CN105997835B - A kind of preparation method and products thereof of the biological beauty raw material in immunocyte source - Google Patents
A kind of preparation method and products thereof of the biological beauty raw material in immunocyte source Download PDFInfo
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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
- 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
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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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Abstract
The present invention provides a kind of preparation methods of the biological beauty raw material in immunocyte source, wherein, the protein suspension that the method includes being obtained by the culture solution from culture immunocyte with cracking immunocyte, immunoblast Essence is prepared after hyperconcetration, rich in anti-inflammatory antioxidant content, there is anti-inflammatory oxidation resistant effect；It further is prepared into freeze-dried powder after freeze-drying, can be saved for a long time in -80 DEG C of ultra low temperature freezers, become the anti-inflammatory oxidation resistant biological beauty raw material of skin；Anti-inflammatory anti-oxidant face cream product can be prepared by being added in face cream, skin cream and toner, anti-inflammatory anti-oxidant face mask product can be prepared into and directly be prepared into anti-inflammatory anti-oxidant essence liquid product by being added in facial mask, can be used for acne, except spot, sun-proof and anti-oxidant etc. beauty.
Description
Technical field
It is obtained the present invention relates to a kind of method of biological beauty raw material for preparing immunocyte source and by the method
The biological beauty product in the immunocyte source obtained.Particularly, the present invention relates to using from culture immunocyte culture solution and
The protein suspension that immunocyte obtains is cracked, immunoblast Essence is prepared after hyperconcetration, prepares gelation after freeze-drying
The biological beauty raw material of dry powder is rich in anti-inflammatory antioxidant content, has anti-inflammatory oxidation resistant effect, can be prepared into face cream, profit
The products such as skin dew, toner, facial mask and Essence can be used for acne, except spot, sun-proof and anti-oxidant etc. beauty.
Background technique
Currently, various cosmetics are mainly based on chemical component, then be added to some plant extract elites elements or other
The beauty product that deep-sea nutriment is prepared into, to skin play moisturizing, whitening and oil-control the effects of.Although these cosmetics contain
There are some nutriments, but the problems such as because of its active constituent content and validity, in terms of skin is anti-inflammatory also much
It is insufficient.
In recent years, due to the development of biotechnology, cytokine gene recombinant vector is obtained using technique for gene engineering,
It ferments in Escherichia coli or yeast, largely obtains relevant cell factor.It is added in cosmetics as trophic factors or directly makes
With, skin repair, it is anti-inflammatory and anti-oxidant in play certain effect.But since these cell factors are in Escherichia coli or yeast
Etc. what is expressed in low cell, lack protein transcription posttranslational modification in mammalian cells, including glycosylation, methyl
Change and processes, function and the bioactivity such as end modified are very limited.So using fermenting in Escherichia coli or yeast
The cell factor of preparation is applied in cosmetics, and much possible bioactivity is not high or even invalid, or since immunogenicity is asked
Topic skin causes allergic phenomena.
The present invention is continuously cultivated under Cytokine using people's immunocyte, these immunocytes can be expressed natively
With a large amount of cell factor of secretion, the albumen of the expression of people's immunocyte and secretion is modified after having good transcription and translation, is had
Fine bioactivity.So, it also avoids carrying out genetic recombination building, immunocyte can be expressed and be secreted a large amount of natural
Cell factor, the problem for causing allogeneic immune originality high without any gene modification, these cell factors are rich in anti-immunity
Inflammation and anti-oxidant relevant molecule.In conclusion the cell factor that the present invention is expressed and secreted using people's immunocyte, passes through
Cracking, concentration and freeze-drying after be prepared into finished product, within biology validity period more preferably storage timely use, will to whelk,
The optimal anti-inflammatory and anti-oxidant equal beautification functions of the skins such as acne and fash generation.
Summary of the invention
By the research of the cell culture of scientific research personnel's permanent immunity and cosmetic applications of the present invention, for the youth to skin
Preferably anti-inflammatory and anti-oxidant etc. the effect of the generations such as acne, acne and fash, the cell of finder's immunocyte expression and secretion
The factor generates good anti-inflammatory and goes the effects of swelling, going erythema and repair, and joint beauty physical therapy will generate more preferable effect.
The protein suspension that the present invention is obtained by the culture solution from culture immunocyte with cracking immunocyte, is prepared into after hyperconcetration
Immunocyte Essence is rich in anti-inflammatory antioxidant content, has anti-inflammatory oxidation resistant effect.These anti-inflammatory molecules mainly include
Interleukin 12 (interleukin 12, IL-12), interleukin 8 (interleukin 8, IL-8), interleukin 1 receptor are short of money
Anti-agent (interleukin1 receptor antagonist, IL-1Ra), interferon-' alpha ' (interferon α, TFN- α) and dry
Disturb plain γ (interferon γ, TFN- γ) etc.；Antioxidant molecule mainly includes transforminggrowthfactor-β1 (transforming
Growth factor- β 1, TGF-β 1), platelet derived growth factor (Platelet derived growth factor,
) and superoxide dismutase (Superoxide Dismutase, SOD) etc. PDGF；Immunocyte lysate also contains nuclear factor phase
It closes the factor 2 (nuclear factor E2-related factor 2, Nrf2), there is anti-inflammatory and anti-oxidant dual function；
Grow molecule: intercellular adhesion molecule-1 (ICAM-1) and the neutrophil leucocyte endochylema factor 2 (NCF-2).
The anti-inflammatory molecules of immunocyte secretion can specifically inhibit inflammation, the approach that truncation inflammation generates, reduction
Inflammatory reaction；Corium confluent monolayer cells normal physiological context is maintained, inhibits the adherency of damaged cell and epidermal cell, prevents inflammation thin
The diffusion of intercellular promotes impaired cellular metabolism to peel off naturally, restores skin health；Being able to suppress skin histology generation simultaneously can swash
The toxic components of inflammation disease；The inflammatory reaction induced immune complex equally has effects that alleviation.Immunocyte secretion
Antioxidant molecule can be removed and generate excessive ultra-oxygen anion free radical in body metabolic process, improve skin to radical damage
And it induces an illness and to the resistance of free radical external world incitant.
Nrf2 is the basic transcription factor in conjunction with Antioxidant responsive element, induces the target gene table of Antioxidant responsive element
It reaches, including glutathione (glutathione, r-glutamyl cysteingl+glycine, GSH), can induce a variety of
The gst gene of type is expressed, and GST is that have king that is anti-oxidant and integrating detoxication.And Nrf2 high expression can inhibit inflammation
Correlation factor such as (IL) -1 β, IL-6, the expression of TNF-α, iNOS and COX2 etc., thus Nrf2 is not only the total of antioxidant system
Commandant also plays key effect in terms of anti-inflammatory.
Simultaneously we have found that ICAM-1 and NCF-2, which has, advantageously promotes skin fibroblasts proliferation and differentiation, expression
Collagen and elastin laminin improve skin skin quality, especially have fine repair to cicatrix of skin and scar.
The present invention provides a kind of preparation methods of biological beauty raw material for cultivating immunocyte source, wherein the side
Method includes the protein suspension obtained by the culture solution from culture immunocyte with cracking immunocyte, is prepared into after hyperconcetration
Immunocyte Essence is rich in anti-inflammatory antioxidant content；Freeze-dried powder is prepared into after freeze-dried, it can be super at -80 DEG C for a long time
Low temperature refrigerator saves, and becomes the anti-inflammatory oxidation resistant biological beauty raw material of skin.
People's immunocyte provided by the invention is Cytokine-induced killer cells, natural killer cells, natural killer T leaching
Bar cell, bone-marrow-derived lymphocyte and Dendritic Cells etc., it is preferable that for Cytokine-induced killer cells, natural killer cells and from
Right cytotoxic T lymphocyte.
Immunocyte source human peripheral or Cord blood obtain single after lymphocyte separation medium density gradient centrifugation
A nucleus is connected in serum free medium using cytokines interferon-gamma, anti-human CD3 monoclonal antibody, interleukin-11 α and interleukin-22
20 days acquisition Cytokine-induced killer cells of continuous culture and its culture solution；Or pass through cytokine interleukin element 15, interleukin 18
20 days acquisition natural killer cells and its culture solution are continuously cultivated in serum free medium with interleukin-22；Or by cell because
Sub- interleukin 15 and α-GalCer continuously cultivate 20 days acquisition Natural killer T cells and its culture in serum free medium
Liquid.
Preferably, the MSC serum free medium g/ml of μ containing the 0.1-200 hydrocortisone, 1-500 μ g/ml Vitamin C
Acid and 1-1000 μ g/ml actrapid monotard, it has been found that the secretion and expression of Porcine HGF can be promoted, improve cell growth
The content of the factor has especially promoted the expression of ICAM-1 and NCF-2.
It cultivates adaptive immune cell high-speed low temperature after ultrasonication to be centrifuged, cell cracking supernatant is obtained, with immunocyte
Culture solution is concentrated after mixing, and then freeze-drying obtains freeze-dried powder in freeze drier, and labeling after gland sealing places -80
DEG C refrigerator saves.
Immunocyte culture solution prepared by the present invention, the culture solution of the lysate containing immunocyte and its concentrate are exempted from through enzyme-linked
The detection of epidemic disease determining adsorption ELISA kit is rich in anti-inflammatory antioxidant molecule, it may be assumed that cultivate secreted in the culture solution of immunocyte it is anti-
Inflammatory molecule interleukin 12 content is more than 10000pg/ml, interleukin 8 content be more than 100000pg/ml and interleukin 1 by
Body antagonism agent content is more than 5000pg/ml；Antioxidative Factors transforminggrowthfactor-β1 content is more than 7000pg/ml and super oxygen discrimination
Change enzyme content be more than 2000pg/ml and intercellular adhesion molecule-1 content be more than 1500pg/ml and neutrophil leucocyte endochylema because
Sub 2 contents are more than 4000pg/ml.
Anti-inflammatory molecules interleukin 12 content is more than 20ng/ml in the culture solution of the lysate containing immunocyte, and interleukin 8 contains
Amount is more than 150ng/ml and interleukin-1 receptor antagonist content is more than 15ng/ml；Antioxidative Factors transforming growth factor β
1 content is more than 13ng/ml and superoxide dismutase content is more than 5ng/ml；Anti-inflammatory and anti-oxidant double action molecule nuclear factor
2 content of correlation factor is more than 5ng/ml；And intercellular adhesion molecule-1 content be more than 3ng/ml and neutrophil leucocyte endochylema because
Sub 2 contents are more than 6ng/ml.
Anti-inflammatory molecules interleukin 12 content is more than 100ng/ml in concentrate, interleukin 8 content be more than 400ng/ml and
Interleukin-1 receptor antagonist content is more than 60ng/ml；Antioxidative Factors transforminggrowthfactor-β1 content is more than 50ng/ml
It is more than 20ng/ml with superoxide dismutase content；Anti-inflammatory and anti-oxidant 2 content of double action molecule nuclear factor correlation factor are super
It crosses 20ng/ml and intercellular adhesion molecule-1 content is more than 6ng/ml and 2 content of the neutrophil leucocyte endochylema factor is more than
11ng/ml。
Preferably select the concentrate of the culture solution of the lysate containing immunocyte as beauty raw material.
The culture solution freeze-dried powder of the lysate provided by the invention containing immunocyte, can be used as biological beauty raw material, addition
Related product is produced into cosmetics such as face cream, skin cream and toners, after solvent or physiological saline solution is added in freeze-dried powder,
It is added in face cream, skin cream and toner etc. and mixes.
The anti-inflammatory anti-oxidant beauty mask in a kind of immunocyte source provided by the invention, which is characterized in that it is by as follows
At being grouped as:
1) concentrate of the 5-50ml culture solution of the lysate containing immunocyte as described above；
2) 1 non-woven fabrics, membrane of biological fibers, silk film or aquagel membrane etc.；
Facial mask is fitted into the tinfoil paper paper bag of the concentrate containing 10ml, pyrocondensation sealed package, forms product；The biology facial mask produces
Product are stored under the conditions of 4 DEG C, and the shelf-life is 1 month.
Preferably, silk film or aquagel membrane is selected to make facial mask raw material.
The anti-inflammatory anti-oxidant beauty essence in a kind of immunocyte source provided by the invention, which is characterized in that it is by such as
It is lower at being grouped as:
The concentrate 10ml of the culture solution of the lysate containing immunocyte as described above is taken, is packed into 10ml drop bottle or with nozzle
In bottle, it is produced into the anti-inflammatory anti-oxidant Essence of skin.
Or 1 bottle of freeze-dried powder is taken to be dissolved with 10ml solvent, it is fitted into 10ml drop bottle or anti-with skin in nozzle bottle, is produced into
Scorching anti-oxidant Essence.
Product is formed after mounted box packaging, is stored under the conditions of 4 DEG C, the shelf-life is 1 month.
By means of the present invention, by the immunocyte culture solution of acquisition, the lysate containing immunocyte culture solution and
Its concentrate is rich in anti-inflammatory and antioxidant molecule, can be prepared into the production such as face cream, skin cream, toner, facial mask and Essence
Product can have anti-wrinkle, acne scar and scar reparation to skin, remove spot and the beautification functions such as anti-oxidant.Especially when joint
Physics beauty curing will be in use, more preferably beauty curative effect, the i.e. dark blue RF beauty therapy of high temperature, microneedle therapy etc., energy will be generated
Epidermis pore or minimally invasive epidermis cortex are opened, the channel of skin is entered for cell factor.
Detailed description of the invention
Culture solution and concentrate Fig. 1 shows the culture solution of the natural killer cells as prepared by embodiment 2, containing lysate
In each cytokine-expressing and secretion level figure
Fig. 2 indicates the natural killer cells Essence as prepared by embodiment 4 to variation before and after face acne subject use
Figure
Specific embodiment
The present invention provides a kind of preparation methods of biological beauty raw material for cultivating immunocyte source, wherein the side
Method includes the protein suspension obtained by the culture solution from culture immunocyte with cracking immunocyte, is prepared into after hyperconcetration
Immunocyte Essence is rich in anti-inflammatory antioxidant content；Freeze-dried powder is prepared into after freeze-dried, it can be super at -80 DEG C for a long time
Low temperature refrigerator saves, and becomes the anti-inflammatory oxidation resistant biological beauty raw material of skin.
People's immunocyte is Cytokine-induced killer cells, natural killer cells, Natural killer T cells, B
Lymphocyte and Dendritic Cells etc., it is preferable that be Cytokine-induced killer cells, natural killer cells and natural killer T
Lymphocyte.
Single core of the immunocyte from people's venous blood, marrow, Cord blood and placental blood etc. in the method for the invention
Cell.Preferably, fresh peripheral blood or Cord blood are derived from.
Immunocyte source human peripheral or Cord blood obtain single after lymphocyte separation medium density gradient centrifugation
A nucleus uses 100-5000IU/ml cytokines interferon-gamma, the anti-human CD3 monoclonal antibody of 5-500ng/ml, 10-1000IU/ml
Interleukin-11 α and 50-5000IU/ml interleukin-22 continuously cultivates 20 days acquisition cytokine-induced killers in serum free medium
Cell and its culture solution；Or pass through cell factor 5-500ng/ml interleukin 15,5-500ng/ml interleukin 18 and 50-
5000IU/ml interleukin-22 continuously cultivates 20 days acquisition natural killer cells and its culture solution in serum free medium；Or pass through
Cell factor 5-500ng/ml interleukin 15 and 5-1000ng/ml α-GalCer are continuously cultivated 20 days in serum free medium to be obtained
Obtain Natural killer T cells and its culture solution.
The cell culture medium that the present invention uses is cell non-serum culture medium, such as CellGro SCGM, AIM-V, X-VIVO
With the commercial prods such as GT-T551.Preferably, immunocyte serum free medium μ containing 0.1-200 g/ml hydrogenation can ground
Pine, 1-500 μ g/ml ascorbic acid and 1-1000 μ g/ml actrapid monotard.
Adaptive immune cell is counted, 109 cells is taken to be added after 1-10ml1 × PBS (pH=7.4) is mixed and is added
0.1-5ug protease inhibitors is placed in and is crushed 3-10 minutes in Ultrasonic Cell Disruptor on ice, 15000 revolutions per minute kinds of high speed
(rpm) it is centrifuged 20min, takes supernatant to be added to 1L and cultivated in the culture solution of immunocyte.Again by the immunocyte lysate
Culture solution carries out dialysis concentration, or the acetate fiber by 100-1000 molecular weight by the bag filter of 100-1000 molecular weight
Plain film is concentrated by ultrafiltration, and is concentrated 1-10 times, obtains 100ml-500ml concentrate.
1-5ml concentrate is taken to be added in cillin bottle, freeze-drying obtains freeze-dried powder, gland sealing in freeze drier
Labeling afterwards is placed -80 DEG C of refrigerators and is saved, is prepared into cosmetics raw material.
Immunocyte culture solution prepared by the present invention, the culture solution of the lysate containing immunocyte and its concentrate are exempted from through enzyme-linked
The detection of epidemic disease determining adsorption ELISA kit is rich in anti-inflammatory antioxidant molecule, it may be assumed that cultivate secreted in the culture solution of immunocyte it is anti-
Inflammatory molecule interleukin 12 content is more than 10000pg/ml, interleukin 8 content be more than 100000pg/ml and interleukin 1 by
Body antagonism agent content is more than 5000pg/ml；Antioxidative Factors transforminggrowthfactor-β1 content is more than 7000pg/ml and super oxygen discrimination
Change enzyme content be more than 2000pg/ml and intercellular adhesion molecule-1 content be more than 1500pg/ml and neutrophil leucocyte endochylema because
Sub 2 contents are more than 4000pg/ml.
Anti-inflammatory molecules interleukin 12 content is more than 20ng/ml in the culture solution of the lysate containing immunocyte, and interleukin 8 contains
Amount is more than 150ng/ml and interleukin-1 receptor antagonist content is more than 15ng/ml；Antioxidative Factors transforming growth factor β
1 content is more than 13ng/ml and superoxide dismutase content is more than 5ng/ml；Anti-inflammatory and anti-oxidant double action molecule nuclear factor
2 content of correlation factor is more than 5ng/ml；And intercellular adhesion molecule-1 content be more than 3ng/ml and neutrophil leucocyte endochylema because
Sub 2 contents are more than 6ng/ml.
Anti-inflammatory molecules interleukin 12 content is more than 100ng/ml in concentrate, interleukin 8 content be more than 400ng/ml and
Interleukin-1 receptor antagonist content is more than 60ng/ml；Antioxidative Factors transforminggrowthfactor-β1 content is more than 50ng/ml
It is more than 20ng/ml with superoxide dismutase content；Anti-inflammatory and anti-oxidant 2 content of double action molecule nuclear factor correlation factor are super
It crosses 20ng/ml and intercellular adhesion molecule-1 content is more than 6ng/ml and 2 content of the neutrophil leucocyte endochylema factor is more than
11ng/ml。
Preferably select the concentrate of the culture solution of the lysate containing immunocyte as beauty raw material.
The culture solution freeze-dried powder of the lysate provided by the invention containing immunocyte, can be used as biological beauty raw material, addition
It is produced into Related product into cosmetics such as face cream, skin cream and toners, that is, takes 1 bottle of freeze-dried powder that 1ml solvent or physiology is added
After salt water dissolution, it is added in 50ml face cream, skin cream and toner etc. and mixes.
The anti-inflammatory anti-oxidant beauty mask in a kind of immunocyte source provided by the invention, which is characterized in that it is by as follows
At being grouped as:
1) concentrate of the 5-50ml culture solution of the lysate containing immunocyte as described above；
2) 1 non-woven fabrics, membrane of biological fibers, silk film or aquagel membrane etc.；
Facial mask is fitted into the tinfoil paper paper bag of the concentrate containing 10ml, pyrocondensation sealed package, forms product；The biology facial mask produces
Product are stored under the conditions of 4 DEG C, and the shelf-life is 1 month.
Preferably, silk film or aquagel membrane is selected to make facial mask raw material.
The anti-inflammatory anti-oxidant beauty essence in a kind of immunocyte source provided by the invention, which is characterized in that it is by such as
It is lower at being grouped as:
The concentrate 10ml of the culture solution of the lysate containing immunocyte as described above is taken, is packed into 10ml drop bottle or with nozzle
In bottle, it is produced into the anti-inflammatory anti-oxidant Essence of skin.
Or 1 bottle of freeze-dried powder is taken to be dissolved with 10ml solvent, it is fitted into 10ml drop bottle or anti-with skin in nozzle bottle, is produced into
Scorching anti-oxidant Essence.
Product is formed after mounted box packaging, is stored under the conditions of 4 DEG C, the shelf-life is 1 month.
The present invention provides the immunocyte culture solution by obtaining, the culture solution of the lysate containing immunocyte and its concentrations
Liquid is rich in anti-inflammatory and antioxidant molecule, can be prepared into the products such as face cream, skin cream, toner, facial mask and Essence, can
There is anti-wrinkle, acne scar and scar reparation to skin, remove spot and the beautification functions such as anti-oxidant.The present invention also provides joints
Physics beauty curing uses, i.e. the dark blue RF beauty therapy of high temperature, microneedle therapy etc., can open epidermis pore or minimally invasive epidermis skin
Layer enters the channel of skin for cell factor, will generate more preferably beauty curative effect.
Hereinafter, being illustrated to specific embodiments of the present invention, but technical scope of the invention is not limited to these examples.
The culture of 1 people's immunocyte of embodiment
It acquires volunteer 100ml peripheral blood (signing informed consent form with it), it is diluted with 1 × PBS (pH value 7.4)
After 1 times, mononuclearcell is obtained after lymphocyte separation medium density gradient centrifugation, uses 1000IU/ml cytokines interferon
The anti-human CD3 monoclonal antibody of γ, 50ng/ml, 100IU/ml interleukin-11 α and 500IU/ml interleukin-22 (being purchased from German Mei Tian Ni company)
(contain 50 μ g/ml hydrocortisones, 10 μ g/ml ascorbic acid and 50 μ g/ml actrapid monotards in serum free medium AIM-V, purchase
From life company, the U.S.) in continuously cultivate 20 days, obtain 5.0 × 109Cytokine-induced killer cells and 3.0L culture
Liquid.
It (is purchased using cell factor 200ng/ml interleukin 15,100ng/ml interleukin 18 and 1000IU/ml interleukin-22
From German Mei Tian Ni company) it is continuously cultivated in serum free medium AIM-V 20 days, obtain 3.0 × 109Natural killer cells with
And 2.0L culture solution.
Use cell factor 100ng/ml interleukin 15 and 1000ng/ml α-GalCer (being purchased from German Mei Tian Ni company)
It is continuously cultivated in serum free medium AIM-V 20 days, obtains 3.6 × 109Natural killer T cells and 2.4L culture
Liquid.
The preparation of 2 people's immunocyte source beauty raw material of embodiment
The immunocyte obtained in Example 1 is added after 1 × PBS of 10ml (pH=7.4) is mixed and 1ug albumen is added
Enzyme inhibitor (Dalian treasured biotech firm) is placed in and is crushed 8 minutes in Ultrasonic Cell Disruptor (power & light company, the U.S.) on ice, high speed
15000 turns of rpm are centrifuged 20min, and supernatant is taken to be added in the culture solution for cultivating immunocyte.
The culture solution of the lysate containing immunocyte is added in the bag filter (spectrum company, the U.S.) of 500 molecular weight, is put
It sets and carries out dialysis concentration in refrigerator cold-storage, 5 times of volume concentration.
Alternatively, it is also possible to which the cellulose acetate film (U.S. that the culture solution of immunocyte lysate uses 500 molecular weight will be contained
Spectrum company) it is concentrated by ultrafiltration, 5 times of volume concentration.
It is obtained respectively after Cytokine-induced killer cells, natural killer cells and Natural killer T cells concentration
600ml, 400ml and 480ml concentrate take 5ml immunocyte concentrate to be added in cillin bottle, in the freeze drier (U.S.
Power & light company) in freeze-drying obtain freeze-dried powder, labeling after gland sealing is placed -80 DEG C of refrigerators and is saved, is prepared into beauty and make-up
Product raw material.
Cytokine-expressing and secretion level detect in 3 people's immunocyte of embodiment
The culture solution and its concentrate of the immunocyte culture solution, the lysate containing immunocyte that prepare in Example 1 pass through
ELISA kit (R&D system house, the U.S.) detects wherein cell expression and secretion level, is referred to explanation using step
Book, to do blank control with the AIM-V culture medium for not cultivating immunocyte.For the culture solution of immunocyte, containing exempting from table 1
Each cytokine-expressing and secretion level in the culture solution and concentrate of epidemic disease cell pyrolysis liquid, the unit are ng/ml.
Table 1
Shown in table 1 culture solution of immunocyte, secrete in the culture solution and concentrate containing lysate it is high-caliber
Anti-inflammatory molecules and antioxidant molecule, the wherein Nrf2 molecule in cell pyrolysis liquid containing double action, show these cells because
Son will play anti-inflammatory and oxidation resistant effect in terms of beautifying skin.It is found especially that in cell culture fluid and cell pyrolysis liquid
Equal high expressing cell growth factor I CAM-1 and NCF-2 functionally have the function of promoting cell Proliferation and skin repair.
Fig. 1 is the culture solution of natural killer cells, each cytokine-expressing in culture solution and concentrate containing lysate
With secretion level figure, compared with original AIM-V culture medium, natural killer cells has good levels of cytokine secretion.
Embodiment 4 prepares the beauty product of immunocyte culture solution
The anti-inflammatory anti-oxidant beauty mask in immunocyte source is prepared, as follows at being grouped as:
1) concentrate of the culture solution of the lysate containing immunocyte prepared by 10ml embodiment 2；
2) 1 aquagel membrane；
Facial mask is fitted into the tinfoil paper paper bag of the concentrate containing 10ml, pyrocondensation sealed package, forms product；The biology facial mask produces
Product are stored under the conditions of 4 DEG C, and the shelf-life is 1 month.
The anti-inflammatory anti-oxidant beauty essence for preparing immunocyte source, by as follows at being grouped as:
The concentrate 10ml of the culture solution of the lysate containing immunocyte prepared by Example 2 is packed into 10ml drop bottle or band
In nozzle bottle, it is produced into the anti-inflammatory anti-oxidant Essence of skin.
Or 1 bottle of freeze-dried powder prepared by Example 2 is dissolved with 10ml solvent, is packed into 10ml drop bottle or with nozzle bottle
In, it is produced into the anti-inflammatory anti-oxidant Essence of skin.
Product is formed after mounted box packaging, is stored under the conditions of 4 DEG C, the shelf-life is 1 month.
5 immunocyte beauty essence of embodiment
Recruit subject's (signature informed consent form) that 10 faces have erythema and acne, every morning and perfect sleep
Before, it is washed one's face with face cleaning dew, the natural killer cells beauty essence prepared in slightly dry rear smearing or sprinkling embodiment 4 continuously makes
With 14 day time.
There is erythema person all to disappear in whole subjects, there are acne person's acne to mitigate or subside completely, wherein face in Fig. 2
Variation diagram before and after portion's acne subject use, A are using the picture before natural killer cells beauty essence, and B is natural kill
Cell beauty essence uses the picture after 14 days；It can be found that its acne is improved well, skin becomes smooth, bleaches
With it is fine and glossy.
Claims (10)
1. a kind of preparation method of the biological beauty raw material in immunocyte source, which is characterized in that the method includes by from
The culture solution of people's immunocyte, i.e. immunocyte culture solution, and the protein suspension that cracking people's immunocyte obtains are cultivated, that is, contains and exempts from
The culture solution of epidemic disease cell pyrolysis liquid prepares immunoblast Essence, the i.e. culture of the lysate containing immunocyte after hyperconcetration
The concentrate of liquid；It is prepared into freeze-dried powder after freeze-dried, can be saved for a long time in -80 DEG C of ultra low temperature freezers；
Wherein, the culture of immunocyte uses serum free medium, the g/ml of μ containing 0.1-200 hydrocortisone, 1-500 μ g/ml
Ascorbic acid and 1-1000 μ g/ml actrapid monotard；
The immunocyte Essence or freeze-dried powder can be used as biological beauty raw material；
The immunocyte culture solution, the culture solution of the lysate containing immunocyte and its concentrate are rich in interleukin 12
(interleukin 12, IL-12), interleukin 8 (interleukin 8, IL-8), interleukin-1 receptor antagonist
(interleukin 1 receptor antagonist, IL-1Ra), transforminggrowthfactor-β1 (transforming growth
Factor- β 1, TGF-β 1), superoxide dismutase (Superoxide Dismutase, SOD), nuclear factor correlation factor 2
(nuclear factor E2-related factor 2, Nrf2), intercellular adhesion molecule-1 (ICAM-1), neutrophil leucocyte
The endochylema factor 2 (NCF-2)；
The culture solution of culture people's immunocyte is detected through enzyme linked immunosorbent assay (ELISA) ELISA kit, wherein anti-inflammatory molecules are white
12 content of interleukin is more than 10000pg/ml, and interleukin 8 content is more than that 100000pg/ml and interleukin-1 receptor antagonist contain
Amount is more than 5000pg/ml；Antioxidative Factors transforming growth factor content is more than 7000pg/ml and superoxide dismutase content is more than
2000pg/ml；Immunocyte expression anti-inflammatory and anti-oxidant 2 content of double action molecule nuclear factor correlation factor are more than
2000pg/ml；And intercellular adhesion molecule-1 content is more than 1500pg/ml and 2 content of the neutrophil leucocyte endochylema factor is more than
4000pg/ml；
People's immunocyte is Cytokine-induced killer cells, natural killer cells, Natural killer T cells, B lymph
Cell or Dendritic Cells.
2. the method according to claim 1, wherein
It is detected through enzyme linked immunosorbent assay (ELISA) ELISA kit, anti-inflammatory molecules are white in the culture solution of the lysate containing immunocyte
12 content of interleukin is more than 20ng/ml, and interleukin 8 content is more than 150ng/ml and interleukin-1 receptor antagonist content is more than
15ng/ml；Antioxidative Factors transforminggrowthfactor-β1 content is more than 13ng/ml and superoxide dismutase content is more than 5ng/ml；
Anti-inflammatory and anti-oxidant 2 content of double action molecule nuclear factor correlation factor are more than 5ng/ml；And intercellular adhesion molecule-1
Content is more than 3ng/ml and 2 content of the neutrophil leucocyte endochylema factor is more than 6ng/ml.
3. the method according to claim 1, wherein detected through enzyme linked immunosorbent assay (ELISA) ELISA kit,
Cultivating the anti-inflammatory molecules interleukin 12 content secreted in the culture solution of immunocyte is more than 10000pg/ml, interleukin 8
Content is more than 100000pg/ml and interleukin-1 receptor antagonist content is more than 5000pg/ml；Antioxidative Factors conversion life
Long 1 content of factor-beta is more than 7000pg/ml and superoxide dismutase content is more than 2000pg/ml and intercellular adhesion molecule-1
Content is more than 1500pg/ml and 2 content of the neutrophil leucocyte endochylema factor is more than 4000pg/ml；
Or/and anti-inflammatory molecules interleukin 12 content is more than 20ng/ml, interleukin 8 in the culture solution of the lysate containing immunocyte
Content is more than 150ng/ml and interleukin-1 receptor antagonist content is more than 15ng/ml；Antioxidative Factors conversion growth because
Sub- 1 content of β is more than 13ng/ml and superoxide dismutase content is more than 5ng/ml；Anti-inflammatory and anti-oxidant double action molecular core
2 content of factor correlation factor is more than 5ng/ml and intercellular adhesion molecule-1 content is more than 3ng/ml and neutrophil leucocyte born of the same parents
Starching 2 content of the factor is more than 6ng/ml；
Or/and anti-inflammatory molecules interleukin 12 content is more than 100ng/ml in concentrate, interleukin 8 content is more than 400ng/ml
It is more than 60ng/ml with interleukin-1 receptor antagonist content；Antioxidative Factors transforminggrowthfactor-β1 content is more than 50ng/
Ml and superoxide dismutase content are more than 20ng/ml；Anti-inflammatory and anti-oxidant 2 content of double action molecule nuclear factor correlation factor
More than 20ng/ml and intercellular adhesion molecule-1 content is more than 6ng/ml and 2 content of the neutrophil leucocyte endochylema factor is more than
11ng/ml。
4. method according to claim 1-3, which is characterized in that immunocyte source human peripheral or navel
Band blood, obtains mononuclearcell after lymphocyte separation medium density gradient centrifugation；
Cultural method are as follows:
It is continuously trained in serum free medium using cytokines interferon-gamma, anti-human CD3 monoclonal antibody, interleukin-11 α and interleukin-22
Support 20 days acquisition Cytokine-induced killer cells and its culture solution；
Or it is continuously cultivated 20 days and is obtained in serum free medium by cytokine interleukin element 15, interleukin 18 and interleukin-22
Natural killer cells and its culture solution；
Or 20 days acquisition natural killer Ts are continuously cultivated in serum free medium by 15 and α-GalCer of cytokine interleukin element
Lymphocyte and its culture solution；
Serum free medium μ containing the 0.1-200 g/ml hydrocortisone, 1-500 μ g/ml ascorbic acid and 1-1000 μ g/ml
Actrapid monotard.
5. method according to claim 1-3, which is characterized in that described method includes following steps:
Adaptive immune cell is counted, 109 cells is taken to be added after 1 × PBS of 1-10ml is mixed and 0.1-5ug albumen is added
Enzyme inhibitor is placed in and is crushed 3-10 minutes in Ultrasonic Cell Disruptor on ice, high speed 15000 revolutions per minute kinds (rpm) centrifugation
20min takes supernatant to be added to 1L and cultivated in the culture solution of immunocyte；The culture solution of the immunocyte lysate is passed through again
The bag filter of 100-1000 molecular weight carries out dialysis concentration, or is surpassed by the cellulose acetate film of 100-1000 molecular weight
Filter concentration, is concentrated 1-10 times, obtains 100ml-500ml concentrate；
1-5ml concentrate is taken to be added in cillin bottle, freeze-drying obtains freeze-dried powder in freeze drier, pastes after gland sealing
Label are placed -80 DEG C of refrigerators and are saved.
6. method according to claim 1-3, which is characterized in that people's immunocyte is cytokine induction
Kill cell, natural killer cells or Natural killer T cells；
The immunocyte culture solution, for the concentrate of the culture solution of the lysate containing immunocyte.
7. method according to claim 1-3, which is characterized in that the freeze-dried powder as biological beauty raw material,
It is added in face cream, skin cream or toner and is produced into Related product；Preparation method is to take 1 bottle of freeze-dried powder that 1ml solvent is added
Or after physiological saline solution, it is added in 50ml face cream, skin cream or toner and mixes.
8. a kind of anti-inflammatory anti-oxidant beauty mask in immunocyte source, which is characterized in that it is by as follows at being grouped as:
1) concentrate of the culture solution of the described in any item lysates containing immunocyte of 5-50ml claim 1-7；
2) facial mask raw material: selection silk film or aquagel membrane make facial mask raw material；Preparation method are as follows: fill facial mask raw material
Enter in the tinfoil paper paper bag containing concentrate, pyrocondensation sealed package, forms product；The mask product is stored under the conditions of 4 DEG C, the shelf-life
It is 1 month.
9. a kind of anti-inflammatory anti-oxidant beauty essence in immunocyte source, which is characterized in that preparation method are as follows:
The concentrate 10ml of the culture solution of the lysate containing immunocyte described in claim any one of 1-7 is taken, 10ml is packed into
Drop bottle or in nozzle bottle, being produced into the anti-inflammatory anti-oxidant beauty essence of skin；
Or freeze-dried powder described in 1 bottle of claim any one of 1-7 is taken to be dissolved with 10ml solvent, it is packed into 10ml drop bottle or with spray
In mouth bottle, it is produced into the anti-inflammatory anti-oxidant beauty essence of skin；
Product is formed after mounted box packaging, is stored under the conditions of 4 DEG C, the shelf-life is 1 month.
10. a kind of preparation method of the biological beauty raw material in immunocyte source, which is characterized in that the method includes walking as follows
Suddenly
Step 1: the culture of people's immunocyte
Volunteer 100ml peripheral blood is acquired, after it is diluted 1 times with 1 × PBS, after lymphocyte separation medium density gradient centrifugation
Obtain mononuclearcell；
The cultural method are as follows:
Use 1000IU/ml cytokines interferon-gamma, the anti-human CD3 monoclonal antibody of 50ng/ml, 100IU/ml interleukin-11 α and 500IU/
Ml interleukin-22 is continuously cultivated 20 days in serum free medium AIM-V, obtains 5.0 × 109Cytokine-induced killer cells,
And 3.0L culture solution；
Alternatively, using cell factor 200ng/ml interleukin 15,100ng/ml interleukin 18 and 1000IU/ml interleukin-22 in nothing
It is continuously cultivated in blood serum medium AIM-V 20 days, obtains 3.0 × 109Natural killer cells and 2.0L culture solution；
Alternatively, using cell factor 100ng/ml interleukin 15 and 1000ng/ml α-GalCer in serum free medium AIM-V
Continuous culture 20 days, obtains 3.6 × 109Natural killer T cells and 2.4L culture solution；
Wherein, the serum free medium AIM-V contains 50 μ g/ml hydrocortisones, 10 μ g/ml ascorbic acid and 50 μ g/ml people
Insulin；
Step 2: prepared by people's immunocyte source beauty raw material
It takes the immunocyte obtained in step 1 to be added after 1 × PBS of 10ml is mixed and 1ug protease inhibitors is added, be placed in
Be crushed 8 minutes in Ultrasonic Cell Disruptor on ice, 15000 turns of rpm of high speed are centrifuged 20min, take supernatant be added to cultivated it is immune thin
In the culture solution of born of the same parents；
The culture solution of the lysate containing immunocyte is added in the bag filter of 500 molecular weight, places in refrigerator cold-storage and carries out thoroughly
Analysis concentration, 5 times of volume concentration；
Alternatively, the culture solution of the lysate containing immunocyte is concentrated by ultrafiltration using the cellulose acetate film of 500 molecular weight, body
5 times of product concentration；
Cytokine-induced killer cells, natural killer cells or Natural killer T cells concentration after obtain respectively 600ml,
400ml and 480ml concentrate takes 5ml immunocyte concentrate to be added in cillin bottle, is freeze-dried in freeze drier
To freeze-dried powder, labeling after gland sealing is placed -80 DEG C of refrigerators and is saved, is prepared into cosmetics raw material；
Step 3: cytokine-expressing and secretion level detect in people's immunocyte
The immunocyte culture solution of preparation, the culture solution of the lysate containing immunocyte and its concentrate is taken to examine through ELISA kit
Survey wherein cell expression and secretion level；It is rich in following cell factor: IL-12, IL-8, IL-1Ra, TGF-β 1, SOD,
Nrf2, ICAM-1 and NCF-2；
When the immunocyte is Cytokine-induced killer cells, IL-12, IL-8, IL- in the immunocyte culture solution
1Ra, TGF-β 1, the content of SOD, Nrf2, ICAM-1 and NCF-2 be respectively 12.06,101.93,7.57,8.42,2.57,
0.02,1.76 and 4.32；
IL-12, IL-8 in the culture solution of the lysate containing immunocyte, IL-1Ra, TGF-β 1, SOD, Nrf2, ICAM-1 and
The content of NCF-2 is respectively 24.51,169.13,17.66,15.9,6.5,5.65,4.46 and 7.57；
IL-12, IL-8, IL-1Ra, TGF-β 1, the content of SOD, Nrf2, ICAM-1 and NCF-2 are respectively in the concentrate
149.81,560.56,78.64,66.42,33.56,45.24,8.76 and 12.13；
When the immunocyte is natural killer cells, IL-12, IL-8, IL-1Ra, TGF-β in the immunocyte culture solution
1, the content of SOD, Nrf2, ICAM-1 and NCF-2 are respectively 14.43,113.42,5.48,7.56,3.16,0.04,1.65 and
3.75；IL-12, IL-8 in the culture solution of the lysate containing immunocyte, IL-1Ra, TGF-β 1, SOD, Nrf2, ICAM-1 and
The content of NCF-2 is respectively 21.71,170.21,16.67,14.32,7.01,5.24,5.08 and 6.69；
IL-12, IL-8, IL-1Ra, TGF-β 1, the content of SOD, Nrf2, ICAM-1 and NCF-2 are respectively in the concentrate
149.81,560.56,78.64,66.42,33.56,45.24,8.76 and 12.13；
When the immunocyte is Natural killer T cells, IL-12, IL-8 in the immunocyte culture solution, IL-1Ra,
TGF-β 1, the content of SOD, Nrf2, ICAM-1 and NCF-2 are respectively 13.76,132.78,6.23,7.8,3.09,0.01,2.06
With 3.98；
IL-12, IL-8 in the culture solution of the lysate containing immunocyte, IL-1Ra, TGF-β 1, SOD, Nrf2, ICAM-1 and
The content of NCF-2 is respectively 25.81,181.43,18.52,16.45,7.71,6.16,3.84 and 7.51；
IL-12, IL-8, IL-1Ra, TGF-β 1, the content of SOD, Nrf2, ICAM-1 and NCF-2 are respectively in the concentrate
179.34,590.57,78.26,62.41,30.51,47.86,6.80 and 13.05；
The unit of above-mentioned factor content is ng/ml.
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| KR101969045B1 (en) * | 2016-11-22 | 2019-04-19 | 신동혁 | Culture media kits for NK cell Cultivation, NK cell culture method using the same, NK Cell media solution without blood serum, and cosmetic composition comprising the serum free conditioned media |
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| KR102014467B1 (en) * | 2017-12-14 | 2019-08-26 | (주)녹십자웰빙 | Cosmetic composition and pharmaceutical composition for improving atopic dermatis, alopetic, wound, or skin wrinkle |
| CN109620771B (en) * | 2018-12-19 | 2022-08-26 | 浙江奥比特生物科技有限公司 | Cosmetic composition for resisting inflammation and removing acne of skin and product thereof |
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| CN110804607B (en) * | 2019-11-18 | 2021-09-21 | 深圳市润科生物科技有限公司 | Preparation method of high-concentration human mesenchymal stem cell lysate |
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