniotic fluid and fetal tissue.Difference is come The mescenchymal stem cell in source has similar form, expresses identical surface markers, in terms of having similar biological characteristics, such as Amplification can be cultivated in vitro, and can be divided under given conditions thin including nerve cell, osteoblast, cartilage cell, muscle The cell of more organization systems including born of the same parents, fat cell.MSC is also making other than with multidirectional histocyte differentiation capability Important regulative is played in blood, 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.
UC-MSC is exactly paracrine there are one important function other than it can be divided into the damage of histocyte repair tissue Cell factor carries out tissue repair, such as secretory nerve Cell health factor, angiogenic factor, the hematopoiesis support factor, immune tune Save the factor, anti-apoptosis factor and chemotactic factor (CF) etc..In the highly expressed factor of UC-MSC, G-CSF, GM-CSF, HGF, LIF, IL- 11 belong to Hemopoietic factor, there is promotion hemoposieis.HGF, IL-1 α, IL-6, IL-8 belong to immune-regulating factor.There are also EGF, A variety of active factors that can adjust skin function such as FGF, VEGF, TGF-β, KGF.
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 preparation method of umbilical cord mesenchymal stem cells factor coniplexes, the party The umbilical cord mesenchymal stem cells factor coniplexes that method obtains mainly contain epidermal growth factor (EGF), Desmocyte growth factor Sub (FGF), keratinocyte growth factor (KGF), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF) etc., The cytokine complex has significant improvement result to skin.
The second object of the present invention is to provide umbilical cord mesenchymal stem cells factor coniplexes in improving skin condition Using being accelerated using rear skin renewal, become fine and smooth, glossy, nose, the area T pore obviously become smaller, and crow's foot is reduced, method Line is enabled to shoal, thin out through instrument detection ultraviolet light color spot, brown spot is thin out.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of preparation method of umbilical cord mesenchymal stem cells factor coniplexes, comprising the following steps:
Umbilical cord mesenchymal stem cells carry out adhere-wall culture；
Liquid is changed, continues to cultivate, until cell is covered with to 85%-95%, culture solution is collected, obtains containing a large amount of umbilical cord mesenchymas The culture solution of the factor of stem cell secretion；
The culture solution filtering removal cell fragment, obtains the liquid containing the umbilical cord mesenchymal stem cells factor coniplexes Body.
The preparation method of umbilical cord mesenchymal stem cells factor coniplexes provided by the invention, after culture appropriate, cell Factor content is abundant, and method is easy.
Further, the umbilical cord mesenchymal stem cells select P3-P7 for any one or more of.
P3-P7 is good for umbilical cord mesenchymal stem cells growth conditions, is easy culture, and activity is strong, what is be obtained by culture is thin Intracellular cytokine content is more.
As in various embodiments, umbilical cord mesenchymal stem cells can select P3 generation, P4 generation, P5 generation, P6 generation, P7 generation etc. Deng.
Further, the component for cultivating culture medium used is as follows:
On the basis of without phenol red α-MEM, add following components: FBS percentage by volume is 10% ± 2%, glutamine Liquor capacity percentage is that 1% ± 0.05%, bFGF concentration is 20 ± 2ng/mL, and EGF concentration is 20 ± 2ng/mL；
Wherein, the concentration of the glutamine solution is 200mM.
The present invention selects the culture medium to cultivate umbilical cord mesenchymal stem cells, is conducive to umbilical cord mesenchymal stem cells and secretes Trophic factors.
Further, the adhere-wall culture carries out in culture bottle, in terms of T75 culture bottle, is inoculated with 1.0 in each culture bottle ×106-2.0×106A cell, wherein the volume of the culture medium added is 8-12mL.
Further, condition of culture is equal are as follows: and 37 DEG C, 5%CO2, saturated humidity.
Culture carries out in the incubator.
Further, after the adhere-wall culture 8-15h, culture solution is sucked to remove not adherent cell and cell fragment, Culture solution is rejoined, continues to cultivate.
Further, described to continue after cultivating 2-3 days, collect culture solution.Continue culture 2-3 days after, cell cover with to 90% or so, at this point, collecting culture solution, culture solution contains more cell factor.
Further, the filtering is carried out using not less than 20 μm of aperture sieve.
Preferably, it is carried out using 20-40 μm of aperture sieve.
After the screen to filtrate, remove cell debris or fragment and some cells not removed, it is dry to obtain umbilical cord mesenchyma Cytokine complex.
Further, the liquid containing the umbilical cord mesenchymal stem cells factor coniplexes obtained further includes the step of packing Suddenly, cryo-conservation is then carried out.Generally saved at -20 DEG C.
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, stem cell factor are exactly one type, and skin nursing can be turned to cellular level by the various factors that stem cell is rich in, Fundamentally realize the rejuvenation of skin condition.
Stem cell factor mainly includes the following factor:
1) epidermal growth factor (EGF)
EGF is that the stimulation of epithelial cell division growth is former, and EGF activates a series of lifes in conjunction with the EGF receptor on cell membrane Change reaction, change intracellular calcium concentration, starts gene related with cell division, so that akinete is entered division stage, have Effect promotes and adjusts epidermal growth, proliferation.In addition to this, EGF can also increase the absorption for promoting other endogenous factors, Promote cell secretion hyaluronic acid and glycoprotein, adjust skin texture, make skin moisturizing, smooth, soft, delay skin aging, Because being referred to as " the beautiful factor ".
2) fibroblast growth factor (FGF)
FGF is a kind of multi-functional nonspecific activity substance, and the various kinds of cell such as Human Umbilical Vein Endothelial Cells, fibroblast have promotion Cell division effect.FGF can improve the microenvironment of cell growth, promote fibroblastic growth and development, constantly with new thin Born of the same parents replace aged cells, promote elastomer and collagen synthesis, constantly update skin, keep skin elasticity, allow skin The state tender in cunning.In addition, FGF can also reduce melanin content, coloured corpuscle quantity is reduced, the pigment for mitigating skin is heavy ?.The rush new life function of FGF, moreover it is possible to which after-sun recovery are played the role of in the reparation for promoting damaged cell.
3) keratinocyte growth factor (KGF)
KGF can promote epithelial cell proliferation and differentiation, moreover it is possible to keep cytoskeleton to stablize, organize the disconnected of actin It splits, keeps cell results complete.Thus, KGF can be avoided Apoptosis aging caused by active oxygen.KGF can induce cutin shape It is internalized by cell, mediating melanin particle is transferred to keratinocyte from melanocyte, avoids skin by UVB's Damage.KGF can also be by the metabolism of acceleration keratinocyte, the problem that hair follicle can be prevented coarse.
4) transforming growth factor β (TGF-β)
TGF-β is fibroblastic major chemokine, can stimulate fibroblast division growth.TGF-β at The inducing chemotactic of fibrocyte is conducive to the progress of process of tissue reparation.TGF-β can also inducing macrophage secretion blood vessel hair The raw factor, accelerates vascularization, can also promote the synthesis of fibronectin, hyaluronic acid etc., therefore skin is available It repairs well, becomes smooth ruddy.
5) vascular endothelial growth factor (VEGF)
VEGF is that presently found effect is most strong, the highest angiogenic factors of specificity.Skin epidermis tissue is without blood Pipe structure, it is main to provide nutriment by dermal microvascular and metabolic waste object is discharged.The blood vessel number of old application on human skin is sharply It reduces, it is insufficient so as to cause the nutrition supply of skin, hinder the metabolism of cell.It is logical that VEGF can effectively improve local vascular Permeability provides sufficient nutriment and growth factor for fibroblastic proliferation and collage synthesis, promotes cell division, change Kind skin microcirculation, is that skin metabolism product is easy to be discharged, it is not easy to form dark sore, blackspot and chloasma etc..
The preparation method of umbilical cord mesenchymal stem cells factor coniplexes provided by the invention, what is obtained contains umbilical cord mesenchyma The liquid of stem cell factor compound is rich in above-mentioned substance.
The present invention also provides the liquid containing umbilical cord mesenchymal stem cells factor coniplexes made from above-mentioned preparation method Body is improving the application in skin condition.
It is specifically used that steps are as follows:
1) face cleaning facial cleanser wash clean, naturally dry.
2) dedicated one layer of anaesthetic of face, after smearing uniformly is smeared, one layer of covering preservative film leads to thickness to prevent anaesthetic flowing Unevenly, expose the positions such as eye, nose, mouth, wait 60-70min.
3) clear water cleans anaesthetic, and operator puts on sterile gloves, opens sterile gauze, dips appropriate skin degerming with newly Geramine, wiping face twice, carry out facial disinfection.
4) remaining bromogeramine is wiped out with dry sterile gauze, until facial no moisture residual.
5) adjusting electronic micropin length is 1mm, carries out micropin needle thorn in Face and cheek precedence sequence, guarantees that every piece of skin all relates to And there is micro bleeding category normal phenomenon.
6) liquid produced by the present invention containing umbilical cord mesenchymal stem cells factor coniplexes dissolved in advance is added dropwise simultaneously, It is smeared uniformly with hand, is slowly absorbed compound.
7) the several sensitive parts of eyelid, forehead, the bridge of the nose, upper lip suitably reduce micropin length to 0.7-0.9mm.
8) order needle is pierced to covering full face, while compound is added dropwise, and is constantly applied to absorption.
9) it is coated with the moisture saver mask of 4 DEG C of cryo-conservations in advance 20 minutes, makes skin calm.
With traditional skin care condition ratio, umbilical cord mesenchymal stem cells factor coniplexes can activate Skin Cell from inside to outside, from Skin corium improves skin function, and by this and face, improvement can more persistently.Micropin can effectively do umbilical cord mesenchyma Cell complexes are sent to epidermis once, are more advantageous to the absorption of the factor, can excite skin regeneration from inside to outside, thus can more accommodate Improve skin condition long.
The present invention also provides a kind of cosmetics, containing containing umbilical cord mesenchymal stem cells made from above-mentioned preparation method The liquid of factor coniplexes.
Further, the cosmetics include lotion, face cream, eye cream.
Compared with prior art, the invention has the benefit that
(1) preparation method of umbilical cord mesenchymal stem cells factor coniplexes provided by the invention, method is easy, and what is obtained is thin Intracellular cytokine type is abundant, and content is high.
(2) umbilical cord mesenchymal stem cells factor coniplexes provided by the invention can activate Skin Cell from inside to outside, from true Cortex improves skin function, and by this and face, improvement can more persistently.
(3) umbilical cord mesenchymal stem cells factor coniplexes provided by the invention provide bigger development for the nursing of skin Space.
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 skin sophistication variation comparison photo in experimental example 1 of the present invention；
Fig. 2 is skin shine variation comparison photo in experimental example 1 of the present invention；
Fig. 3 is eye circumference wrinkle variation comparison photo in experimental example 1 of the present invention；
Fig. 4 is that 1 middle-ultraviolet lamp Colour patterns of experimental example of the present invention detect comparison diagram；
Fig. 5 is the pretherapy and post-treatment picture of chloasma in experimental example 1 of the present invention；
Fig. 6 is that chloasma detects comparison diagram before and after treatment in experimental example 1 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, the preparation of umbilical cord mesenchymal stem cells culture medium
On the basis of without phenol red α-MEM, add following components: FBS percentage by volume is 10%, glutamine solution body Product percentage is that 1%, bFGF concentration is 20ng/mL, and EGF concentration is 20ng/mL；
Wherein, the concentration of the glutamine solution is 200mM.
2, the preparation method containing umbilical cord mesenchymal stem cells factor coniplexes
1) it P3 generation, the good umbilical cord mesenchymal stem cells of growth conditions, is resuspended with complete medium.Each T75 bottles of inoculation 1.5×106A cell, complete medium dosage are 10mL.Jiggling is uniformly distributed cell, is steadily put into 37 DEG C, 5%CO2 It is overnight adherent in incubator.
2) culture solution is sucked within second day to remove not adherent cell and some cell fragments, rejoin 10mL and train completely Base is supported, 37 DEG C, 5%CO are put into2, cultivate in saturated humidity incubator.
3) visual cell's growth conditions, general 2-3 days cells are covered with to 90% or so, have contained a large amount of navels in culture solution at this time The factor with mescenchymal stem cell secretion.The culture solution that each culture bottle is collected is focused in clean and sterile bottle.
4) culture solution of collection is filtered with 40 μm of cell screen clothes, removes cell fragment, detects component therein, as a result such as Shown in table 1.
1 component situation of table
EGF(ng/mL) FGF(pg/mL) KGF(ng/mL) TGF-β(pg/mL) VEGF(ng/mL)
63.20±5.47 22.39±3.75 39.03±2.99 30.42±5.84 673.80±50.14
Obtained liquid is dispensed with sterile 5mL EP pipe, and -20 DEG C freeze, spare.
Embodiment 2
1, the preparation of umbilical cord mesenchymal stem cells culture medium
On the basis of without phenol red α-MEM, add following components: FBS percentage by volume is 10% ± 2%, glutamine Liquor capacity percentage is that 1% ± 0.05%, bFGF concentration is 20 ± 2ng/mL, and EGF concentration is 20 ± 2ng/mL；
Wherein, the concentration of the glutamine solution is 200mM.
2, the preparation method containing umbilical cord mesenchymal stem cells factor coniplexes
1) it P4 generation, the good umbilical cord mesenchymal stem cells of growth conditions, is resuspended with complete medium.Each T75 bottles of inoculation 1.5×106A cell, complete medium dosage are 10mL.Jiggling is uniformly distributed cell, is steadily put into 37 DEG C, 5%CO2 It is overnight adherent in incubator.
2) culture solution is sucked within second day to remove not adherent cell and some cell fragments, rejoin 10mL and train completely Base is supported, 37 DEG C, 5%CO are put into2, cultivate in saturated humidity incubator.
3) visual cell's growth conditions, general 2-3 days cells are covered with to 90% or so, have contained a large amount of navels in culture solution at this time The factor with mescenchymal stem cell secretion.The culture solution that each culture bottle is collected is focused in clean and sterile bottle.
4) culture solution of collection is filtered with 40 μm of cell screen clothes, removes cell fragment, detects component therein, as a result such as Shown in table 2.
2 component situation of table
EGF(ng/mL) FGF(pg/mL) KGF(ng/mL) TGF-β(pg/mL) VEGF(ng/mL)
62.80±4.78 23.15±3.67 38.52±2.73 30.22±5.56 668.09±45.01
Obtained liquid is dispensed with sterile 5mL EP pipe, and -20 DEG C freeze, spare.
Embodiment 3
1, the preparation of umbilical cord mesenchymal stem cells culture medium
On the basis of without phenol red α-MEM, add following components: FBS percentage by volume is 10%, glutamine solution body Product percentage is that 1%, bFGF concentration is 20ng/mL, and EGF concentration is 20ng/mL；
Wherein, the concentration of the glutamine solution is 200mM.
2, the preparation method containing umbilical cord mesenchymal stem cells factor coniplexes
1) it P7 generation, the good umbilical cord mesenchymal stem cells of growth conditions, is resuspended with complete medium.Each T75 bottles of inoculation 1.5×106A cell, complete medium dosage are 10mL.Jiggling is uniformly distributed cell, is steadily put into 37 DEG C, 5% CO2, overnight adherent in saturated humidity incubator.
2) culture solution is sucked within second day to remove not adherent cell and some cell fragments, rejoin 10mL and train completely Base is supported, 37 DEG C, 5%CO are put into2, cultivate in saturated humidity incubator.
3) visual cell's growth conditions, general 2-3 days cells are covered with to 90% or so, have contained a large amount of navels in culture solution at this time The factor with mescenchymal stem cell secretion.The culture solution that each culture bottle is collected is focused in clean and sterile bottle.
4) culture solution of collection is filtered with 40 μm of cell screen clothes, removes cell fragment, detects component therein, as a result such as Shown in table 3.
3 component situation of table
EGF(ng/mL) FGF(pg/mL) KGF(ng/mL) TGF-β(pg/mL) VEGF(ng/mL)
62.03±4.96 21.87±3.32 38.75±2.82 29.67±6.01 657.45±53.05
Obtained liquid is dispensed with sterile 5mL EP pipe, and -20 DEG C freeze, spare.
Comparative example 1
1) it is counted after digesting human umbilical cord mesenchymal stem cells, using mesenchymal stem cell serum-free culture medium by people's umbilical cord Mescenchymal stem cell suspends, and adjustment cell concentration is 1.5 × 106A/mL.
2) by above-mentioned inoculation of suspension liquid into T75 culture bottle, culture overnight
3) it the liquid in culture bottle is absorbed, washs culture bottle with α-MEM, then add the α-MEM of 10mL, 6000U RhPDGF-BB-the BB of recombinant human interferon alpha 2 and 600ng.
4) in 37 DEG C, 5%CO2, cultivate for 24 hours under the conditions of saturated humidity, then add the recombinant human epo of 60IU, continue Culture 72 hours collects supernatant, obtains human umbilical cord mesenchymal stem cells culture supernatant.
5) culture solution of collection is filtered with 40 μm of cell screen clothes, removes cell fragment, detect component therein, cell because The results are shown in Table 4 for the content detection of son.
4 component situation of table
EGF(ng/mL) FGF(pg/mL) KGF(ng/mL) TGF-β(pg/mL) VEGF(ng/mL)
35.34±3.25 12.15±4.73 20.08±2.56 15.12±5.78 200.45±37.54
Comparative example 2
P4 is directly selected to be cultivated for umbilical cord mesenchymal stem cells using the method for comparative example 1, the content of cell factor Testing result is as shown in table 5.
5 component situation of table
EGF(ng/mL) FGF(pg/mL) KGF(ng/mL) TGF-β(pg/mL) VEGF(ng/mL)
38.044±2.97 16.42±3.78 24.12±2.46 16.01±4.38 270.45±32.15
Said components testing result finds that EGF, FGF, KGF in 1-3 of the embodiment of the present invention, TGF-β, VEGF component contain No notable difference is measured, and EGF, FGF of comparative example 2, KGF, TGF-β, VEGF constituent content are slightly above comparative example 1.
Experimental example 1
The present invention also provides the liquid containing umbilical cord mesenchymal stem cells factor coniplexes made from above-mentioned preparation method Body is improving the application in skin condition.
It is specifically used that steps are as follows:
1) face cleaning facial cleanser wash clean, naturally dry.
2) dedicated one layer of anaesthetic of face, after smearing uniformly is smeared, one layer of covering preservative film leads to thickness to prevent anaesthetic flowing Unevenly, expose the positions such as eye, nose, mouth, wait 60-70min.
3) clear water cleans anaesthetic, and operator puts on sterile gloves, opens sterile gauze, dips appropriate skin degerming with newly Geramine, wiping face twice, carry out facial disinfection.
4) remaining bromogeramine is wiped out with dry sterile gauze, until facial no moisture residual.
5) adjusting electronic micropin length is 1mm, carries out micropin needle thorn in Face and cheek precedence sequence, guarantees that every piece of skin all relates to And there is micro bleeding category normal phenomenon.
6) liquid produced by the present invention containing umbilical cord mesenchymal stem cells factor coniplexes dissolved in advance is added dropwise simultaneously, It is smeared uniformly with hand, is slowly absorbed compound.
7) the several sensitive parts of eyelid, forehead, the bridge of the nose, upper lip suitably reduce micropin length to 0.7-0.9mm.
8) order needle is pierced to covering full face, while compound is added dropwise, and is constantly applied to absorption.
9) it is coated with the moisture saver mask of 4 DEG C of cryo-conservations in advance 20 minutes, makes skin calm.
Experimental group 1: male or female, the age 33-60 years old.After liquid undergoing treatment 5 times provided using the embodiment of the present invention 1, into Row effect compares, and each treatment interval time is 3 weeks, and detection uses the Visia skinanalysis apparatus of U.S. Canfield company, machine Device provides related content scoring, and concrete outcome is as shown in table 6- table 7.
6 wrinkle of table and pore treatment are compared
The treatment of 7 color spot of table is compared
Example picture control section result is as shown in Figs 1-4.
Wherein, as shown in Figure 1, in Fig. 1, A is picture before treating for sophistication variation, and B is picture after treatment.
As shown in Fig. 2, in Fig. 2, A is picture before treating for glossiness variation, and B is picture after treatment.
For the variation of eye circumference wrinkle as shown in figure 3, in Fig. 3, A is picture before treating, and B is picture after treatment.
For the detection of ultraviolet light Colour patterns as shown in figure 4, in Fig. 4, A is picture before treating, and B is picture after treatment.Fig. 3 and figure 4 be the detection of the different parameters of same people.
Chloasma variation picture detection and instrument, which are taken pictures, to be detected as shown in Figure 5 and Figure 6, and in Figures 5 and 6, A is figure before treatment Piece, B are pictures after treatment.
Above content variance analysis, it is found that liquid provided by the invention is accelerated using rear skin renewal, becomes fine and smooth, has Gloss, nose, the area T pore obviously become smaller, and crow's foot is reduced, and decree line shoals, thin out through instrument detection ultraviolet light color spot, yellow Foxiness is thin out, and there were significant differences for therapeutic effect.
Experimental group 2:
Male or female, the age 33-60 years old.After liquid undergoing treatment 5 times of comparative example 2 of the present invention offer, effect ratio is carried out Compared with each treatment interval time is 3 weeks, as a result as shown in table 8 and table 9.
8 wrinkle of table and pore treatment are compared
The treatment of 9 color spot of table is compared
Through data difference analysis, the reduction degree mean value of the right and left wrinkle is 60% or more in table 6, the right and left hair The reduction degree mean value in hole is 35% or more；The reduction degree mean value of the right and left ultraviolet light color spot is 40% or so in table 7, The reduction degree mean value of the right and left chloasma is 30% or so.
For the reduction degree mean value of the right and left wrinkle within 25%, the reduction degree of the right and left pore is equal in table 8 Value is in 20%-25%；For the reduction degree mean value of the right and left ultraviolet light color spot 15% or so, the right and left is yellowish-brown in table 9 The reduction degree mean value of spot is within 10%.
That is, the liquid that comparative example 2 provides has certain effect after, it is still, provided in an embodiment of the present invention Therapeutic effect is substantially better than comparative example 2.
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 preparation method of umbilical cord mesenchymal stem cells factor coniplexes, which comprises the following steps:
Umbilical cord mesenchymal stem cells carry out adhere-wall culture；
Liquid is changed, continues to cultivate, until cell is covered with to 85%-95%, collects culture solution, is obtained dry thin containing a large amount of umbilical cord mesenchymas The culture solution of the factor of intracrine；
The culture solution filtering removal cell fragment, obtains the liquid containing the umbilical cord mesenchymal stem cells factor coniplexes.
2. preparation method according to claim 1, which is characterized in that the umbilical cord mesenchymal stem cells select P3-P7 generation It is any one or more of.
3. preparation method according to claim 1, which is characterized in that cultivate the following institute of component of culture medium used Show:
On the basis of without phenol red α-MEM, add following components: FBS percentage by volume is 10% ± 2%, glutamine solution Percentage by volume is that 1% ± 0.05%, bFGF concentration is 20 ± 2ng/mL, and EGF concentration is 20 ± 2ng/mL；
Wherein, the concentration of the glutamine solution is 200mM.
4. preparation method according to claim 3, which is characterized in that the adhere-wall culture carries out in culture bottle, with T75 Culture bottle meter is inoculated with 1.0 × 10 in each culture bottle6-2.0×106A cell, wherein the volume of the culture medium added is 8- 12mL。
5. the preparation method according to claim 4, which is characterized in that condition of culture is equal are as follows: 37 DEG C, 5%CO2, it is saturated wet Degree.
6. preparation method according to claim 1, which is characterized in that after the adhere-wall culture 8-15h, suck culture solution with Remove not adherent cell and cell fragment, rejoin culture solution, continues to cultivate.
7. preparation method according to claim 6, which is characterized in that it is described to continue after cultivating 2-3 days, collect culture solution.
8. preparation method according to claim 1-7, which is characterized in that the filtering is not less than 20 using aperture μm sieve carries out, it is preferred to use 20-40 μm of aperture sieve carries out；
Further, the liquid containing the umbilical cord mesenchymal stem cells factor coniplexes obtained further includes the steps that packing, Then cryo-conservation is carried out.
9. the liquid containing umbilical cord mesenchymal stem cells factor coniplexes made from the described in any item preparation methods of claim 1-8 Body is improving the application in skin condition.
10. a kind of cosmetics, which is characterized in that containing containing navel made from the described in any item preparation methods of claim 1-8 Liquid with mescenchymal stem cell factor coniplexes；
Further, the cosmetics include lotion, face cream, eye cream.
CN201811409240.1A 2018-11-23 2018-11-23 A kind of preparation method and application of umbilical cord mesenchymal stem cells factor coniplexes Withdrawn CN109316373A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111956670A (en) * 2020-08-31 2020-11-20 杭州伊瑟奇生物科技有限公司 Preparation method of mesenchymal stem cells and active factor compound freeze-dried product thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111956670A (en) * 2020-08-31 2020-11-20 杭州伊瑟奇生物科技有限公司 Preparation method of mesenchymal stem cells and active factor compound freeze-dried product thereof

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