CN105997835A - Preparing method of immune-cell-derived biological-beautifying raw materials and products thereof - Google Patents
Preparing method of immune-cell-derived biological-beautifying raw materials and products thereof Download PDFInfo
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- CN105997835A CN105997835A CN201610303513.9A CN201610303513A CN105997835A CN 105997835 A CN105997835 A CN 105997835A CN 201610303513 A CN201610303513 A CN 201610303513A CN 105997835 A CN105997835 A CN 105997835A
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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 invention provides a preparing method of immune-cell-derived biological-beautifying raw materials. The method includes the steps that a culturing solution for culturing immune cells and a protein suspension obtained by splitting immune cells are subjected to hyperconcentration to obtain immune-cell essence, wherein the essence is rich in anti-inflammatory and anti-oxidation ingredients and has the anti-inflammatory and anti-oxidation effects; the essence is further subjected to freezing drying to obtain freezing-dried powder which can be stored in a -80-DEG-C ultra-low-temperature refrigerator for a long time, and the anti-inflammatory and anti-oxidation skin biological-beautifying raw materials are obtained. When the biological-beautifying raw materials are added into face cream, skin cream and toner, anti-inflammatory and anti-oxidation face cream products can be prepared; when the biological-beautifying raw materials are added into masks, anti-inflammatory and anti-oxidation mask products can be prepared; the biological-beautifying raw materials can be directly prepared into anti-inflammatory and anti-oxidation essence products and can be used for treating skin acne, removing freckles, blocking the sun, resisting oxidation and the like.
Description
Technical field
The present invention relates to a kind of method of biological beauty raw material preparing immunocyte source and by described
The biological beauty product in the immunocyte source that method obtains.Especially, the present invention relates to use exempt from from cultivation
The protein suspension that the culture fluid of epidemic disease cell and cracking immunocyte obtain, is prepared as immunocyte after ultra concentration
Essence, is prepared as the biological beauty raw material of lyophilized powder after lyophilization, rich in antiinflammatory antioxidant content, tool
There is antiinflammatory antioxidative effect, can be prepared as facial cream, skin cream, cosmetic water, facial film and essence etc. and produce
Product, can be used for acne, except beauty treatments such as speckle, sun-proof and antioxidation.
Background technology
At present, various cosmetics mainly based on chemical composition, then with the addition of some plant extract elite element or
The cosmetics that other deep-sea nutrient substance of person are prepared as, play the effects such as moisturizing, whitening and control oil to skin.
Although these cosmetics contain some nutrient substance, but because of its problem such as active constituent content and effectiveness,
The aspects such as skin antiinflammatory antioxidation are the most not enough.
In recent years, due to the development of biotechnology, technique for gene engineering is used to obtain cytokine gene restructuring
Carrier, ferments in escherichia coli or yeast, obtains relevant cell factor in a large number.Add as trophic factors
Or directly use in cosmetics, in skin repair, antiinflammatory and antioxidation, play certain effect.But due to
These cytokines are expressed in the cells such as escherichia coli or yeast etc. be low, and it lacks mammal thin
Protein transcription post translational modification in born of the same parents, including glycosylation, methylates and the process such as end modified, its function
It is very limited with biological activity.So using the cytokine of preparation of fermenting in escherichia coli or yeast to answer
Being used in cosmetics, the most possible biological activity is the highest the most invalid, or due to Immunogenicity skin
Cause allergic phenomena.
The present invention uses people's immunocyte to cultivate continuously under Cytokine, and these immunocytes can be natural
Substantial amounts of cytokine is expressed and is secreted on ground, and people's immunocyte is expressed and the albumen of secretion has good transcribing
Post translational modification, has fine biological activity.So, it also avoid and carry out gene recombinaton structure, exempt from
Epidemic disease cell can be expressed and secrete the most natural cytokine, does not has any genetic modification to cause heterologous to exempt from
The problem that epidemic focus is high, these cytokines are rich in the anti-immune inflammation molecule relevant with antioxidation.To sum up institute
Stating, the present invention utilizes people's immunocyte to express and the cytokine of secretion, is done by cracking, concentration and freezing
Be prepared as finished product after dry, more preferably store within effect duration biology and timely use, will to comedo, acne and
The skins such as erythra produce the beautification function such as optimal antiinflammatory and antioxidation.
Summary of the invention
The research with cosmetic applications is cultivated, in order to skin through scientific research personnel's permanent immunity cell of the present invention
Comedo, acne and erythra etc. produce the effect of preferably antiinflammatory and antioxidation etc., finder's immunocyte table
The cytokine reaching and secreting produces good antiinflammatory and goes to swell, go erythema and reparation etc. to act on, and combines U.S.
Hold physiotherapy and will produce more preferable effect.The present invention is by from culture fluid and the cracking immunity cultivating immunocyte
The protein suspension that cell obtains, is prepared as immunocyte essence after ultra concentration, is melted into rich in antiinflammatory antioxygen
Point, there is antiinflammatory antioxidative effect.These anti-inflammatory molecules mainly include interleukin 12 (interleukin 12,
IL-12), interleukin 8 (interleukin 8, IL-8), interleukin-1 receptor antagonist (interleukin
1 receptor antagonist, IL-1Ra), interferon-ALPHA (interferon α, TFN-α) and interferon gamma
(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
Have Nrf 2 (nuclear factor E2-related factor 2, Nrf2), have anti-inflammatory and
Antioxidation dual-use function；Growth molecule: intercellular adhesion molecule-1 (ICAM-1) and neutrophilic granulocyte born of the same parents
The slurry factor 2 (NCF-2).
The anti-inflammatory molecules of immunocyte secretion can specifically suppress inflammation, blocks the way that inflammation produces
Footpath, weakens inflammatory reaction；Maintain skin corium cell normal physiological context, suppression damaged cell and epidermis cell
Adhesion, prevent the intercellular diffusion of inflammation, promote that impaired cellular metabolism peels off naturally, recovery skin is strong
Health；Skin histology can be suppressed to produce the toxic components that can excite inflammation simultaneously；Immune complex is induced
Inflammatory reaction there is alleviation effect equally.The antioxidant molecule of immunocyte secretion can remove organism metabolism mistake
Journey produces the ultra-oxygen anion free radical of excess, improves skin and radical damage is induced an illness and to certainly
Resistance by base external world incitant.
Nrf2 is the basic transcription factor combining Antioxidant responsive element, the mesh of induction Antioxidant responsive element
Mark gene expression, including glutathion (glutathione, r-glutamyl cysteingl+glycine,
GSH), polytype gst gene can be induced to express, and GST is to have antioxidation and integrate removing toxic substances
The king of effect.And Nrf2 high expressed can suppress inflammation-related factor such as (IL)-1 β, IL-6, TNF-α, iNOS
With the expression of COX2 etc., thus Nrf2 is not only the commander-in-chief of antioxidant system, in terms of anti-inflammatory also
Play pivotal role.
We have found that simultaneously ICAM-1 and NCF-2 have advantageously promote skin flbroblast propagation and
Differentiation, expresses collagen protein and elastin laminin, improves skin skin quality, particularly has cicatrix of skin and cicatrix
There is fine repair.
The invention provides a kind of preparation method of biological beauty raw material cultivating immunocyte source, wherein,
Described method includes the protein suspension by obtaining from the culture fluid and cracking immunocyte of cultivating immunocyte,
Immunocyte essence it is prepared as, rich in antiinflammatory antioxidant content after ultra concentration；Preparation after freeze-dried
Become lyophilized powder, can preserve at-80 DEG C of ultra cold storage freezers for a long time, become skin antiinflammatory antioxidative biology beautiful
Hold raw material.
People's immunocyte that the present invention provides is Cytokine-induced killer cells, natural killer cell, nature
Cytotoxic T lymphocyte, bone-marrow-derived lymphocyte and dendritic cell etc., it is preferable that for cytokine-induced killer
Cell, natural killer cell and Natural killer T cells.
Described immunocyte source human peripheral or Cord blood, after lymphocyte separation medium density gradient centrifugation
Obtain mononuclearcell, use cytokines interferon-gamma, anti-human CD3 monoclonal antibody, interleukin-11 α and Bai Jie
Element 2 is cultivated continuously 20 days in serum-free medium and is obtained Cytokine-induced killer cells and culture fluid thereof；
Or cultivated continuously in serum-free medium by cytokine interleukin element 15, interleukin 18 and interleukin-22
Within 20 days, it is derived from Natural killer cell and culture fluid thereof；Or by cytokine interleukin element 15 and α-GalCer
Serum-free medium is cultivated 20 days continuously and obtains Natural killer T cells and culture fluid thereof.
Preferably, described MSC serum-free medium containing 0.1-200 μ g/ml hydrocortisone,
1-500 μ g/ml ascorbic acid and 1-1000 μ g/ml insulin human, it has been found that cell growth factor can be promoted
The secretion of son and expression, improve the content of cell growth factor, particularly promoted ICAM-1 and NCF-2
Expression.
Cultivate adaptive immune cell high-speed low temperature after ultrasonication to be centrifuged, it is thus achieved that cell cracking supernatant, and exempt from
Concentrating after the mixing of epidemic disease cell culture fluid, then in freeze drier, lyophilization obtains lyophilized powder, and gland is close
It is honored as a queen labeling, places-80 DEG C of Refrigerator stores.
Immunocyte culture fluid prepared by the present invention, the culture fluid containing immunocyte lysate and concentrated solution warp thereof
Enzyme-linked immunosorbent assay ELISA kit detects rich in antiinflammatory antioxidant molecule, it may be assumed that cultivate immunity thin
In the culture fluid of born of the same parents, the anti-inflammatory molecules interleukin 12 content of secretion contains more than 10000pg/ml, interleukin 8
Measure more than 100000pg/ml and interleukin-1 receptor antagonist content more than 5000pg/ml；Antioxidation
Factor converting growth factor beta 1 content exceedes more than 7000pg/ml and superoxide dismutase content
2000pg/ml, and intercellular adhesion molecule-1 content more than 1500pg/ml and neutrophilic granulocyte endochylema because of
Son 2 content are more than 4000pg/ml.
In culture fluid containing immunocyte lysate, anti-inflammatory molecules interleukin 12 content is more than 20ng/ml, in vain
Interleukin 8 content more than 150ng/ml and interleukin-1 receptor antagonist content more than 15ng/ml；Antioxygen
Change factor converting growth factor beta 1 content more than 13ng/ml and superoxide dismutase content more than 5ng/ml；
Anti-inflammatory and antioxidation dual function molecule Nrf 2 content are more than 5ng/ml；And iuntercellular
Adhesion molecule1 content more than 3ng/ml and the neutrophilic granulocyte endochylema factor 2 content more than 6ng/ml.
In concentrated solution, anti-inflammatory molecules interleukin 12 content exceedes more than 100ng/ml, interleukin 8 content
400ng/ml and interleukin-1 receptor antagonist content are more than 60ng/ml；Antioxidative Factors converts growth
Factor-beta 1 content more than 50ng/ml and superoxide dismutase content more than 20ng/ml；Anti-inflammatory and antioxidation
Dual function molecule Nrf 2 content is more than 20ng/ml, and intercellular adhesion molecule-1 contains
Measure more than 6ng/ml and the neutrophilic granulocyte endochylema factor 2 content more than 11ng/ml.
It is preferably the concentrated solution selecting the culture fluid containing immunocyte lysate as beauty treatment raw material.
The culture fluid lyophilized powder containing immunocyte lysate that the present invention provides, can be former as biological beauty
Material, adds to and produces into Related product in the cosmetics such as facial cream, skin cream and cosmetic water, and lyophilized powder adds molten
After matchmaker or physiological saline solution, join in facial cream, skin cream and cosmetic water etc. and mix.
The antiinflammatory antioxidation beauty mask in a kind of immunocyte source that the present invention provides, it is characterised in that its
Become to be grouped into by following:
1) the 5-50ml concentrated solution of culture fluid containing immunocyte lysate as above；
2) 1 non-woven fabrics, membrane of biological fibers, silkworm silk film or aquagel membrane etc.；
Being loaded in the tinfoil paper paper bag containing 10ml concentrated solution by facial film, pyrocondensation packs, and forms product；Should
Under the conditions of biological mask product is saved in 4 DEG C, the shelf-life is 1 month.
Preferably, silkworm silk film or aquagel membrane is selected to make facial film raw material.
The antiinflammatory antioxidation beauty essence in a kind of immunocyte source that the present invention provides, it is characterised in that
It is become to be grouped into by following:
Take the concentrated solution 10ml of the culture fluid containing immunocyte lysate as above, load 10ml drop bottle
Or in band nozzle bottle, produce into skin antiinflammatory antioxidation essence.
Or take 1 bottle of lyophilized powder 10ml solvent and dissolve, load in 10ml drop bottle or band nozzle bottle,
Produce into skin antiinflammatory antioxidation essence.
Forming product after mounted box packaging, under the conditions of being saved in 4 DEG C, the shelf-life is 1 month.
By the method for the present invention, by the immunocyte culture fluid obtained, training containing immunocyte lysate
Nutrient solution and concentrated solution thereof rich in anti-inflammatory and antioxidant molecule, can be prepared as facial cream, skin cream, cosmetic water,
The product such as facial film and essence, it is possible to skin is had wrinkle removal, acne cicatrix and cicatrix reparation, except speckle and
The beautification functions such as antioxidation.Especially when combining physics beauty curing and using, curative effect that generation is more preferably improved looks,
I.e. high temperature dark blue RF beauty therapy, microneedle therapy etc., can open epidermis pore or Wicresoft's epidermis cortex, for
Cytokine enters the passage of skin.
Accompanying drawing explanation
Fig. 1 represent the culture fluid by natural killer cell prepared by embodiment 2, culture fluid containing lysate with
And each cytokine-expressing and secretion level figure in concentrated solution
Fig. 2 represent by natural killer cell essence prepared by embodiment 4 to face acne experimenter use before
Rear variation diagram
Detailed description of the invention
The invention provides a kind of preparation method of biological beauty raw material cultivating immunocyte source, wherein,
Described method includes the protein suspension by obtaining from the culture fluid and cracking immunocyte of cultivating immunocyte,
Immunocyte essence it is prepared as, rich in antiinflammatory antioxidant content after ultra concentration；Preparation after freeze-dried
Become lyophilized powder, can preserve at-80 DEG C of ultra cold storage freezers for a long time, become skin antiinflammatory antioxidative biology beautiful
Hold raw material.
Described people's immunocyte is Cytokine-induced killer cells, natural killer cell, natural killer T
Lymphocyte, bone-marrow-derived lymphocyte and dendritic cell etc., it is preferable that for Cytokine-induced killer cells,
Natural killer cell and Natural killer T cells.
In the method for the invention, immunocyte derives from people's venous blood, bone marrow, Cord blood and placental blood etc.
Mononuclearcell.Preferably, fresh peripheral blood or Cord blood are derived from.
Described immunocyte source human peripheral or Cord blood, after lymphocyte separation medium density gradient centrifugation
Obtain mononuclearcell, use 100-5000IU/ml cytokines interferon-gamma, the anti-human CD3 of 5-500ng/ml
Monoclonal antibody, 10-1000IU/ml interleukin-11 α and 50-5000IU/ml interleukin-22 connect in serum-free medium
Continuous cultivation 20 days obtains Cytokine-induced killer cells and culture fluid thereof；Or pass through cytokine
5-500ng/ml interleukin 15,5-500ng/ml interleukin 18 and 50-5000IU/ml interleukin-22 are at depletion of blood
Clear culture medium is cultivated continuously and within 20 days, is derived from Natural killer cell and culture fluid thereof；Or pass through cytokine
5-500ng/ml interleukin 15 and 5-1000ng/ml α-GalCer cultivate 20 in serum-free medium continuously
It obtains Natural killer T cells and culture fluid thereof.
The present invention use cell culture medium be cell non-serum culture medium, as CellGro SCGM,
The commercial prods such as AIM-V, X-VIVO and GT-T551.Preferably, described immunocyte serum-free
Culture medium is containing 0.1-200 μ g/ml hydrocortisone, 1-500 μ g/ml ascorbic acid and 1-1000 μ g/ml people's pancreas
Island element.
Adaptive immune cell is counted, takes 109 cells and add 1-10ml1 × PBS (pH=7.4) mixing
Rear and add 0.1-5ug protease inhibitor, it is positioned in Ultrasonic Cell Disruptor broken 3-10 minute on ice,
The centrifugal 20min of 15000 turns of every point of kinds (rpm) at a high speed, takes supernatant and joins 1L and cultivated immunocyte
In culture fluid.Again the culture fluid of this immunocyte lysate is entered by the bag filter of 100-1000 molecular weight
Row dialysis concentrates, or is concentrated by ultrafiltration by the cellulose acetate membrane of 100-1000 molecular weight, concentrates
1-10 times, it is thus achieved that 100ml-500ml concentrated solution.
Taking 1-5ml concentrated solution and join in cillin bottle, in freeze drier, lyophilization obtains lyophilized powder,
Labeling after gland sealing, places-80 DEG C of Refrigerator stores, is prepared as cosmetics raw material.
Immunocyte culture fluid prepared by the present invention, the culture fluid containing immunocyte lysate and concentrated solution warp thereof
Enzyme-linked immunosorbent assay ELISA kit detects rich in antiinflammatory antioxidant molecule, it may be assumed that cultivate immunity thin
In the culture fluid of born of the same parents, the anti-inflammatory molecules interleukin 12 content of secretion contains more than 10000pg/ml, interleukin 8
Measure more than 100000pg/ml and interleukin-1 receptor antagonist content more than 5000pg/ml；Antioxidation
Factor converting growth factor beta 1 content exceedes more than 7000pg/ml and superoxide dismutase content
2000pg/ml, and intercellular adhesion molecule-1 content more than 1500pg/ml and neutrophilic granulocyte endochylema because of
Son 2 content are more than 4000pg/ml.
In culture fluid containing immunocyte lysate, anti-inflammatory molecules interleukin 12 content is more than 20ng/ml, in vain
Interleukin 8 content more than 150ng/ml and interleukin-1 receptor antagonist content more than 15ng/ml；Antioxygen
Change factor converting growth factor beta 1 content more than 13ng/ml and superoxide dismutase content more than 5ng/ml；
Anti-inflammatory and antioxidation dual function molecule Nrf 2 content are more than 5ng/ml；And iuntercellular
Adhesion molecule1 content more than 3ng/ml and the neutrophilic granulocyte endochylema factor 2 content more than 6ng/ml.
In concentrated solution, anti-inflammatory molecules interleukin 12 content exceedes more than 100ng/ml, interleukin 8 content
400ng/ml and interleukin-1 receptor antagonist content are more than 60ng/ml；Antioxidative Factors converts growth
Factor-beta 1 content more than 50ng/ml and superoxide dismutase content more than 20ng/ml；Anti-inflammatory and antioxidation
Dual function molecule Nrf 2 content is more than 20ng/ml, and intercellular adhesion molecule-1 contains
Measure more than 6ng/ml and the neutrophilic granulocyte endochylema factor 2 content more than 11ng/ml.
It is preferably the concentrated solution selecting the culture fluid containing immunocyte lysate as beauty treatment raw material.
The culture fluid lyophilized powder containing immunocyte lysate that the present invention provides, can be former as biological beauty
Material, adds in the cosmetics such as facial cream, skin cream and cosmetic water and produces into Related product, i.e. take 1 bottle of lyophilizing
After powder adds 1ml solvent or physiological saline solution, join in 50ml facial cream, skin cream and cosmetic water etc.
Mix.
The antiinflammatory antioxidation beauty mask in a kind of immunocyte source that the present invention provides, it is characterised in that its
Become to be grouped into by following:
1) the 5-50ml concentrated solution of culture fluid containing immunocyte lysate as above；
2) 1 non-woven fabrics, membrane of biological fibers, silkworm silk film or aquagel membrane etc.；
Being loaded in the tinfoil paper paper bag containing 10ml concentrated solution by facial film, pyrocondensation packs, and forms product；Should
Under the conditions of biological mask product is saved in 4 DEG C, the shelf-life is 1 month.
Preferably, silkworm silk film or aquagel membrane is selected to make facial film raw material.
The antiinflammatory antioxidation beauty essence in a kind of immunocyte source that the present invention provides, it is characterised in that
It is become to be grouped into by following:
Take the concentrated solution 10ml of the culture fluid containing immunocyte lysate as above, load 10ml drop bottle
Or in band nozzle bottle, produce into skin antiinflammatory antioxidation essence.
Or take 1 bottle of lyophilized powder 10ml solvent and dissolve, load in 10ml drop bottle or band nozzle bottle,
Produce into skin antiinflammatory antioxidation essence.
Forming product after mounted box packaging, under the conditions of being saved in 4 DEG C, the shelf-life is 1 month.
The invention provides the immunocyte culture fluid by obtaining, culture fluid containing immunocyte lysate and
Its concentrated solution, rich in anti-inflammatory and antioxidant molecule, can be prepared as facial cream, skin cream, cosmetic water, facial film
With the product such as essence, it is possible to skin is had wrinkle removal, acne cicatrix and cicatrix reparation, except speckle and antioxygen
The beautification functions such as change.Present invention also offers associating physics beauty curing to use, i.e. the dark blue RF beauty of high temperature
Therapy, microneedle therapy etc., can open epidermis pore or Wicresoft's epidermis cortex, enters skin for cytokine
Passage, curative effect that generation is more preferably improved looks.
Hereinafter, the specific embodiment of the present invention is illustrated, but the technical scope of the present invention is not limited to these
Example.
The cultivation of embodiment 1 people's immunocyte
Gather volunteer's 100ml peripheral blood (signing Informed Consent Form with it), used 1 × PBS (pH
Value is 7.4) dilution 1 times after, after lymphocyte separation medium density gradient centrifugation obtain mononuclearcell, make
With 1000IU/ml cytokines interferon-gamma, 50ng/ml anti-human CD3 monoclonal antibody, 100IU/ml interleukin-11 α
(contain at serum-free medium AIM-V with 500IU/ml interleukin-22 (being purchased from Mei Tian Ni company of Germany)
50 μ g/ml hydrocortisones, 10 μ g/ml ascorbic acid and 50 μ g/ml insulin humans, be purchased from U.S.'s life
Company) in cultivate 20 days continuously, it is thus achieved that 5.0 × 109Cytokine-induced killer cells, and 3.0L training
Nutrient solution.
Cytokine 200ng/ml interleukin 15,100ng/ml interleukin 18 and 1000IU/ml is used to be situated between in vain
Element 2 (being purchased from Mei Tian Ni company of Germany) is cultivated 20 days in serum-free medium AIM-V continuously, obtains
Obtain 3.0 × 109Natural killer cell and 2.0L culture fluid.
Cytokine 100ng/ml interleukin 15 and 1000ng/ml α-GalCer is used (to be purchased from Germany beautiful
It Ni company) cultivate 20 days continuously in serum-free medium AIM-V, it is thus achieved that 3.6 × 109NKT
T lymphocyte and 2.4L culture fluid.
Prepared by embodiment 2 people's immunocyte source beauty treatment raw material
The immunocyte obtained in Example 1 adds after 10ml 1 × PBS (pH=7.4) mixes and adds
1ug protease inhibitor (Dalian treasured biotech firm), is positioned on ice at Ultrasonic Cell Disruptor (U.S.'s thermoelectricity
Company) in broken 8 minutes, 15000 turns of rpm are centrifuged 20min at a high speed, take supernatant and join to cultivate and exempt from
In the culture fluid of epidemic disease cell.
Culture fluid containing immunocyte lysate is joined bag filter (U.S.'s spectrum public affairs of 500 molecular weight
Department) in, place and refrigerator cold-storage carries out dialysis concentration, volume concentration 5 times.
Alternatively, it is also possible to the culture fluid containing immunocyte lysate to be used the cellulose acetate of 500 molecular weight
Film (spectrum company of the U.S.) is concentrated by ultrafiltration, volume concentration 5 times.
Cytokine-induced killer cells, natural killer cell and Natural killer T cells divide after concentrating
Do not obtain 600ml, 400ml and 480ml concentrated solution, take 5ml immunocyte concentrated solution and join cillin bottle
In, in freeze drier (power & light company of the U.S.), lyophilization obtains lyophilized powder, gland seal after labeling,
Place-80 DEG C of Refrigerator stores, be prepared as cosmetics raw material.
In embodiment 3 people's immunocyte, cytokine-expressing detects with secretion level
The immunocyte culture fluid of preparation in Example 1, culture fluid containing immunocyte lysate and dense
Contracting liquid detects wherein cell through ELISA kit (R&D system house of the U.S.) and expresses and secretion level,
Step is used to consult and use description, all to do blank with the AIM-V culture medium not cultivating immunocyte
Comparison.Table 1 is each in the culture fluid of immunocyte, the culture fluid containing immunocyte lysate and concentrated solution
Cytokine-expressing and secretion level, described unit is ng/ml.
Table 1
Table 1 shows in the culture fluid of immunocyte, the culture fluid containing lysate and concentrated solution and all secrete height
The anti-inflammatory molecules of level and antioxidant molecule, the Nrf2 wherein containing dual function in cell pyrolysis liquid divides
Son, shows that these cytokines will play anti-inflammatory and antioxidative effect in terms of beautifying skin.Send out especially
All high expressing cell growth factor I CAM-1 and NCF-2 in present cell culture fluid and cell pyrolysis liquid, its
Functionally there is promotion cell proliferation and the effect of skin repair.
Fig. 1 be in the culture fluid of natural killer cell, the culture fluid containing lysate and concentrated solution each cell because of
Sublist reaches and secretion level figure, and compared with original AIM-V culture medium, natural killer cell has well
Levels of cytokine secretion.
Embodiment 4 prepares the cosmetics of immunocyte culture fluid
Preparing the antiinflammatory antioxidation beauty mask in immunocyte source, following one-tenth is grouped into:
1) concentrated solution of the culture fluid containing immunocyte lysate of 10ml embodiment 2 preparation；
2) 1 aquagel membrane；
Being loaded in the tinfoil paper paper bag containing 10ml concentrated solution by facial film, pyrocondensation packs, and forms product；Should
Under the conditions of biological mask product is saved in 4 DEG C, the shelf-life is 1 month.
Prepare the antiinflammatory antioxidation beauty essence in immunocyte source, be following become to be grouped into:
The concentrated solution 10ml of the culture fluid containing immunocyte lysate of Example 2 preparation, loads 10ml
In drop bottle or band nozzle bottle, produce into skin antiinflammatory antioxidation essence.
Or 1 bottle of lyophilized powder 10ml solvent of Example 2 preparation dissolves, load 10ml drop bottle
Or in band nozzle bottle, produce into skin antiinflammatory antioxidation essence.
Forming product after mounted box packaging, under the conditions of being saved in 4 DEG C, the shelf-life is 1 month.
Embodiment 5 immunocyte beauty essence
Recruit 10 faces and have experimenter's (signature Informed Consent Form) of erythema and acne, every morning and complete
Before kind sleep, wash one's face with face cleaning dew, the most dry after smear or spray the natural killer cell of preparation in embodiment 4
Beauty essence, uses 14 day time continuously.
All experimenter there is erythema person the most all to disappear, have acne person's acne alleviate or disappear completely, Fig. 2
In variation diagram before and after wherein face acne experimenter uses, A is for using before natural killer cell beauty essence
Picture, B is that natural killer cell beauty essence uses the picture after 14 days；It appeared that its acne
Well being improved, skin becomes smooth, bleaches and fine and glossy.
Claims (10)
1. the preparation method of the biological beauty raw material in an immunocyte source, it is characterised in that described side
Method includes the protein suspension by obtaining, warp from the culture fluid and cracking people's immunocyte of cultivating people's immunocyte
Immunocyte essence it is prepared as after ultra concentration；It is prepared as lyophilized powder after freeze-dried, can exist for a long time
-80 DEG C of ultra cold storage freezers preserve；
Wherein, the cultivation of immunocyte uses serum-free medium, and preferably it can containing 0.1-200 μ g/ml hydrogenation
Ground pine, 1-500 μ g/ml ascorbic acid and 1-1000 μ g/ml insulin human.
Described immunocyte essence or lyophilized powder can be used as biological beauty raw material；
Described immunocyte culture fluid, culture fluid containing immunocyte lysate and concentrated solution thereof 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), Nrf 2 (nuclear factor E2-related factor 2, Nrf2), iuntercellular glue
Attached molecule-1 (ICAM-1), the neutrophilic granulocyte endochylema factor 2 (NCF-2)；
The culture fluid cultivating people's immunocyte detects through enzyme-linked immunosorbent assay ELISA kit, wherein,
Anti-inflammatory molecules interleukin 12 content is more than 10000pg/ml, and interleukin 8 content is more than 100000pg/ml
With interleukin-1 receptor antagonist content more than 5000pg/ml；Antioxidative Factors transforming growth factor contains
Measure more than 7000pg/ml and superoxide dismutase content more than 2000pg/ml；Immunocyte expresses anti-inflammatory
With antioxidation dual function molecule Nrf 2 content more than 2000pg/ml；And iuntercellular glues
Attached molecule-1 content more than 1500pg/ml and the neutrophilic granulocyte endochylema factor 2 content more than 4000pg/ml.
Method the most according to claim 1, it is characterised in that
Detect through enzyme-linked immunosorbent assay ELISA kit, in the culture fluid containing immunocyte lysate
Anti-inflammatory molecules interleukin 12 content is more than 20ng/ml, and interleukin 8 content is thin with white more than 150ng/ml
Born of the same parents' interleukin-1 receptor antagonist agent content is more than 15ng/ml；Antioxidative Factors transforminggrowthfactor-β1 content surpasses
Cross 13ng/ml and superoxide dismutase content more than 5ng/ml；Anti-inflammatory and antioxidation dual function molecular core
Factor correlation factor 2 content is more than 5ng/ml；And intercellular adhesion molecule-1 content more than 3ng/ml and
The neutrophilic granulocyte endochylema factor 2 content is more than 6ng/ml.
3. according to the method described in any one of claim 1-2, it is characterised in that through Enzyme-linked Immunosorbent Assay
Mensuration ELISA kit detects,
Cultivate the anti-inflammatory molecules interleukin 12 content of secretion in the culture fluid of immunocyte to exceed
10000pg/ml, interleukin 8 content is more than 100000pg/ml and interleukin-1 receptor antagonist content
More than 5000pg/ml；Antioxidative Factors transforminggrowthfactor-β1 content is more than 7000pg/ml and super oxidation discrimination
Change enzyme content is more than 2000pg/ml, and intercellular adhesion molecule-1 content is more than 1500pg/ml and neutrality
The granulocyte endochylema factor 2 content is more than 4000pg/ml.
Or/and, in the culture fluid containing immunocyte lysate, anti-inflammatory molecules interleukin 12 content exceedes
20ng/ml, interleukin 8 content exceedes more than 150ng/ml and interleukin-1 receptor antagonist content
15ng/ml；Antioxidative Factors transforminggrowthfactor-β1 content is more than 13ng/ml and superoxide dismutase content
More than 5ng/ml；Anti-inflammatory and antioxidation dual function molecule Nrf 2 content exceed
5ng/ml, and intercellular adhesion molecule-1 content contains more than 3ng/ml and the neutrophilic granulocyte endochylema factor 2
Amount is more than 6ng/ml.
Or/and, in concentrated solution, anti-inflammatory molecules interleukin 12 content is more than 100ng/ml, interleukin 8 content
More than 400ng/ml and interleukin-1 receptor antagonist content more than 60ng/ml；Antioxidative Factors converts
Growth factor beta 1 content more than 50ng/ml and superoxide dismutase content more than 20ng/ml；Anti-inflammatory is with anti-
Oxidation dual function molecule Nrf 2 content is more than 20ng/ml, and ICAIU
-1 content more than 6ng/ml and the neutrophilic granulocyte endochylema factor 2 content more than 11ng/ml.
4. according to the method described in any one of claim 1-3, it is characterised in that described immunocyte comes
Source human peripheral or Cord blood, obtain mononuclearcell after lymphocyte separation medium density gradient centrifugation；
Cultural method is:
Use cytokines interferon-gamma, anti-human CD3 monoclonal antibody, interleukin-11 α and interleukin-22 at serum-free
Culture medium is cultivated 20 days continuously and obtains Cytokine-induced killer cells and culture fluid thereof；
Or it is continuous in serum-free medium to pass through cytokine interleukin element 15, interleukin 18 and interleukin-22
Cultivate and be derived from Natural killer cell and culture fluid thereof in 20 days；
Or cultivated 20 days continuously in serum-free medium by cytokine interleukin element 15 and α-GalCer
Obtain Natural killer T cells and culture fluid thereof.
Described serum-free medium containing 0.1-200 μ g/ml hydrocortisone, 1-500 μ g/ml ascorbic acid and
1-1000 μ g/ml insulin human.
5. according to the method described in any one of claim 1-4, it is characterised in that described method include as
Lower step:
Adaptive immune cell is counted, takes 109Cell adds after 1-10ml 1 × PBS mixes and adds
0.1-5ug protease inhibitor, is positioned in Ultrasonic Cell Disruptor broken 3-10 minute on ice, and at a high speed 15000
Turn the centrifugal 20min of every point of kind (rpm), take supernatant and join in the culture fluid that 1L cultivated immunocyte；
Again by the bag filter of 100-1000 molecular weight, the culture fluid of this immunocyte lysate is carried out dialysis to concentrate,
Or it is concentrated by ultrafiltration by the cellulose acetate membrane of 100-1000 molecular weight, concentrates 1-10 times, it is thus achieved that
100ml-500ml concentrated solution；
Taking 1-5ml concentrated solution and join in cillin bottle, in freeze drier, lyophilization obtains lyophilized powder,
Labeling after gland sealing, places-80 DEG C of Refrigerator stores.
6. according to the method described in any one of claim 1-5, it is characterised in that described people's immunocyte
For Cytokine-induced killer cells, natural killer cell, Natural killer T cells, bone-marrow-derived lymphocyte
Or dendritic cell；Preferably, it is Cytokine-induced killer cells, natural killer cell or naturally kills
Hinder T lymphocyte；
Described immunocyte culture fluid, it is therefore preferable to containing the concentrated solution of the culture fluid of immunocyte lysate.
7. according to the method described in any one of claim 1-6, it is characterised in that described lyophilized powder conduct
Biological beauty raw material, adds in facial cream, skin cream or cosmetic water and produces into Related product；Its preparation method
For, after taking 1 bottle of lyophilized powder addition 1ml solvent or physiological saline solution, join 50ml facial cream, skin moistening
Dew or cosmetic water mix.
8. the antiinflammatory antioxidation beauty mask in an immunocyte source, it is characterised in that it is by becoming as follows
It is grouped into:
1) concentration of the culture fluid containing immunocyte lysate described in any one of 5-50ml claim 1-7
Liquid；
2) facial film raw material: non-woven fabrics, membrane of biological fibers, silkworm silk film or aquagel membrane；
Its preparation method is: being loaded in the tinfoil paper paper bag containing concentrated solution by facial film raw material, pyrocondensation packs,
Form product；Under the conditions of this mask product is saved in 4 DEG C, the shelf-life is 1 month；
Preferably, silkworm silk film or aquagel membrane is selected to make facial film raw material.
9. the antiinflammatory antioxidation beauty essence in an immunocyte source, it is characterised in that its preparation side
Method is:
Take the concentrated solution 10ml of the culture fluid containing immunocyte lysate described in any one of claim 1-7,
Load in 10ml drop bottle or band nozzle bottle, produce into skin antiinflammatory antioxidation beauty essence；
Or take 1 bottle of described lyophilized powder 10ml solvent and dissolve, load 10ml drop bottle or band nozzle bottle
In, produce into skin antiinflammatory antioxidation beauty essence；
Forming product after mounted box packaging, under the conditions of being saved in 4 DEG C, the shelf-life is 1 month.
10. the preparation method of the biological beauty raw material in an immunocyte source, it is characterised in that described side
Method comprises the steps
Step one, the cultivation of people's immunocyte
Gather volunteer's 100ml peripheral blood, after being used 1 × PBS to dilute 1 times, lymphocyte separation medium
Mononuclearcell is obtained after density gradient centrifugation；
Described cultural method is:
Use 1000IU/ml cytokines interferon-gamma, 50ng/ml anti-human CD3 monoclonal antibody, 100IU/ml white
Interleukin 1 α and 500IU/ml interleukin-22 is cultivated 20 days in serum-free medium AIM-V continuously, it is thus achieved that
5.0×109Cytokine-induced killer cells, and 3.0L culture fluid；Wherein, described serum-free medium
Containing 50 μ g/ml hydrocortisones, 10 μ g/ml ascorbic acid and 50 μ g/ml insulin humans；
Or, use cytokine 200ng/ml interleukin 15,100ng/ml interleukin 18 and 1000IU/ml
Interleukin-22 is cultivated 20 days in serum-free medium AIM-V continuously, it is thus achieved that 3.0 × 109NKT is thin
Born of the same parents and 2.0L culture fluid；
Or, use cytokine 100ng/ml interleukin 15 and 1000ng/ml α-GalCer at serum-free
Culture medium A IM-V is cultivated 20 days continuously, it is thus achieved that 3.6 × 109Natural killer T cells and 2.4L
Culture fluid；
Wherein, described serum-free medium AIM-V contains 50 μ g/ml hydrocortisones, 10 μ g/ml Vitamin Cs
Acid and 50 μ g/ml insulin humans.
Prepared by step 2, people's immunocyte source beauty treatment raw material
Take after the immunocyte obtained in step one adds 10ml 1 × PBS mixing and add 1ug protease and press down
Preparation, is positioned over and crushes 8 minutes in Ultrasonic Cell Disruptor on ice, and 15000 turns of rpm are centrifuged 20min at a high speed,
Take supernatant to join in the culture fluid cultivating immunocyte；
Culture fluid containing immunocyte lysate is joined in the bag filter of 500 molecular weight, place refrigerator cold
Tibetan carries out dialysis concentrate, volume concentration 5 times；
Or, the culture fluid containing immunocyte lysate uses the cellulose acetate membrane of 500 molecular weight carry out
It is concentrated by ultrafiltration, volume concentration 5 times；
Cytokine-induced killer cells, natural killer cell or Natural killer T cells divide after concentrating
Do not obtain 600ml, 400ml and 480ml concentrated solution, take 5ml immunocyte concentrated solution and join cillin bottle
In, in freeze drier, lyophilization obtains lyophilized powder, labeling after gland sealing, places-80 DEG C of refrigerators
Preserve, be prepared as cosmetics raw material.
In step 3, people's immunocyte, cytokine-expressing detects with secretion level
Take the immunocyte culture fluid of preparation in step one, the culture fluid containing immunocyte lysate and concentration thereof
Liquid detects wherein cell through ELISA kit and expresses and secretion level；It is all rich in following cytokine:
IL-12, IL-8, IL-1Ra, TGF-β 1, SOD, Nrf2, ICAM-1 and NCF-2；
Preferably, when described immunocyte is Cytokine-induced killer cells, in described immunocyte culture fluid
IL-12, IL-8, IL-1Ra, TGF-β 1, the content of SOD, Nrf2, ICAM-1 and NCF-2 are respectively
12.06,101.93,7.57,8.42,2.57,0.02,1.76 and 4.32；
IL-12 in the described culture fluid containing immunocyte lysate, IL-8, IL-1Ra, TGF-β 1, SOD,
The content of Nrf2, ICAM-1 and NCF-2 is respectively 24.51,169.13,17.66,15.9,6.5,5.65,
4.46 and 7.57；
IL-12 in described concentrated solution, IL-8, IL-1Ra, TGF-β 1, SOD, Nrf2, ICAM-1 and
The content of NCF-2 is respectively 149.81,560.56,78.64,66.42,33.56,45.24,8.76 and 12.13；
Further preferably, when described immunocyte is natural killer cell, in described immunocyte culture fluid
IL-12, IL-8, IL-1Ra, TGF-β 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 in the described culture fluid containing immunocyte lysate, IL-8, IL-1Ra, TGF-β 1, SOD,
The content of Nrf2, ICAM-1 and NCF-2 is respectively 21.71,170.21,16.67,14.32,7.01,
5.24,5.08 and 6.69；
IL-12 in described concentrated solution, IL-8, IL-1Ra, TGF-β 1, SOD, Nrf2, ICAM-1 and
The content of NCF-2 is respectively 149.81,560.56,78.64,66.42,33.56,45.24,8.76 and 12.13；
Further preferably, when described immunocyte is Natural killer T cells, described immunocyte is trained
IL-12 in nutrient solution, IL-8, IL-1Ra, TGF-β 1, the containing of SOD, Nrf2, ICAM-1 and NCF-2
Amount is respectively 13.76,132.78,6.23,7.8,3.09,0.01,2.06 and 3.98；
IL-12 in the described culture fluid containing immunocyte lysate, IL-8, IL-1Ra, TGF-β 1, SOD,
The content of Nrf2, ICAM-1 and NCF-2 is respectively 25.81,181.43,18.52,16.45,7.71,
6.16,3.84 and 7.51；
IL-12 in described concentrated solution, IL-8, IL-1Ra, TGF-β 1, SOD, Nrf2, ICAM-1 and
The content of NCF-2 is respectively 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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