CN109136180A - Human umbilical cord's blood mescenchymal stem cell extract and its preparation method and application - Google Patents
Human umbilical cord's blood mescenchymal stem cell extract and its preparation method and application Download PDFInfo
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- CN109136180A CN109136180A CN201810794798.XA CN201810794798A CN109136180A CN 109136180 A CN109136180 A CN 109136180A CN 201810794798 A CN201810794798 A CN 201810794798A CN 109136180 A CN109136180 A CN 109136180A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
- A61K8/983—Blood, e.g. plasma
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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
- A61Q19/08—Anti-ageing preparations
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/02—Atmosphere, e.g. low oxygen conditions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
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- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Birds (AREA)
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- Developmental Biology & Embryology (AREA)
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- Organic Chemistry (AREA)
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Abstract
The invention discloses a kind of human umbilical cord's blood mescenchymal stem cell extracts and its preparation method and application.The somatic umbilicus blood mescenchymal stem cell extract is prepared by following step: 1) separating mesenchymal stem cells derived from human umbilical blood from people's agent band blood, and cultivate mesenchymal stem cells derived from human umbilical blood in being added with ascorbic culture medium；2) the secreting type content of the mesenchymal stem cells derived from human umbilical blood is obtained from the culture medium for cultivating the mesenchymal stem cells derived from human umbilical blood；3) it will be crushed by the mesenchymal stem cells derived from human umbilical blood of culture, obtain the nonsecreting type content of mesenchymal stem cells derived from human umbilical blood；4) the secreting type content is mixed with the nonsecreting type content, takes full advantage of people's agent with extract of the separation mesenchymal stem cells derived from human umbilical blood preparation including secreting type content and nonsecreting type content in blood.It additionally include the preparation method and its application in cosmetics of above-mentioned human umbilical cord's blood mescenchymal stem cell extract.
Description
Technical field
The present invention relates to biomedical stem cell and organizational engineering technical fields, more particularly to a kind of human umbilical cord
Blood mescenchymal stem cell extract and its preparation method and application.
Background technique
Mescenchymal stem cell (MSCs) is a kind of adult stem cell with self-renewing and multi-lineage potential, is present in
Marrow, adipose tissue, bleeding of the umbilicus and a variety of fetal tissues.It can secrete cytokine profiles and growth factor, promote Hematopoietic Stem thin
The proliferation and differentiation of born of the same parents (HSC).MSCs also has immunological regulation, anti-inflammatory and tissue repair effect, can reduce graft-versus-host
Sick (GVHD) and other transplanting related complications.Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is main at present
If being extracted from spinal cord and bleeding of the umbilicus.Cord blood is remained in placenta and umbilical cord after delivery of baby, ligature of the cord and detachment
Blood.Human umbilical cord's blood mescenchymal stem cell, which refers to, is present in one of neonatal umbilical cord tissue versatile stem cell, it
Many kinds of histocytes can be divided into, there is wide potential applicability in clinical practice.
There is clinical trial to prove human umbilical cord's blood mescenchymal stem cell yield and most biological properties at present
Similar to bone marrow mescenchymal stem cell, compared to mesenchymal stem cell, umbilical cord blood mesenchymal stem cells are had the advantage that
(1) from a wealth of sources without ethics problem, it easily acquires, easily expands.(2) there are stronger competence for added value, several times faster than marrow MSC.
It (3) is a kind of more original MSC group.(4) lower HLA-ABC (classical Human leucocyte antigen-Ⅰ class antigen) and HLA-DR table
It reaches.Therefore human umbilical cord's blood mescenchymal stem cell is an analogy mesenchymal stem cell more preferably cell population.But currently,
Its effective component can only can be obtained from cell by human umbilical cord's blood mescenchymal stem cell, or passes through human umbilical cord's blood mesenchyma
Stem cell prepares its secreting type content, and lacks while obtaining the research of its effective component and secreting type content.
People especially women also increasingly payes attention to the cosmetology in daily life, thin for that can slow down skin
The demand of the product of born of the same parents' aging and ultraviolet damage is also constantly enhancing.And people increasingly pay attention to the quality and safety of skin care item
Property.It is necessary to the effective components of combination mesenchymal stem cells in umbilical cord blood and secreting type content to study oxidation resistant product.
Summary of the invention
In order to solve the single technical problem of the effective component obtained above by human umbilical cord's blood mescenchymal stem cell, this
Invention proposes a kind of somatic umbilicus blood mescenchymal stem cell extract and its preparation method and application.
Technical problem of the invention is resolved by technical solution below:
A kind of human umbilical cord's blood mescenchymal stem cell extract, is prepared by following step:
1) mesenchymal stem cells derived from human umbilical blood is separated from people's agent band blood, and is trained in being added with ascorbic culture medium
Support mesenchymal stem cells derived from human umbilical blood；
2) it is dry that the human cord blood mesenchyma is obtained from the culture medium for cultivating the mesenchymal stem cells derived from human umbilical blood
The secreting type content of cell；
3) it will be crushed by the mesenchymal stem cells derived from human umbilical blood of culture, obtain the overstepping one's bounds of mesenchymal stem cells derived from human umbilical blood
Secrete type content；
4) the secreting type content is mixed with the nonsecreting type content.
Preferably, the secreting type content is obtained from the culture medium that in vitro culture was no more than for five generations；And/or from body
Human umbilical cord's blood mescenchymal stem cell that outer culture was no more than for five generations obtains the nonsecreting type content.
The present invention also provides a kind of preparation methods of above-mentioned human umbilical cord's blood mescenchymal stem cell extract, including following step
It is rapid:
1) mesenchymal stem cells derived from human umbilical blood is separated from people's agent band blood, and is trained in being added with ascorbic culture medium
Support mesenchymal stem cells derived from human umbilical blood；
2) it is dry that the human cord blood mesenchyma is obtained from the culture medium for cultivating the mesenchymal stem cells derived from human umbilical blood
The secreting type content of cell；
3) it will be crushed by the mesenchymal stem cells derived from human umbilical blood of culture, obtain the overstepping one's bounds of mesenchymal stem cells derived from human umbilical blood
Secrete type content；
4) the secreting type content is mixed with the nonsecreting type content.
Preferably, in step 1), the concentration of the vitamin C in the medium is 40uM/ml-60uM/ml.
Preferably, in step 1), in step 1), the culture medium is in the carbon dioxide and body that volume ratio is 5-7%
Product is than cultivating the mesenchymal stem cells derived from human umbilical blood under conditions of the oxygen for 5-7%.
It preferably, further include identifying the mesenchymal stem cells derived from human umbilical blood after separation in step 1).
In addition, the present invention also proposes prepared by above-mentioned human umbilical cord's blood mescenchymal stem cell extract or above-mentioned preparation method
Application of human umbilical cord's blood mescenchymal stem cell extract in cosmetics.
In addition, the present invention also proposes a kind of anti-apolexis composition, it is dry thin including human umbilical cord's blood mesenchyma described above
Born of the same parents' extract or human umbilical cord's blood mescenchymal stem cell extract of preparation method described above preparation.
It preferably, further include epithelical cell growth factor, glutathione, niacinamide and palmitinic acid SymPeptide.
It is highly preferred that the volume ratio of human umbilical cord's blood mescenchymal stem cell extract is 0.05-5%, the epidermis
The concentration of Porcine HGF is 10-30ug/ml, and the concentration of the glutathione is 2-8ug/ml, the concentration of the niacinamide
Concentration for 2-8mg/ml and the palmitinic acid SymPeptide is 1-3mg/ml.
In one of the embodiments, the volume ratio of human umbilical cord's blood mescenchymal stem cell extract be 1%, it is described
The concentration of epithelical cell growth factor is 20ug/ml, and the concentration of the glutathione is 5ug/ml, and the concentration of the niacinamide is
The concentration of 5mg/ml and the palmitinic acid SymPeptide is 2mg/ml.
The beneficial effect of the present invention compared with the prior art includes: to separate human cord blood mesenchyma from people's agent band blood to do carefully
Born of the same parents, and added with mesenchymal stem cells derived from human umbilical blood is cultivated in ascorbic culture medium, stem cell media is obtained, is improved
The yield of the secreting type content of human umbilical cord blood mescenchymal stem cell, furthermore also accelerates cell in stem cell media
Proliferative speed；The secreting type content of the mesenchymal stem cells derived from human umbilical blood is obtained from the stem cell media；It will be through
The mesenchymal stem cells derived from human umbilical blood for crossing culture is broken, obtains the nonsecreting type content of mesenchymal stem cells derived from human umbilical blood；It will
The secreting type content is mixed with the nonsecreting type content, is obtained human umbilical cord's blood mescenchymal stem cell extract, is filled
It includes secreting type content and nonsecreting type content that separation mesenchymal stem cells derived from human umbilical blood preparation in people's agent band blood, which point is utilized,
The extract of object.
Detailed description of the invention
Fig. 1 is the human umbilical cord's blood mescenchymal stem cell aspect graph being separately cultured in the embodiment of the present invention 1.
Fig. 2 is human umbilical cord's blood mescenchymal stem cell flow analysis chart in the embodiment of the present invention 2.
Fig. 3 is human umbilical cord's blood mescenchymal stem cell content Sterility testing effect picture in the embodiment of the present invention 3.
Fig. 4 be anti-apolexis composition containing human umbilical cord's blood mescenchymal stem cell extract in the embodiment of the present invention 4 without
Bacterial examination surveys effect picture.
Fig. 5 is the anti-ageing of human umbilical cord's blood mescenchymal stem cell extract of the ratio containing different volumes in the embodiment of the present invention 5
Old composition compares figure for slowing down the measurement of human skin cell's aging effects.
Fig. 6 is that proliferation of human dermal fibroblasts is fast after the ultraviolet treatment with irradiation of different time in the embodiment of the present invention 5
Figure is compared in the measurement of rate.
Fig. 7 be the mescenchymal stem cell of blood containing human umbilical cord extract in the embodiment of the present invention 5 anti-apolexis composition for
Slow down human skin cell's aging and figure is compared in the measurement of ultraviolet damage effect.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to the accompanying drawing to the present invention
Specific embodiment be described in detail.Many details are explained in the following description in order to fully understand this hair
It is bright.But the invention can be embodied in many other ways as described herein, those skilled in the art can be not
Similar improvement is done in the case where violating intension of the present invention, therefore the present invention is not limited to the specific embodiments disclosed below.
Embodiment 1 separates and cultivates mesenchymal stem cells derived from human umbilical blood
1.1 umbilical cord blood collection
By there is the assistance of the hospital for obstetrics and gynaecology of Long-term Collaboration with our company, 30 years old following healthy, no heredity disease is selected
Disease, free from infection, AntiHIV1 RT activity and hepatitis B virus serological markers detection all acquire bleeding of the umbilicus for negative Cesarean esction woman.It will after the completion of acquisition
Bleeding of the umbilicus, which is placed in ice chest and is transported immediately to laboratory, carries out separating treatment.Wherein, the puerpera being related to is real to isolated Cord blood
Proved recipe case is known and is agreed to.
The separation and culture of 1.2 mesenchymal stem cells in umbilical cord blood
The Cord blood of acquisition is diluted in equal volume with phosphate buffer (abbreviation PBS), uses lymph separating liquid later
Density gradient centrifugation is carried out, tunica albuginea layer is collected, obtains mononuclearcell.With the ascorbic DMEM training for being 50uM/ml containing concentration
It supports base to wash 2 times, then, is inoculated in the Tissue Culture Flask of T25 according to the density of 2x106/ml, is placed in 37 DEG C, contains volume
Than be for 5% carbon dioxide and volume ratio 5% oxygen hypoxemia incubator in cultivated, people can be improved in the environment of hypoxemia
The multiplication rate of somatic umbilicus blood mescenchymal stem cell and the yield of secreting type content are obtained and are filled between more human umbilical cord's blood
Matter stem cell and secreting type content, culture medium still use the ascorbic DMEM culture medium containing concentration for 50uM/ml；Vc
Injury of the free radical to cell can be helped prevent, cell viability is kept, further increases human umbilical cord's blood mescenchymal stem cell
Multiplication rate and secreting type content yield.It carries out changing liquid after 72 hours, removes not adherent cell.Every 3 days later
A not good liquor is changed, and collects cell culture medium before changing liquid, -20 degree refrigerators is placed in and saves.About at 12 days, cell density reaches
80% fusion.It is digested with 0.25% pancreatin, and is passed on according to the ratio of 1:3, it is daily later thin under the microscope
Intracellular growth situation.Isolated mesenchymal stem cells in umbilical cord blood form is shown in attached drawing 1.
As can be seen from Figure 1: being uniformly distributed by the cell that embodiment 1 obtains in shuttle shape, have typical mesenchyma dry
The cellular morphology of cell.
Human umbilical cord's blood mescenchymal stem cell of 2 pairs of embodiment separation carries out cell type identification
2.1 take in vitro culture to the mesenchymal stem cells in umbilical cord blood of the third generation, digest, centrifugation, and with PBS by cell density
It is adjusted to 1x106/ml, is dispensed in multiple pipes.
2.2 draw following antibody respectively: ITC-anti-human-IgG FC (purchased from Biolegend, article No. 409309),
FITC-anti-hunman-CD44 (being purchased from Biolegend, article No. 338803), FITC-anti-hunman-CD105 (are purchased from
Biolegend, article No. 323203), PE-anti-human-IgGFC (be purchased from Biolegend, article No. 409311), PE-anti-
Hunman-CD14 (being purchased from Biolegend, article No. 367103), PE-anti-hunman-CD34 (are purchased from Biolegend, article No.
343505), PE-anti-hunman-CD31 (being purchased from Biolegend, article No. 303105) 5ul；
Antibody is added to addition in the cell suspension into step 2.1 in different pipes by 2.3, and room temperature is protected from light incubation
Every pipe volume is added to 500ul with binding buffer by 15min.
The sample handled well is carried out cell flow cytometer showed by 2.4.It is as shown in Figure 2 to analyze result.
Flow cytometric analysis results show: the cell height expression mescenchymal stem cell for separating and cultivating by embodiment 1 is special
Sign property cell surface marker CD44 and CD105, do not express candidate stem cell characteristic surface marker CD34 and CD14, not yet
Endothelial cell characteristics surface marker CD31 is expressed, therefore, the cell for separating and cultivating by embodiment 1 is mescenchymal stem cell
Monoid.
Embodiment 3 extracts mesenchymal stem cells derived from human umbilical blood content
3.1 extract stem cell content in mesenchymal stem cells derived from human umbilical blood culture medium；
3.1.1 the culture medium saved in -20 degree refrigerators collected in cell cultivation process 4 degree of refrigerators are placed in thaw；
3.1.2 temperature is controlled using refrigerated centrifuge.When refrigerated centrifuge temperature reaches 4 degree, the cell that will thaw
Culture medium moves into 50ml centrifuge tube, and is centrifuged 5min using 1000r/min, and supernatant is filtered with the filter of 0.45uM.
3.1.3 filtered supernatant is moved on in a new 50ml high speed centrifugation pipe, 10000r/min is centrifuged 20min.
Liquid is discarded supernatant, the sediment of centrifugation bottom of the tube is resuspended with the sterile water of 2% volume ratio, was carried out with the filter of 0.45uM
Filter obtains the secreting type content of mesenchymal stem cells derived from human umbilical blood in culture medium.
3.2 extract the nonsecreting type content in mesenchymal stem cells derived from human umbilical blood
3.2.1 cell precipitation is collected, is counted；
3.2.2 every 1x107 cell precipitation is resuspended with 1ml sterile water；
3.2.3 the cell suspension of resuspension is placed in 2min in liquid nitrogen, makes its fast freezing, then place it in 37 degree of constant temperature
Metal bath 5min, makes it thaw, and then quickly shakes 3min with whirlpool concussion instrument, makes cell rupture；
3.2.4 3.2.3 step is repeated three times；
3.2.5 temperature is controlled using refrigerated centrifuge.When refrigerated centrifuge temperature reaches 4 degree, 3.2.4 is obtained thin
Born of the same parents' mixture is placed in a centrifuge 1000r/min centrifugation 5min.Aspirate supernatant body carried out supernatant with the filter of 0.45uM
Filter, obtains the nonsecreting type content of mesenchymal stem cells derived from human umbilical blood；
3.2.6 the 3.1.3 secreting type content obtained and 3.2.5 the nonsecreting type content obtained are mixed,
Human umbilical cord's blood mescenchymal stem cell extract is obtained, and is dispensed by the volume of every pipe 100ul, and take a progress nothing
Bacterial examination is surveyed.2:5 is mixed secreting type content by volume with nonsecreting type content in the present embodiment.
The Sterility testing of 3.3 human umbilical cord's blood mescenchymal stem cell extracts
Human umbilical cord's blood mescenchymal stem cell extract that 3.2.6 is obtained dilutes 5 times with sterile water, is coated on non-resistant
LB plate on, take isometric sterile water as negative control, take isometric non-aqua sterilisa as positive control.72 hours
After observe result.As a result see example diagram 3.Wherein, 1 is positive control；2 be negative control；3 be human umbilical cord's blood mescenchymal stem cell
The stem cell composition of extract.
The result shows that: the human umbilical cord's blood mescenchymal stem cell extract extracted through embodiment 3 be it is sterile, can carry out
Subsequent operation.
Embodiment 4 prepares anti-apolexis composition
The preparation of 4.1 epithelical cell growth factors: the recombination epithelical cell growth factor albumen buying of source of people is certainly
ThermoFisher.Before preparation, simply it is centrifuged with centrifuge, powder is centrifuged to bottom of bottle, be configured to 1000ug/ml with sterile water
Mother liquor, be distributed into 50ul/ pipe, be placed in -20 degree refrigerators save.
The preparation of 4.2 niacinamide: niacinamide is purchased from sigma.Suitable niacinamide powder is weighed, is configured to sterile water
The mother liquor of 100mg/ml is distributed into 50ul/ pipe with 0.22uM filter filtration sterilization, is placed in -20 degree and saves.
The preparation of 4.3 palmitinic acid SymPeptides: palmitinic acid SymPeptide is purchased from Nanjing peptide industry.Weigh suitable five victory of palmitinic acid
Peptide powder is configured to the mother liquor of 50mg/ml with sterile water, with 0.22uM filter filtration sterilization, is distributed into 50ul/ pipe, is placed in -20
Refrigerator is spent to save.
The preparation of 4.4 glutathione: reduced glutathione is purchased from sigma.Weigh suitable L- reduced form gluathione
Peptide powder is configured to the mother liquor of 100ug/ml with sterile water, with 0.22uM filter filtration sterilization, is distributed into 50ul/ pipe, be placed in-
20 degree of refrigerators save.
It is as follows using the foundation of above-mentioned several substances:
Epithelical cell growth factor (Epidermal growth factor, EGF) is that one kind of human endocrine is important
Growth factor, denier can strongly facilitate the division and growth of Skin Cell, in addition, it can also stimulate extracellular some big points
The synthesis and secretion of sub (such as hyaluronic acid and glycoprotein) moisturize the skin, thus it can not only be used for cosmetics additive and
It performs the operation for facial plasty, promotes the metabolism of application on human skin, reduces skin deformity；Can also treat in medicine skin injury,
Dermatitis caused by postoperative wound, bedsore, canker sore and gangrene and radiotherapy etc..It can accelerate wound and ulcer surface
Healing.
Collagen can clear up the harmful substance during skin oxidative, repair the elastic fibers cell of skin damage,
Have the function that prevent skin ageing and enhancing skin elasticity.Meanwhile collagen can promote skin activity cell to generate, and increase
Add skin tightness.In addition, collagen can accelerate the rate of metabolism of Skin Cell, promotes cutin to fall off naturally, disappear
Except melanin deposition desalinates skin splash.
Glutathione (glutathione) is widely present in animal and plant, is by glutamic acid, cysteine and glycine
In conjunction with the tripeptides containing sulfydryl.What body newly metabolism of old generation generated crosses polyradical meeting damage biological film, and invasion life divides greatly
Son accelerates body aging, and the generation of induced tumor or atherosclerosis.Glutathione is in the intracorporal chemical, biological defense system of people
It plays an important role, there are various physiological functions.Its major physiological effect is can to dispose the intracorporal free radical of people, is done
For a kind of important antioxidant in vivo, the sulfydryl in the molecules such as many protein and enzyme is protected, the removing of interior free yl is made
Reaction can continue to carry out.Glutathione can not only eliminate human free radical, can also improve the immunity of the human body.Glutathione dimension
Shield health, anti-aging, than the young National People's Congress the effect of played on the cell of old man's slowization.
Niacinamide is also vitamin B3, is the generally acknowledged skin anti-aging composition in cosmetic dermatology field, most important function
Effect is the colour of skin dimness for mitigating and preventing skin and generate in early stage aging course, can also repair impaired Stratum corneum lipids screen
Barrier improves skin resistance, and has effects that powerful deep layer water lock.
SymPeptide can promote collagen, elastic fibers and hyaluronic acid synthesis, improve the water content of skin, increase skin
Skin thickness and reduce microgroove, conducive to damage or inflammation skin reparation,.
Epidermal growth factor, collagen, glutathione, niacinamide and SymPeptide have certain slowing down skin aging
Function, itself does not damage skin, uses in different beauty products respectively.Show in conjunction with following experimental datas
The present invention facilitates these types of substance combination human umbilical cord blood mescenchymal stem cell extract to alleviate Skin Cell aging and purple
Outer damage.
The preparation of 4.5 stem cell compositions containing human cord blood stem cell content: according to epithelical cell growth factor
20ug/ml, glutathione 5ug/ml, niacinamide 5mg/ml, palmitinic acid SymPeptide 2mg/ml prepare mixed liquor, are separately added into simultaneously
Human umbilical cord's blood mesenchyma that the embodiment 3 that volume ratio is 4%, 1%, 0.25%, 0.063%, 0.016% and 0% obtains is dry
Cell extract is finally configured to the anti-aging combination containing human umbilical cord's blood mescenchymal stem cell extract of various concentration
Object, and dry powder is made in it, to detect human umbilical cord's blood mescenchymal stem cell extract containing different volumes ratio
Effect of the anti-apolexis composition for alleviation human skin cell's aging and ultraviolet radiation damage.
The 4.6 stem cell composition Sterility testings containing mesenchymal stem cells derived from human umbilical blood content obtained: 4.5 are obtained
The anti-apolexis composition (volume ratio of human umbilical cord's blood mescenchymal stem cell extract is 4%) obtained is dissolved with sterile water, is coated with
In on the LB plate of non-resistant, taking isometric sterile water as negative control, take isometric non-aqua sterilisa as positive right
According to.Result is observed after 72 hours.As a result as shown in Figure 4.Wherein, 4 be positive control；5 be negative control；6 be to contain human body navel
Anti-apolexis composition with blood mescenchymal stem cell extract.
The result shows that: it is combined through the anti-aging containing human umbilical cord's blood mescenchymal stem cell extract that embodiment 4 obtains
Object be it is sterile, subsequent operation can be carried out.
Embodiment 5 detects the anti-apolexis composition prepared by the present invention containing human umbilical cord's blood mescenchymal stem cell extract
The effect of slowing down Skin Cell aging and ultraviolet damage
5.1 anti-apolexis compositions containing human umbilical cord's blood mescenchymal stem cell extract decline for slowing down human skin cell
The measurement of old effect
It, will in the anti-apolexis composition grouping containing human umbilical cord's blood mescenchymal stem cell extract of various various concentrations
As a control group without the group containing human umbilical cord's blood mescenchymal stem cell extract).
The anti-apolexis composition grouping sheet containing human umbilical cord's blood mescenchymal stem cell extract of 1. various concentration of table
| Group | Mesenchymal stem cells derived from human umbilical blood content content (percentage) |
| A | 0 |
| B | 0.016% |
| C | 0.063% |
| D | 0.25% |
| E | 1% |
| F | 4% |
Using human skin fibroblasts, 96 orifice plates are inoculated according to the hole 1x103/, the inoculation of 5 multiple holes is inoculated with 3 piece of 96 hole altogether
Plate.Difference the 3rd day (i.e. D3) after inoculation, progress MTT test in the 5th day (i.e. D5) and the 7th day (i.e. D7).Testing result is shown in Table 2
And attached drawing 5.
Table 2.MTT detects the stem cell composition processing of the mesenchymal stem cells derived from human umbilical blood content containing various concentration
Human skin cell's multiplication rate
In conjunction with table 2 and attached drawing 5, MTT testing result shows:
1. the anti-apolexis composition prepared by the present invention containing human umbilical cord's blood mescenchymal stem cell extract can effectively delay
Solve cell ageing；
2. declining when being 0.016% containing human umbilical cord's blood mescenchymal stem cell extractive content to human skin cell
Old relaxation effect is unobvious.
3. when the content containing human umbilical cord's blood mescenchymal stem cell extract reaches 0.064% or more, cell number
Mesh has a certain upgrade compared to control group；In conjunction with the testing result of D3 and D5, mentioned containing human umbilical cord's blood mescenchymal stem cell
It takes the volume ratio of object bigger, the promotion cultivation effect of human skin cell is more obvious；In conjunction with the testing result of D7, simultaneously for slow
The aging effects for solving the human skin cell of test are also more obvious.
Human skin cell's damage model of 5.2 building ultraviolet light irradiations
Using human skin fibroblasts, 96 orifice plates are inoculated according to the hole 1x103/, the inoculation of 5 multiple holes is inoculated with 4 piece of 96 hole altogether
Plate.The 1st day after inoculation, after ultraviolet light difference irradiating cell adherent 96 orifice plate 0h (control), 1h, 2h, 3h, 4h
It is then incubated for.Then, difference the 3rd day (i.e. D3) after inoculation, progress MTT test in the 5th day (i.e. D5) and the 7th day (i.e. D7).Inspection
Survey the results are shown in Table 3 and attached drawing 6.
Table 3.MTT detects different time ultraviolet treatment with irradiation proliferation of human dermal fibroblasts rate
| 0h | 1h | 2h | 3h | 4h | |
| D3 | 100.03±0.013 | 96.05% ± 0.012 | 90.38% ± 0.035 | 85.21% ± 0.029 | 85.49% ± 0.038 |
| D5 | 99.99% ± 0.015 | 94.06% ± 0.010 | 88.18% ± 0.030 | 73.94% ± 0.073 | 74.38% ± 0.042 |
| D7 | 100.01±0.017 | 94.05% ± 0.007 | 88.04% ± 0.027 | 76.89% ± 0.029 | 74.92% ± 0.028 |
MTT testing result shows: 1. combine the testing result of D3, D5 and D7, and it is small to be in 03 in time of ultraviolet irradiation range
When it is interior, with the extension of time of ultraviolet irradiation, cellular damage degree increases；2. ultraviolet light irradiates more than 3 hours, to application on human skin
The proliferation of cell causes damage to reach critical value；3. the testing result of D5 and D7 is combined, with the decline of cell proliferation rate, this
Kind impaired performance must be especially apparent.Therefore, the modeling side provided by the invention for damaging human skin cell by ultraviolet light irradiation
Method is feasible.
5.3 anti-apolexis compositions containing human umbilical cord's blood mescenchymal stem cell extract decline for slowing down human skin cell
Old and ultraviolet damage effect measurement
It, will not in the anti-apolexis composition grouping of human umbilical cord's blood mescenchymal stem cell extract of several different volumes ratios
The mescenchymal stem cell extract of blood containing human umbilical cord, and do not receive ultraviolet light irradiation, as a control group.Detection is shone in ultraviolet light
In the cellular damage model penetrated, the anti-apolexis composition of the mescenchymal stem cell extract of blood containing human umbilical cord is for human skin cell
Injury repair ability.
Using human skin fibroblasts, 96 orifice plates are inoculated according to the hole 1x103/, the inoculation of 5 multiple holes is inoculated with 5 piece of 96 hole altogether
Plate.The 1st day after inoculation, using ultraviolet light, (wavelength was the ultraviolet B radiation of 280nm~320nm, energy dose 80mJ/
cm2) 96 adherent orifice plate 1h, 2h, 3h of irradiating cell, 4h are followed by cultivated respectively.Progress MTT survey in 7th day after inoculation
Examination.Testing result is shown in Table 2 and attached drawing 7.
Table 4.MTT is detected in ultraviolet damage model and is extracted using human umbilical cord's blood mescenchymal stem cell of the ratio containing different volumes
Influence of the anti-apolexis composition of object to human skin cell's multiplication rate
MTT testing result shows:
1. being in 0-3 hours in time of ultraviolet irradiation range, with the extension of time of ultraviolet irradiation, cellular damage degree
Increase；Ultraviolet light irradiates more than 3 hours, and the damage caused by the proliferation of human skin cell reaches critical value.This with 5.2 in
Data are identical, therefore on the one hand illustrate the stability of ultraviolet damage model provided by the invention, while this data illustrates people
The anti-apolexis composition of somatic umbilicus blood mescenchymal stem cell extract has preferable repairing effect to ultraviolet MODEL DAMAGE.
2. the anti-apolexis composition using human umbilical cord's blood mescenchymal stem cell extract prepared by the present invention can effectively delay
Solve the damage that the ultraviolet irradiation of human skin cell generates；Wherein, when human umbilical cord's blood mescenchymal stem cell extract volume ratio is
When 0.016%, alleviating, human skin cell's damage effect as caused by ultraviolet light is unobvious.But when between human umbilical cord's blood
When the content of mesenchymal stem cells extract reaches 0.064% or more, using anti-aging composition prepared by the present invention for alleviating
The damage effect of the human skin cell as caused by ultraviolet irradiation gradually enhances, and human umbilical cord's blood mescenchymal stem cell extracts object
Product ratio is bigger, and effect is more obvious.Even, damage within limits that (damage effect is less than continuous 3 caused by the ultraviolet irradiation
Hour ultraviolet irradiation, such as ultraviolet irradiation 1 to 2 hour), the human umbilical cord's blood mescenchymal stem cell for using the present invention to prepare extracts
(wherein, the volume ratio of human umbilical cord's blood mescenchymal stem cell extract is more than and 1%) can not only alleviate the anti-aging composition of object, very
To the damage that can repair the human skin cell as caused by ultraviolet light irradiation.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Each technical characteristic of embodiment described above can carry out arbitrarily
Combination, for simplicity of description, it is not all possible to each technical characteristic in above-described embodiment combination be all described, so
And as long as there is no contradiction in the combination of these technical features, it all should be considered as described in this specification.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those skilled in the art to which the present invention belongs, it is not taking off
Under the premise of from present inventive concept, several equivalent substitute or obvious modifications can also be made, and performance or use is identical, all answered
When being considered as belonging to protection scope of the present invention.
Claims (10)
1. a kind of human umbilical cord's blood mescenchymal stem cell extract, which is characterized in that prepared by following step:
1) mesenchymal stem cells derived from human umbilical blood is separated from people's agent band blood, and cultivates people in being added with ascorbic culture medium
Mesenchymal stem cells in umbilical cord blood；
2) mesenchymal stem cells derived from human umbilical blood is obtained from the culture medium for cultivating the mesenchymal stem cells derived from human umbilical blood
Secreting type content；
3) it will be crushed by the mesenchymal stem cells derived from human umbilical blood of culture, obtain the nonsecreting type of mesenchymal stem cells derived from human umbilical blood
Content；
4) the secreting type content is mixed with the nonsecreting type content.
2. human umbilical cord's blood mescenchymal stem cell extract as described in claim 1, which is characterized in that do not surpass from vitro culture
After obtaining the secreting type content in the culture medium in five generations；And/or between human umbilical cord's blood that in vitro culture was no more than for five generations
Mesenchymal stem cells obtain the nonsecreting type content.
3. a kind of preparation method of such as described in any item human umbilical cord's blood mescenchymal stem cell extracts of claim 1-2,
It is characterized in that, comprising the following steps:
1) mesenchymal stem cells derived from human umbilical blood is separated from people's agent band blood, and cultivates people in being added with ascorbic culture medium
Mesenchymal stem cells in umbilical cord blood；
2) mesenchymal stem cells derived from human umbilical blood is obtained from the culture medium for cultivating the mesenchymal stem cells derived from human umbilical blood
Secreting type content；
3) it will be crushed by the mesenchymal stem cells derived from human umbilical blood of culture, obtain the nonsecreting type of mesenchymal stem cells derived from human umbilical blood
Content；
4) the secreting type content is mixed with the nonsecreting type content.
4. preparation method as claimed in claim 3, which is characterized in that in step 1), the vitamin C is in the culture medium
In concentration be 40uM/ml-60uM/ml.
5. preparation method as claimed in claim 3, which is characterized in that in step 1), the culture medium is 5- in volume ratio
The mesenchymal stem cells derived from human umbilical blood is cultivated under conditions of the oxygen that 7% carbon dioxide and volume ratio is 5-7%.
6. preparation method as claimed in claim 3, which is characterized in that further include to people's umbilical cord after separation in step 1)
Blood mescenchymal stem cell is identified.
7. any one of the described in any item human umbilical cord's blood mescenchymal stem cell extracts of claim 1-2 or claim 3-6
Application of the human umbilical cord's blood mescenchymal stem cell extract of the preparation method preparation in cosmetics.
8. a kind of anti-apolexis composition, which is characterized in that including the described in any item human umbilical cord's blood mesenchymas of claim 1-2
Stem cell extract or human umbilical cord's blood mescenchymal stem cell of the described in any item preparation method preparations of claim 3-6 extract
Object.
9. anti-apolexis composition as claimed in claim 8, which is characterized in that further include epithelical cell growth factor, gluathione
Peptide, niacinamide and palmitinic acid SymPeptide.
10. anti-apolexis composition as claimed in claim 9, which is characterized in that human umbilical cord's blood mescenchymal stem cell mentions
The volume ratio for taking object is 0.05-5%, the concentration of the epithelical cell growth factor is 10-30ug/ml, the glutathione
Concentration is 2-8ug/ml, and the concentration of the niacinamide is 2-8mg/ml and the concentration of the palmitinic acid SymPeptide is 1-3mg/ml.
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