CN110368356A - Skin injury reparative factor composition and preparation method thereof - Google Patents
Skin injury reparative factor composition and preparation method thereof Download PDFInfo
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- CN110368356A CN110368356A CN201910730822.8A CN201910730822A CN110368356A CN 110368356 A CN110368356 A CN 110368356A CN 201910730822 A CN201910730822 A CN 201910730822A CN 110368356 A CN110368356 A CN 110368356A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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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
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
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Abstract
The invention discloses a kind of skin injury reparative factor composition, the skin injury reparative factor composition contains immunocyte culture secretion and immunocyte lysate.Above-mentioned skin injury reparative factor composition, on the one hand Secretion regulation culture is oriented to immunocyte, the bioactive substances such as the multiple growth factor for being conducive to skin injury reparation, protease, micromolecule polypeptide in enrichment culture secretion realize the intercommunication of cell therapy technology and skin regeneration technique technology；On the other hand, stem cell, immunocyte clinical research, the culture supernatant in production process can be made to be fully used again, avoiding these culture supernatants as biologic garbage, there are potential hazards to natural environment and human health, also wherein active constituent is developed and utilized, reduces cell production cost.
Description
Technical field
The invention belongs to cell culture applied technical field, in particular to a kind of skin injury reparative factor composition and its
Preparation method.
Background technique
The Aging Mechanism of skin is that ozone free radical (ROS) leads to that microcirculation disorder, metabolism slow down, cell Proliferation is divided
The main reason for phenomena such as changing reduced capability, further resulting in presentation wrinkle, color spot, this is skin aging.Skin Cell regeneration
The bioactive substances such as regulatory factor, superoxide dismutase (SOD), bioactive micro peptide are the maximally efficient of confrontation ROS
Solution.Bioactive substance pass through following approach realize ageing skin regeneration: by remove oxyradical, repair by
Cell, hyperplasia collagen and elastic fibers are damaged, skin inner supporting structure, activation substrate stem cell regenerating differentiation are strengthened.Such as
The purpose of what realizes the anti-aging of skin using cell therapy is an important research direction.However no matter people are in reality at present
The production preparation for only focusing on leucocyte, immunocyte, mescenchymal stem cell in research or GMP production is tested, relevant cell is trained
Feeding supernatant and extract is discarded as biologic garbage, is not utilized adequately to it, this greatly wastes cell
The bioactive substance secreted during the cultivation process.
How cell culture resource making full use of in skin nursing, reparation is realized, being that this field is urgently to be resolved asks
Topic.
Summary of the invention
Technical problem solved by the invention is to provide a kind of skin injury reparative factor composition and preparation method thereof, and one
Aspect is a variety of by containing in the culture secretion and immunocyte lysate in research discovery immunocyte incubation
Growth factor, protease and micromolecule polypeptide isoreactivity substance are consistent with factor height needed for skin repair, regeneration, thus
The composition can be applied in skin repair, that is, realize the purpose for realizing skin nursing, reparation by cell therapy；It is another
Aspect, and stem cell, immunocyte clinical research, the culture supernatant in production process can be made to be fully used, it avoids
To natural environment and human health, there are potential hazards as biologic garbage for these culture supernatants, also to wherein active constituent into
Row development and utilization, reduce the cost of cell therapy.
The present invention provides a kind of skin injury reparative factor composition, the skin injury reparative factor composition contains
Immunocyte culture secretion and immunocyte lysate.
The skin injury reparative factor composition also contains mescenchymal stem cell culture point in one of the embodiments,
Secretion.
In one of the embodiments, immunocyte culture secretion in the skin injury reparative factor composition, exempt from
The protein content mass ratio of epidemic disease cell lysate and mescenchymal stem cell culture secretion is 2:(1~3): (1~3).
The work that the mescenchymal stem cell culture secretion is 3~10 from algebra in one of the embodiments,
The culture supernatant of cell strain production mescenchymal stem cell.
The immunocyte derives from healthy human peripheral blood, marrow and neonatal umbilical cord in one of the embodiments,
Any one or a few in blood.
The source for mesenchymal stem cells is in the blood of Healthy People, fat, skin and new in one of the embodiments,
Raw youngster is with any one or a few tissue in tissue.
In one of the embodiments, the total protein concentration of the skin injury reparative factor composition be 2mg/ml~
6mg/ml。
The present invention also provides a kind of preparation method of skin injury reparative factor composition as described above, the preparations
Method the following steps are included:
Immunocyte is subjected to induction stress stimulation culture, is collected in the culture induced in stress stimulation incubation
The culture supernatant is obtained the immunocyte culture secretion through separating treatment by clear liquid；
By it is described induction stress stimulation culture after immunocyte carry out 2~4 multigelations, separating treatment obtain described in
Immunocyte lysate；
The immunocyte culture secretion and the immunocyte lysate are mixed the acquisition skin injury to repair
Multifactor composition.
Immunocyte is subjected to induction stress stimulation culture in one of the embodiments, the collection induction stress pierce
Swash the culture supernatant in incubation, comprising the following steps:
The immunocyte of collection is inoculated in Fiber differentiation in the culture medium containing inducing cytokine by Fiber differentiation for the first time
4d~12d collects culture supernatant；
Lipopolysaccharides stress stimulation, by the immunocyte after Fiber differentiation for the first time in contain inducing cytokine and lipopolysaccharides
Culture medium in cultivate 10.0h~12.0h；
Continue Fiber differentiation, by the immunocyte after lipopolysaccharides stress stimulation in the culture medium containing inducing cytokine
Continue Fiber differentiation 5d~13d, collects culture supernatant；
Dioxygen water process, by the immunocyte after continuation Fiber differentiation in the training containing inducing cytokine and hydrogen peroxide
It supports and handles 0.5h~1.0h in base；
Heat stress impact culture, by hydrogen peroxide treated immunocyte in the culture medium containing inducing cytokine in
Heat stress impact culture 4.0h~5.0h, collects culture supernatant under 38 DEG C~40 DEG C of cultivation temperature.
The inducing cytokine includes interleukins IL-2, interleukins IL-1a in one of the embodiments,
And interferon.
Above-mentioned skin injury reparative factor composition, on the one hand by being secreted using the culture in immunocyte incubation
A variety of growth factors, protease and the micromolecule polypeptide isoreactivity substance contained in object and immunocyte lysate, thus
The composition can be applied in skin repair, that is, be able to achieve the purpose for realizing skin nursing, reparation by cell therapy；Separately
On the one hand, and stem cell, immunocyte clinical research, the culture supernatant in production process can be made to be fully used, kept away
Exempting from these culture supernatants as biologic garbage, there are potential hazards to natural environment and human health, also to wherein active constituent
It develops and utilizes, reduces the cost of cell therapy.
Detailed description of the invention
In order to illustrate the technical solutions in the embodiments of the present application or in the prior art more clearly, below will be to institute in embodiment
Attached drawing to be used is needed to be briefly described, it should be apparent that, the accompanying drawings in the following description is only one recorded in the present invention
A little embodiments are also possible to obtain other drawings based on these drawings for those of ordinary skill in the art.
Fig. 1 is the micro- sem observation photo of 1 primary immune cells of the embodiment of the present invention；
Fig. 2 is the micro- sem observation photo that the embodiment of the present invention 1 passes on immunocyte after Fiber differentiation step for the first time；
Fig. 3 is the micro- sem observation photo of passage immunocyte after 1 lipopolysaccharides stress stimulation of the embodiment of the present invention；
Fig. 4 is the micro- sem observation photo of passage immunocyte after 1 dioxygen water treatment steps of the embodiment of the present invention；
Fig. 5 is the micro- sem observation photo that 1 heat stress of the embodiment of the present invention impacts passage immunocyte after incubation step；
Fig. 6 is immunocyte motility rate column diagram under the different cultivation stages of the embodiment of the present invention 1；
Fig. 7 is that the embodiment of the present invention 1 induces immunocyte culture secretion content histogram after stress stimulation culture；
Fig. 8 is the micro- sem observation photo of primary cell of the mescenchymal stem cell of the embodiment of the present invention 1；
Fig. 9 is the micro- sem observation photo of passage cell of the mescenchymal stem cell of the embodiment of the present invention 1；
Figure 10 is the mescenchymal stem cell culture secretion content histogram of the embodiment of the present invention 1；
Figure 11 is that 1 immunocyte of the embodiment of the present invention induces stress stimulation culture different phase and mescenchymal stem cell training
Support skin injury reparative factor total content histogram in supernatant.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment pair
The present invention is further described.It should be appreciated that the specific embodiments described herein are only used to explain the present invention, but simultaneously
It is not used in the restriction present invention.
The first aspect of the present invention provides a kind of skin injury reparative factor composition, the skin injury reparative factor group
It closes object and contains immunocyte culture secretion and immunocyte lysate.
Above-mentioned skin injury reparative factor composition, on the one hand by being secreted using the culture in immunocyte incubation
A variety of growth factors, protease and the micromolecule polypeptide isoreactivity substance contained in object and immunocyte lysate, thus
The composition can be applied in skin repair, that is, be able to achieve the purpose for realizing skin nursing, reparation by cell therapy；Separately
On the one hand, and stem cell, immunocyte clinical research, the culture supernatant in production process can be made to be fully used, kept away
Exempting from these culture supernatants as biologic garbage, there are potential hazards to natural environment and human health, also to wherein active constituent
It develops and utilizes, reduces the cost of cell therapy.
As a kind of optional embodiment, above-mentioned skin injury reparative factor composition also contains mescenchymal stem cell culture
Secretion.Mescenchymal stem cell (mesenchymal stem cells) is a kind of intracorporal seed repair cell.The present invention is logical
Cross the study found that mescenchymal stem cell in vivo can Proliferation, Differentiation to replace the Skin Cell of aging death, while mesenchyma is dry
Cell passes through from/paracrine approach secretion skin injury restoration bioactive molecule, such as fibronectin (Fibronectin) conduct
Wound healing, vascular endothelial growth factor (VEGF) is promoted to promote skin regeneration, fibrous bud Porcine HGF (bFGF)
The recovery of aging tissues, epithelial cell growth factor (EGF) is promoted to promote endothelial cell proliferation movement, hepatocyte growth factor
(HGF) active cell treats wound, transforming growth factor (TGF-β) restores impaired tissue and adjusts skin immune cells proliferation
Deng.The secretion of mescenchymal stem cell during the cultivation process not only has the function of reparation skin injury, further study show that,
It also has the regulation double biological characteristic to immunocyte.Such as a kind of important immunocyte-lymphocyte is in skin
It serves a dual purpose in aging and regeneration, appropriateness activation is the stimulus signal of Skin Cell Regeneration and Repair and over-activity then excites
Oxyradical, which quickly generates, causes a series of biological events such as apoptosis, death, lipid oxidation, DNA break.Benefit of the invention
Regulate and control feature with mildness of the external mescenchymal stem cell to lymphocyte, analog skin repair regenerates physiological status.This hair
It is bright extract derived mesenchymal stem cells in vitro culture, secrete in breeding have answering for skin injury reparation and immunocyte regulation
Active material is closed, and is applied in skin injury reparative factor composition, skin injury reparative factor can be significantly improved
Composition is used to repair the effect of skin injury.
Still optionally further, immunocyte culture secretion, immunocyte cracking in skin injury reparative factor composition
The protein content mass ratio of object and mescenchymal stem cell culture secretion is 2:(1~3): (1~3).It is further preferred that should
Immunocyte culture secretion, immunocyte lysate and mescenchymal stem cell culture in skin injury reparative factor composition
The protein content mass ratio of secretion is 1:1:1.
As a kind of optional embodiment, mescenchymal stem cell culture secretion is thin from the work that algebra is 3~10
The culture supernatant of born of the same parents' strain production mescenchymal stem cell.
As a kind of optional embodiment, source for mesenchymal stem cells is in the blood of Healthy People, fat, skin and new life
Youngster is with any one or a few tissue in tissue.Optionally, newborn may include bleeding of the umbilicus, umbilical cord, amnion, tire with tissue
Any one or a few in disk.
As a kind of optional embodiment, immunocyte derives from healthy human peripheral blood, marrow and umbilical cord blood
In any one or a few.
As a kind of optional embodiment, the total protein concentration of skin injury reparative factor composition is 2mg/ml~6mg/
ml。
Optionally, the skin injury reparative factor composition is as cosmetic material.When being used for the main of cosmetics
When ingredient, the verified cosmetics have fine effect to skin aging problem caused by ozone free radical.And institute of the present invention
It obtains skin injury reparative factor composition and the formula system biofacies content of mainstream cosmetics is good, can be used as original beauty
Active adding ingredient uses in anti-ageing cosmetics.
The second aspect of the present invention provides a kind of preparation method of above-mentioned skin injury reparative factor composition, the preparation
Method the following steps are included:
Immunocyte is subjected to induction stress stimulation culture, is collected in the culture induced in stress stimulation incubation
The culture supernatant is obtained the immunocyte culture secretion through separating treatment by clear liquid；
The immunocyte after stress stimulation culture will be induced to carry out 2~4 multigelations, separating treatment obtains described immune
Cell lysate；
Immunocyte culture secretion and the immunocyte lysate are mixed and obtain skin injury reparative factor group
Close object.
The preparation method of the skin injury reparative factor composition is oriented regulation by induction stress stimulation culture,
Active material needed for promoting immunocyte orientation secretion skin repair regeneration, to improve the yield of active material, make its
ROS removes functionally being further strengthened for component, and contains the skin injury reparative factor composition of preparation with height
Amount has the regenerated active material of skin repair, improves the using effect of the skin injury reparative factor composition.
It should be noted that the immunocyte in the present invention includes natural killer cells or is trained by induction stress stimulation
The killing cell supported and induced.
The present invention is by carrying out induction stress stimulation culture to immunocyte, and strengthened secretory cell growth factor, egg
White enzyme, micromolecule polypeptide composition etc. a variety of bioactive substance solution with ROS scavenging effect, can be used as cosmetic material into
The application in row beautifying and antisenility field.
Optionally, immunocyte derives from healthy human peripheral blood, marrow and umbilical cord blood in the preparation method
Any one or a few tissue, it is single after removing red blood cell and blood platelet in the tissue such as peripheral blood, marrow or Cord blood
Nucleus carries out further induction stress stimulation culture as raw material.
Specifically, mononuclearcell includes lymphocyte and monocyte.
It optionally, is 37 DEG C, 5% by the culture environment that immunocyte carries out induction stress stimulation culture in the preparation method
CO2, saturated humidity, incubation time be 15d~25d.Still optionally further, by immunocyte in the training containing inducing cytokine
It supports in base and carries out induced medium, wherein culture medium is lymphocyte serum.
As a kind of optional embodiment, immunocyte is carried out to induce stress stimulation culture, the collection induction stress
Stimulate the culture supernatant in incubation, comprising the following steps:
Fiber differentiation, by the immunocyte of collection be inoculated in Fiber differentiation 4d in the culture medium containing inducing cytokine~
12d collects culture supernatant.
Lipopolysaccharides stress stimulation, by the immunocyte after Fiber differentiation for the first time in contain inducing cytokine and lipopolysaccharides
Culture medium in cultivate 10.0h~12.0h.
Continue Fiber differentiation, by the immunocyte after lipopolysaccharides stress stimulation in the culture medium containing inducing cytokine
Continue Fiber differentiation 5d~13d, collects culture supernatant.
Hydrogen peroxide treatment will continue immunocyte after Fiber differentiation in containing inducing cytokine and hydrogen peroxide
0.5h~1.0h is handled in culture medium.
Heat stress impact culture, by hydrogen peroxide treated immunocyte in the culture medium containing inducing cytokine in
Heat stress impact culture 4.0h~5.0h, collects culture supernatant under 38 DEG C~40 DEG C of cultivation temperature.
Fiber differentiation is carried out in the culture medium containing inducing cytokine by above-mentioned, and by carrying out in different phase
Different degrees of lipopolysaccharides stress stimulation, dioxygen water process and heat stress impacts three kinds of stress stimulation modes, can make to be immunized
Cell is oriented secretion, its secretion is promoted to have the active material of skin injury repair.
Optionally, in Fiber differentiation step for the first time, the inoculum density of immunocyte is 5 × 106A/ml~1 × 107A/
Ml, and during Fiber differentiation for the first time, half amount is carried out to immunocyte according to immune cell growth situation and changes liquid and passage,
Half amount changes in liquid and needs to add full dose inducing cytokine simultaneously, and the content of inducing cytokine in culture medium is made to keep permanent substantially
It is fixed.The Fiber differentiation time is preferably 4d~12d for the first time, more preferably 6d, at the lipopolysaccharides stress stimulation that hereafter carries out, hydrogen peroxide
Immunocyte is in logarithmic growth phase when reason and heat stress impact culture, immunocyte can be made further to activate.
The culture supernatant collected in Fiber differentiation step for the first time can directly carry out separating treatment, can also save in -20 DEG C
It is spare.
Optionally, in lipopolysaccharides stress stimulation step, final concentration of 80ng/ml~120ng/ of lipopolysaccharides in culture medium
ml；It is highly preferred that in culture medium lipopolysaccharides final concentration of 100ng/ml.
Optionally, in continuing Fiber differentiation step, by the immunocyte after lipopolysaccharides stress stimulation in thin containing induction
Continuing the incubation time of Fiber differentiation in the culture medium of intracellular cytokine is preferably 7d~10d, more preferably 8d.Continue Fiber differentiation step
The culture supernatant collected in rapid can directly carry out separating treatment, can also save backup in -20 DEG C.
Optionally, in dioxygen water treatment steps, final concentration of 10umol/L~30umol/L of hydrogen peroxide in culture medium,
It is highly preferred that in culture medium hydrogen peroxide final concentration of 20umol/L.
Preferably, in heat stress impact incubation step, cultivation temperature is 39 DEG C., heat stress, which impacts in incubation step, to be collected
Culture supernatant can directly carry out separating treatment, can also be saved backup in -20 DEG C.
As a kind of optional embodiment, inducing cytokine include interleukins IL-2, interleukins IL-1a with
And interferon.Still optionally further, interleukins IL-2, interleukins IL-1a and interferon be in the medium
Concentration be respectively 800U/ml~1200U/ml.
As a kind of optional embodiment, the immunocyte after the induction stress stimulation culture is carried out 2 times~4 times instead
Multiple freeze thawing, separating treatment obtain the immunocyte lysate, comprising the following steps:
Immunocyte after induction stress stimulation culture is placed in cryopreservation tube with physiological saline resuspension；By above-mentioned cryopreservation tube,
It is placed in liquid nitrogen and freezes 1min~3min, take out recovery 30s~90s in 37 DEG C of water-baths rapidly afterwards；Repeat the above steps 2~4
It is secondary, it preferably repeats the above steps 3 times；Cell suspension after multigelation is centrifuged with the speed of 2500rpm~3500rpm
10min~20min is preferably centrifuged 15min with the speed of 3000rpm；Centrifuged supernatant is collected, which directly carries out
Separating treatment can also be saved backup in -20 DEG C.
In the present invention, the immunocyte after stress stimulation culture will be induced to pass through multigelation, cracks immunocyte,
The active material in immunocyte is discharged, and then therefrom extracts active material, nitroblue tetrazolium photochemical reduction method, ultraviolet suction can be passed through
Receipts method and measurement substrate glutathione (GSH) speed that is oxidized measure the superoxide dismutase in immunocyte lysate
The vigor of enzyme (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px).
As a kind of optional embodiment, the mescenchymal stem cell culture contained in skin injury reparative factor composition divides
The preparation method of secretion the following steps are included:
Directed differentiation culture, by the primary mescenchymal stem cell of acquisition with 18000/cm2~22000/cm2Cell density
Directed differentiation in the mesenchymal stem cell serum-free culture medium containing 5%~10% concentration human blood platelets lysate is inoculated in train
It supports；
Secondary culture, when growth of mesenchymal stem cells to 70%~90% fusion, by 2500/cm2~3500/cm2It is close
Degree carries out secondary culture, and collection algebra is that cell strain production mescenchymal stem cell culture supernatant, the culture are made in 3 generations~10 foundries
Supernatant directly carries out separating treatment, can also save backup in -20 DEG C.
Optionally, mesenchymal stem cell serum-free culture medium is containing 5%~10% concentration human blood platelets lysate, without blood
The normal saline solution of clear additive.
Still optionally further, obtain the method for primary mescenchymal stem cell the following steps are included:
The tissue such as umbilical cord, amnion, placenta, fat of Healthy People is acquired, phosphate buffer (PBS) washes off the residual in blood vessel
Blood；
Separate and remove vascular tissue and haemocyte；
Residue tissue is shredded to about 1mm2~2mm2Tissue block moves in 0.05%~0.15% clostridiopetidase A II, and 37 DEG C disappear
Change 1.0h~3.0h, 5min~15min is centrifuged with the speed of 2000r/min~3000r/min；
0.20%~0.30% trypsin digestion 10min~30min of the lower sediment after being centrifuged is taken, with 2000r/
The speed of min~3000r/min is centrifuged 5min~15min；
Supernatant after abandoning or adopting centrifugation, the lower sediment after retaining centrifugation, is blown and beaten with PBS into suspension, 100 μm of strainer mistakes
Filter is centrifuged 5min~15min with the speed of 1800r/min~2200r/min, obtains mescenchymal stem cell.Optionally, it is filled between being somebody's turn to do
Matter stem cell is washed 2 times before inoculation with PBS.
Still optionally further, in the directed differentiation incubation step of mescenchymal stem cell, condition of culture is 37 DEG C, 5%CO2、
Saturated humidity environment.
Still optionally further, in the directed differentiation incubation step of mescenchymal stem cell, directed differentiation culture for 24 hours~36h after
Non- attached cell is removed, culture medium is replaced, hereafter every 3d~4d changes fresh culture, until working as growth of mesenchymal stem cells extremely
70%~90% fusion.
As a kind of optional embodiment, the immunocyte saved backup in -20 DEG C induces each step of stress stimulation culture
The centrifuged supernatant and mescenchymal stem cell secondary culture collected in the culture supernatant of middle collection, immunocyte multigelation
Culture supernatant is collected in step to carry out separating treatment by following methods respectively:
Be sub-packed in 50ml centrifuge tube with the every pipe of 40ml, at 4 DEG C, with the speed of 3500rpm centrifugation 15min, with
The speed centrifugation 10min of 4000rpm, 8min is centrifuged with the speed of 5000rpm, removing protein is gone to flocculate；
Supernatant is centrifuged to be filtered with 0.22um filter；
Supernatant is sub-packed in 50ml, in the super filter tube that molecular cut off is 3KD by the every pipe of 15ml/ after filtering,
At 4 DEG C, 10min~40min is centrifuged with the speed ultrafiltration of 3000r/min~4000r/min, is concentrated into final volume
1.5ml~2ml is added the physiological saline of 4 DEG C of pre-coolings, is supplemented to final volume 15ml；
3 above-mentioned ultrafiltration centrifugally operateds are carried out continuously, difference adaptive immune cell culture secretion 3KD concentrate is immunized
Cell lysate 3KD concentrate and mescenchymal stem cell culture secretion 3KD concentrate.
Culture supernatant to be collected in -20 DEG C of immunocyte saved backup induction each steps of stress stimulation culture below
It is further detailed for the separating treatment of liquid:
The culture supernatant of collection is sub-packed in 50ml centrifuge tube with the every pipe of 40ml, at 4 DEG C, with the speed of 3500rpm
Degree centrifugation 15min, it is centrifuged 8min with the speed centrifugation 10min of 4000rpm, with the speed of 5000rpm, removing protein is gone to flocculate；
Supernatant is centrifuged to be filtered with 0.22um filter；
Supernatant is sub-packed in 50ml, in the super filter tube that molecular cut off is 3KD by the every pipe of 15ml/ after filtering；
At 4 DEG C, 10min~40min is centrifuged with the speed ultrafiltration of 3000r/min~4000r/min, is concentrated into final volume
1.5ml~2ml；
The physiological saline of 4 DEG C of pre-coolings is added, is supplemented to final volume 15ml；
At 4 DEG C, 10min~40min is centrifuged with the speed ultrafiltration of 3000r/min~4000r/min, is concentrated into final volume
1.5ml~2ml；
The physiological saline of 4 DEG C of pre-coolings is added, is supplemented to final volume 15ml；
At 4 DEG C, 10min~40min is centrifuged with the speed ultrafiltration of 3000r/min~4000r/min, is concentrated into final volume
1.5ml~2ml；
Merge each pipe 3KD concentrate, adaptive immune cell culture secretion 3KD concentrate.
As a kind of optional embodiment, immunocyte culture secretion 3KD concentrate, immunocyte lysate 3KD are dense
Contracting liquid and mescenchymal stem cell culture secretion 3KD concentrate are with following methods progress final concentration adjusting and protein stabilized body
System establishes:
It by above-mentioned immunocyte culture secretion 3KD concentrate, immunocyte lysate 3KD concentrate and fills respectively
Matter stem cell culture secretion 3KD concentrate is sub-packed in centrifuge tube by 10ml/ pipe；
At 4 DEG C, 10min~30min is centrifuged with the speed of 8000r/min~12000r/min, supernatant is collected, abandons
Albumen flocculation sedimentation；
It is always dense to adjust albumen with the normal saline dilution of the pre-cooling supernatant for the protein content for measuring the supernatant collected
Degree is 2mg/ml~6mg/ml；
With 0.22um filter membrane aseptic filtration, obtains the specific immunocyte culture secretion solution of total protein concentration, is immunized
Cell lysate solution and mescenchymal stem cell culture secretion solution.
Further, by the specific immunocyte culture secretion solution of total protein concentration, immunocyte lysate solution with
And mescenchymal stem cell culture secretion solution mixes in proportion, obtains skin injury reparative factor composition of the invention.
In the preparation method of skin injury reparative factor composition of the invention, the characteristic with following three aspect:
(1) the reinforcing secretion of endogenous skin injury reparative factor, by adjusting condition of in vitro culture, directional induction stress
It stimulates immunocyte to strengthen skin injury reparative factor --- the secretion of radicals scavenging albumen, collects immunocyte culture supernatant
And the active component in immunocyte, obtain endogenous skin injury reparative factor.
(2) the reinforcing regulation of Extraneous skin trauma reduction factors: there is height to lymphocyte according to mescenchymal stem cell
The ability of regulation and control of degree, the active component in separation and Extraction mescenchymal stem cell culture supernatant obtain having regulation to lymphocyte
The Extraneous skin trauma reduction factors of ability；
(3) the comprehensive synergistic effect of skin injury reparative factor composition function: skin injury reparative factor composition
In be not only rich in leukocyte activity oxygen radical removing component, also include stem cell skin regeneration activation factor, and it is exogenous
Skin injury reparative factor is to having synergistic effect, the composition and other cosmetics between endogenous skin injury reparative factor
Material combination provides effective solution for skin oxidative aging problem.
Embodiment 1
The acquisition of high biological safety source of people immunocyte and Fiber differentiation for the first time:
The clinical testing product standard for pressing immunocyte carries out the external extensive expansion of immunocyte among the workshop GMP
Increase, obtains cell culture supernatant, specifically includes the following steps:
Bleeding of the umbilicus, the peripheral blood 50ml or more of Healthy People are acquired, separated plasma is continued to employ；
Remaining cell employment lymphocyte separation medium density gradient centrifugation is separated, it is thin to collect the single core of tunica albuginea layer respectively
Born of the same parents；
After the cleaning twice of tunica albuginea layer, adjustment cell density is 5 × 106~1 × 107A/ml, is inoculated in containing inducing cell
The content of factor IL-2, IL-1a and interferon is respectively in the lymphocyte serum of 1000U/ml；In 37
DEG C, cultivate cell in the environment of 5%CO2, saturated humidity, half amount is carried out to cell according to cell growth status and changes liquid and passage,
3 kinds of inducing cytokines of full dose, Fiber differentiation immune cells are added simultaneously；In passage step, culture supernatant is collected simultaneously
It is saved backup in -20 DEG C.
In the micro- sem observation photo and passage step of the tunica albuginea layer mononuclearcell of collection --- primary immune cells
The micro- sem observation photo difference of immunocyte is as depicted in figs. 1 and 2.
Induce stress stimulation activated immune cell and regulation immunocyte secretion:
When culture was to the 7th day, it is respectively in the content containing inducing cytokine IL-2, IL-1a and interferon
In the lymphocyte serum of 1000U/ml, addition lipopolysaccharides (LPS) to final concentration of 100ng/ml, in 37 DEG C, 5%
CO2, lipopolysaccharides stress stimulation Fiber differentiation immunocyte 10.0h~12.0h is carried out in the environment of saturated humidity；Lipopolysaccharides stress
The micro- sem observation photo that immunocyte is passed on after stimulation is as shown in Figure 3；
The culture medium containing LPS is removed, the content containing inducing cytokine IL-2, IL-1a and interferon is added
The respectively lymphocyte serum of 1000U/ml continues at 37 DEG C, 5%CO2, induces training in the environment of saturated humidity
It supports immunocyte 8 days, collects culture supernatant；
When culture was to the 15th day, it is respectively in the content containing inducing cytokine IL-2, IL-1a and interferon
In the lymphocyte serum of 1000U/ml, H is added2O2To final concentration of 20umol/L, in 37 DEG C, 5%CO2, saturation
0.5-1.0h is handled in the environment of humidity；Micro- sem observation photo such as Fig. 4 institute of immunocyte is passed on after dioxygen water treatment steps
Show；
It will be through LPS and H2O2Immunocyte after stress stimulation culture in logarithmic growth phase is transferred to 39 DEG C, 5%CO2It is full
In the environment of humidity, heat stress impact culture 4.0h~5.0h；Heat stress passes on the aobvious of immunocyte after impacting incubation step
It is as shown in Figure 5 that micro mirror observes photo.
After culture, collects culture supernatant and saved backup in -20 DEG C.
Immunocyte motility rate figure under the different cultivation stages of the embodiment of the present invention 1 is as shown in fig. 6, it can be seen from the figure that former
Each stage of culture is impacted in Fiber differentiation for the first time, lipopolysaccharides stress stimulation, dioxygen water process, heat stress for immunocyte,
Maintain higher Cell viability.Immunocyte is after inducing stress stimulation culture, and immunocyte culture secretion content is such as
Shown in Fig. 7.It can be seen from figure 7 that containing considerable transforming growth factor (TGF-β), γ in immunocyte culture secretion
Interferon (IFN-γ), stem cell factor (SCF), fibrous bud Porcine HGF (bFGF) and a collagen type.
The freezing-thawing and cracking of immunocyte is handled:
Immunocyte after collecting induction stress stimulation culture, is resuspended with physiological saline, is placed in 5ml cryopreservation tube；
It by cryopreservation tube containing cell, is placed in liquid nitrogen and freezes 2min, take out the recovery 1min in 37 DEG C of water-baths rapidly afterwards,
It repeats above-mentioned steps 2 times；
Cell cracking suspension after multigelation merges, and 15min is centrifuged with the speed of 3000rpm, after collecting cell cracking
Supernatant, and saved backup in -20 DEG C.
By nitroblue tetrazolium photochemical reduction method, ultraviolet absorption method, measurement substrate glutathione (GSH) speed that is oxidized come
The measurement induction superoxide dismutase (SOD) of the forward and backward immunocyte lysate of stress stimulation culture, catalase (CAT),
The variation of glutathione peroxidase (GSH-Px) vigor, measurement result are as shown in table 1.
Superoxide dismutase (SOD), the hydrogen peroxide of the induction forward and backward immunocyte lysate of stress stimulation culture of table 1
Enzyme (CAT), glutathione peroxidase (GSH-Px) vigor
As it can be seen from table 1 immunocyte, after stress stimulation culture of the invention, immunocyte lysate surpasses
Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) vigor are obviously improved.
The culture of high biological safety source of people mescenchymal stem cell
By the clinical testing product standard of mescenchymal stem cell, the external of mescenchymal stem cell is carried out among the workshop GMP
Extensive amplification, obtains cell culture supernatant, specifically includes following steps；
The tissue such as umbilical cord, amnion, placenta, fat of Healthy People is acquired, phosphate buffer (PBS) washes off the residual in blood vessel
Blood；
Vascular tissue and haemocyte are separated and removed, residue tissue is shredded to about 1mm2~2mm2Tissue block moves to
In 0.1% clostridiopetidase A II, 37 DEG C of digestion 2h are centrifuged 10min with the speed of 2500r/min；
It takes lower sediment to use 0.25% trypsin digestion 20min again, 10min is centrifuged with the speed of 2500r/min；
Supernatant is abandoned, precipitating is retained, with PBS piping and druming at suspension, 100 μm of strainer filterings are centrifuged with the speed of 2000r/min
10min is obtained unicellular；
The unicellular of acquisition is washed 2 times with PBS, with 20000/cm2Cell density be inoculated in addition 5% concentration people's blood it is small
In the mesenchymal stem cell serum-free culture medium of plate lysate, 37 DEG C are set, 5%CO2It is cultivated under saturated humidity environment；It is removed after 2d
Non- attached cell replaces fresh culture, and every 3d~4d changes fresh culture, when cell grows to about 80% fusion, by 3,
000/cm2Density passed on, use algebra for 3 generations~10 foundries make cell strain produce mescenchymal stem cell culture supernatant
Liquid is collected culture supernatant and is saved backup in -20 DEG C.The primary cell of mescenchymal stem cell and passage through secondary culture are thin
The micro- sem observation photo difference of born of the same parents is as shown in Figure 8 and Figure 9.
The separating treatment of separate sources active factors and protease:
It will be in the culture that collected in the immunocyte induction each step of stress stimulation culture saved backup in above step
Culture is collected in the centrifuged supernatant and mescenchymal stem cell subculture step collected in clear liquid, immunocyte multigelation
Supernatant carries out separating treatment by following methods respectively:
Immunocyte induction stress stimulation culture supernatant, the centrifuged supernatant collected in immunocyte multigelation,
Collection culture supernatant is melted respectively according to type in mesenchymal stem cells subculture step summarizes；
The every pipe of 40ml is sub-packed in 50ml centrifuge tube, at 4 DEG C, with the speed centrifugation 15min of 3500rpm, with 4000rpm
Speed centrifugation 10min, 8min is centrifuged with the speed of 5000rpm, go removing protein to flocculate；
Supernatant is centrifuged to be filtered with 0.22um filter；
Supernatant is sub-packed in 50ml, in the super filter tube that molecular cut off is 3KD by the every pipe of 15ml/ after filtering；
At 4 DEG C, 30min is centrifuged with the speed of 3800r/min, is concentrated into final volume 1.5ml~2.0ml；
The intravenous injection physiological saline (0.9%NaCl) of 4 DEG C of pre-coolings is added, is supplemented to final volume 15ml；
At 4 DEG C, 30min is centrifuged with the speed of 3800r/min, is concentrated into final volume 1.5ml~2.0ml；
The intravenous injection physiological saline (0.9%NaCl) of 4 DEG C of pre-coolings is added, is supplemented to final volume 15ml；
At 4 DEG C, 30min is centrifuged with the speed of 3800r/min, final volume 1.5ml~2.0ml is concentrated into, merges each pipe
3KD concentrate adaptive immune cell culture secretion 3KD concentrate, immunocyte lysate 3KD concentrate and fills respectively
Matter stem cell cultivates secretion 3KD concentrate.
Final concentration adjusts and protein stabilized Establishing:
By three kinds of 3KD concentrates, it is sub-packed in centrifuge tube by 10ml/ pipe；
At 4 DEG C, 20min is centrifuged with the speed of 10000r/min, supernatant is collected, abandons albumen flocculation sedimentation；
The protein content for measuring the supernatant collected, should with intravenous injection physiological saline (0.9%NaCl) dilution of pre-cooling
Supernatant, adjustment total protein concentration are 4mg/ml；
It is molten to obtain the immunocyte culture secretion that total protein concentration is 4mg/ml respectively for 0.22um filter membrane aseptic filtration
Liquid, immunocyte lysate solution and mescenchymal stem cell culture secretion solution extract sample and carry out Quality Control.
High bioactivity skin injury reparative factor composition is mixed with:
Immunocyte culture secretion solution, immunocyte lysate solution and mescenchymal stem cell training after will be quantitative
Nutrient secretion solution is combined according to the ratio of volume ratio 1:1:1, obtains skin injury reparative factor composition of the invention.It should
Skin injury reparative factor composition carry out biochemistry detection, detection project include bacterium, fungi, mycoplasma, Chlamydia, concentration,
Endotoxin, specific detection quality standard are as follows:
Bacterium: negative；
Fungi: negative；
Mycoplasma: negative；
Chlamydia: negative；
Endotoxin: less than 0.25EU/ml；
Concentration: 4mg/ml.
Embodiment 2
The acquisition of high biological safety source of people immunocyte and Fiber differentiation for the first time:
The clinical testing product standard for pressing immunocyte carries out the external extensive expansion of immunocyte among the workshop GMP
Increase, obtains cell culture supernatant, specifically includes the following steps:
Bleeding of the umbilicus, the peripheral blood 50ml or more of Healthy People are acquired, separated plasma is continued to employ；
Remaining cell employment lymphocyte separation medium density gradient centrifugation is separated, it is thin to collect the single core of tunica albuginea layer respectively
Born of the same parents；
After the cleaning twice of tunica albuginea layer, adjustment cell density is 5 × 106~1 × 107A/ml, is inoculated in containing inducing cell
The content of factor IL-2, IL-1a and interferon is respectively in the lymphocyte serum of 800U/ml；In 37 DEG C,
5%CO2, cultivate cell in the environment of saturated humidity, half amount is carried out to cell according to cell growth status and changes liquid and passage, simultaneously
Add 3 kinds of inducing cytokines of full dose, Fiber differentiation immune cells；In passage step, culture supernatant is collected and in -20
It DEG C saves backup.
Induce stress stimulation activated immune cell and regulation immunocyte secretion:
When culture was to the 7th day, it is respectively in the content containing inducing cytokine IL-2, IL-1a and interferon
In the lymphocyte serum of 800U/ml, addition lipopolysaccharides (LPS) to final concentration of 80ng/ml, in 37 DEG C, 5%
CO2, lipopolysaccharides stress stimulation Fiber differentiation immunocyte 10.0h~12.0h is carried out in the environment of saturated humidity；
The culture medium containing LPS is removed, the content containing inducing cytokine IL-2, IL-1a and interferon is added
The respectively lymphocyte serum of 800U/ml continues at 37 DEG C, 5%CO2, induces training in the environment of saturated humidity
It supports immunocyte 4 days, collects culture supernatant；
When culture was to the 10th day, it is respectively in the content containing inducing cytokine IL-2, IL-1a and interferon
In the lymphocyte serum of 800U/ml, H is added2O2To final concentration of 10umol/L, in 37 DEG C, 5%CO2, saturation
0.5-1.0h is handled in the environment of humidity；Micro- sem observation photo such as Fig. 4 institute of immunocyte is passed on after dioxygen water treatment steps
Show；
It will be through LPS and H2O2Immunocyte after stress stimulation culture in logarithmic growth phase is transferred to 38 DEG C, 5%CO2It is full
In the environment of humidity, heat stress impact culture 4.0h~5.0h.After culture, collects culture supernatant and protected in -20 DEG C
It deposits spare.
The freezing-thawing and cracking of immunocyte is handled:
Immunocyte after collecting induction stress stimulation culture, is resuspended with physiological saline, is placed in 5ml cryopreservation tube；
It by cryopreservation tube containing cell, is placed in liquid nitrogen and freezes 2min, take out the recovery 1min in 37 DEG C of water-baths rapidly afterwards,
It repeats above-mentioned steps 1 time；
Cell cracking suspension after multigelation merges, and 10min is centrifuged with the speed of 2500rpm, after collecting cell cracking
Supernatant, and saved backup in -20 DEG C.
The culture of high biological safety source of people mescenchymal stem cell
By the clinical testing product standard of mescenchymal stem cell, the external of mescenchymal stem cell is carried out among the workshop GMP
Extensive amplification, obtains cell culture supernatant, specifically includes following steps；
The tissue such as umbilical cord, amnion, placenta, fat of Healthy People is acquired, phosphate buffer (PBS) washes off the residual in blood vessel
Blood；
Vascular tissue and haemocyte are separated and removed, residue tissue is shredded to about 1mm2~2mm2Tissue block moves to
In 0.05% clostridiopetidase A II, 37 DEG C of digestion 1h are centrifuged 5min with the speed of 2000r/min；
It takes lower sediment to use 0.20% trypsin digestion 10min again, 5min is centrifuged with the speed of 2000r/min；
Supernatant is abandoned, precipitating is retained, with PBS piping and druming at suspension, 100 μm of strainer filterings are centrifuged with the speed of 1800r/min
5min is obtained unicellular；
The unicellular of acquisition is washed 2 times with PBS, with 18000/cm2Cell density be inoculated in addition 5% concentration people's blood it is small
In the mesenchymal stem cell serum-free culture medium of plate lysate, 37 DEG C are set, 5%CO2It is cultivated under saturated humidity environment；It is removed after 2d
Non- attached cell replaces fresh culture, and every 3d~4d changes fresh culture, when cell grows to about 70% fusion, presses
2500/cm2Density passed on, use algebra for 3 generations~10 foundries make cell strain produce mescenchymal stem cell culture supernatant
Liquid is collected culture supernatant and is saved backup in -20 DEG C.
The separating treatment of separate sources active factors and protease:
It will be in the culture that collected in the immunocyte induction each step of stress stimulation culture saved backup in above step
Culture is collected in the centrifuged supernatant and mescenchymal stem cell subculture step collected in clear liquid, immunocyte multigelation
Supernatant carries out separating treatment by following methods respectively:
Immunocyte induction stress stimulation culture supernatant, the centrifuged supernatant collected in immunocyte multigelation,
Collection culture supernatant is melted respectively according to type in mesenchymal stem cells subculture step summarizes；
The every pipe of 40ml is sub-packed in 50ml centrifuge tube, at 4 DEG C, with the speed centrifugation 15min of 3500rpm, with 4000rpm
Speed centrifugation 10min, 8min is centrifuged with the speed of 5000rpm, go removing protein to flocculate；
Supernatant is centrifuged to be filtered with 0.22um filter；
Supernatant is sub-packed in 50ml, in the super filter tube that molecular cut off is 3KD by the every pipe of 15ml/ after filtering；
At 4 DEG C, 10min is centrifuged with the speed of 3000r/min, is concentrated into final volume 1.5ml~2.0ml；
The intravenous injection physiological saline (0.9%NaCl) of 4 DEG C of pre-coolings is added, is supplemented to final volume 15ml；
At 4 DEG C, 10min is centrifuged with the speed of 3000r/min, is concentrated into final volume 1.5ml~2.0ml；
The intravenous injection physiological saline (0.9%NaCl) of 4 DEG C of pre-coolings is added, is supplemented to final volume 15ml；
At 4 DEG C, 10min is centrifuged with the speed of 3000r/min, final volume 1.5ml~2.0ml is concentrated into, merges each pipe
3KD concentrate adaptive immune cell culture secretion 3KD concentrate, immunocyte lysate 3KD concentrate and fills respectively
Matter stem cell cultivates secretion 3KD concentrate.
Final concentration adjusts and protein stabilized Establishing:
By three kinds of 3KD concentrates, it is sub-packed in centrifuge tube by 10ml/ pipe；
At 4 DEG C, 10min is centrifuged with the speed of 8000r/min, supernatant is collected, abandons albumen flocculation sedimentation；
The protein content for measuring the supernatant collected, should with intravenous injection physiological saline (0.9%NaCl) dilution of pre-cooling
Supernatant, adjustment total protein concentration are 2mg/ml；
It is molten to obtain the immunocyte culture secretion that total protein concentration is 2mg/ml respectively for 0.22um filter membrane aseptic filtration
Liquid, immunocyte lysate solution and mescenchymal stem cell culture secretion solution extract sample and carry out Quality Control.
High bioactivity skin injury reparative factor composition is mixed with:
Immunocyte culture secretion solution, immunocyte lysate solution and mescenchymal stem cell training after will be quantitative
Nutrient secretion solution is combined according to the ratio of volume ratio 2:1:1, obtains skin injury reparative factor composition of the invention.It should
Skin injury reparative factor composition carry out biochemistry detection, detection project include bacterium, fungi, mycoplasma, Chlamydia, concentration,
Endotoxin, specific detection quality standard are as follows:
Bacterium: negative；
Fungi: negative；
Mycoplasma: negative；
Chlamydia: negative；
Endotoxin: less than 0.25EU/ml；
Concentration: 2mg/ml.
Embodiment 3
The acquisition of high biological safety source of people immunocyte and Fiber differentiation for the first time:
The clinical testing product standard for pressing immunocyte carries out the external extensive expansion of immunocyte among the workshop GMP
Increase, obtains cell culture supernatant, specifically includes the following steps:
Bleeding of the umbilicus, the peripheral blood 50ml or more of Healthy People are acquired, separated plasma is continued to employ；
Remaining cell employment lymphocyte separation medium density gradient centrifugation is separated, it is thin to collect the single core of tunica albuginea layer respectively
Born of the same parents；
After the cleaning twice of tunica albuginea layer, adjustment cell density is 5 × 106~1 × 107A/ml, is inoculated in containing inducing cell
The content of factor IL-2, IL-1a and interferon is respectively in the lymphocyte serum of 1200U/ml；In 37
DEG C, cultivate cell in the environment of 5%CO2, saturated humidity, half amount is carried out to cell according to cell growth status and changes liquid and passage,
3 kinds of inducing cytokines of full dose, Fiber differentiation immune cells are added simultaneously；In passage step, culture supernatant is collected simultaneously
It is saved backup in -20 DEG C.
Induce stress stimulation activated immune cell and regulation immunocyte secretion:
When culture was to the 12nd day, it is respectively in the content containing inducing cytokine IL-2, IL-1a and interferon
In the lymphocyte serum of 1200U/ml, addition lipopolysaccharides (LPS) to final concentration of 120ng/ml, in 37 DEG C, 5%
CO2, lipopolysaccharides stress stimulation Fiber differentiation immunocyte 10.0h~12.0h is carried out in the environment of saturated humidity；
The culture medium containing LPS is removed, the content containing inducing cytokine IL-2, IL-1a and interferon is added
The respectively lymphocyte serum of 1200U/ml continues at 37 DEG C, 5%CO2, induces training in the environment of saturated humidity
It supports immunocyte 12 days, collects culture supernatant；
When culture was to the 25th day, it is respectively in the content containing inducing cytokine IL-2, IL-1a and interferon
In the lymphocyte serum of 1200U/ml, H is added2O2To final concentration of 30umol/L, in 37 DEG C, 5%CO2, saturation
0.5-1.0h is handled in the environment of humidity；Micro- sem observation photo such as Fig. 4 institute of immunocyte is passed on after dioxygen water treatment steps
Show；
It will be through LPS and H2O2Immunocyte after stress stimulation culture in logarithmic growth phase is transferred to 40 DEG C, 5%CO2It is full
In the environment of humidity, heat stress impact culture 4.0h~5.0h.After culture, collects culture supernatant and protected in -20 DEG C
It deposits spare.
The freezing-thawing and cracking of immunocyte is handled:
Immunocyte after collecting induction stress stimulation culture, is resuspended with physiological saline, is placed in 5ml cryopreservation tube；
It by cryopreservation tube containing cell, is placed in liquid nitrogen and freezes 2min, take out the recovery 1min in 37 DEG C of water-baths rapidly afterwards,
It repeats the above steps 3 times；
Cell cracking suspension after multigelation merges, and 20min is centrifuged with the speed of 3500rpm, after collecting cell cracking
Supernatant, and saved backup in -20 DEG C.
The culture of high biological safety source of people mescenchymal stem cell
By the clinical testing product standard of mescenchymal stem cell, the external of mescenchymal stem cell is carried out among the workshop GMP
Extensive amplification, obtains cell culture supernatant, specifically includes following steps；
The tissue such as umbilical cord, amnion, placenta, fat of Healthy People is acquired, phosphate buffer (PBS) washes off the residual in blood vessel
Blood；
Vascular tissue and haemocyte are separated and removed, residue tissue is shredded to about 1mm2~2mm2Tissue block moves to
In 0.15% clostridiopetidase A II, 37 DEG C of digestion 2h are centrifuged 15min with the speed of 3000r/min；
It takes lower sediment to use 0.30% trypsin digestion 30min again, 15min is centrifuged with the speed of 3000r/min；
Supernatant is abandoned, precipitating is retained, with PBS piping and druming at suspension, 100 μm of strainer filterings are centrifuged with the speed of 2200r/min
15min is obtained unicellular；
The unicellular of acquisition is washed 2 times with PBS, with 22000/cm2Cell density be inoculated in addition 10% concentration people's blood
In the mesenchymal stem cell serum-free culture medium of platelet lysate, 37 DEG C are set, 5%CO2It is cultivated under saturated humidity environment；It is removed after 2d
Non- attached cell is gone, fresh culture is replaced, every 3d~4d changes fresh culture, when cell grows to about 90% fusion, presses
3500/cm2Density passed on, use algebra for 3 generations~10 foundries make cell strain produce mescenchymal stem cell culture supernatant
Liquid is collected culture supernatant and is saved backup in -20 DEG C.The primary cell of mescenchymal stem cell and passage through secondary culture are thin
The micro- sem observation photo difference of born of the same parents is as shown in Figure 8 and Figure 9.
The separating treatment of separate sources active factors and protease:
It will be in the culture that collected in the immunocyte induction each step of stress stimulation culture saved backup in above step
Culture is collected in the centrifuged supernatant and mescenchymal stem cell subculture step collected in clear liquid, immunocyte multigelation
Supernatant carries out separating treatment by following methods respectively:
Immunocyte induction stress stimulation culture supernatant, the centrifuged supernatant collected in immunocyte multigelation,
Collection culture supernatant is melted respectively according to type in mesenchymal stem cells subculture step summarizes；
The every pipe of 40ml is sub-packed in 50ml centrifuge tube, at 4 DEG C, with the speed centrifugation 35min of 4000rpm, with 4000rpm
Speed centrifugation 10min, 8min is centrifuged with the speed of 5000rpm, go removing protein to flocculate；
Supernatant is centrifuged to be filtered with 0.22um filter；
Supernatant is sub-packed in 50ml, in the super filter tube that molecular cut off is 3KD by the every pipe of 15ml/ after filtering；
At 4 DEG C, 40min is centrifuged with the speed of 4000r/min, is concentrated into final volume 1.5ml~2.0ml；
The intravenous injection physiological saline (0.9%NaCl) of 4 DEG C of pre-coolings is added, is supplemented to final volume 15ml；
At 4 DEG C, 40min is centrifuged with the speed of 4000r/min, is concentrated into final volume 1.5ml~2.0ml；
The intravenous injection physiological saline (0.9%NaCl) of 4 DEG C of pre-coolings is added, is supplemented to final volume 15ml；
At 4 DEG C, 40min is centrifuged with the speed of 4000r/min, final volume 1.5ml~2.0ml is concentrated into, merges each pipe
3KD concentrate adaptive immune cell culture secretion 3KD concentrate, immunocyte lysate 3KD concentrate and fills respectively
Matter stem cell cultivates secretion 3KD concentrate.
Final concentration adjusts and protein stabilized Establishing:
By three kinds of 3KD concentrates, it is sub-packed in centrifuge tube by 10ml/ pipe；
At 4 DEG C, 30min is centrifuged with the speed of 12000r/min, supernatant is collected, abandons albumen flocculation sedimentation；
The protein content for measuring the supernatant collected, should with intravenous injection physiological saline (0.9%NaCl) dilution of pre-cooling
Supernatant, adjustment total protein concentration are 6mg/ml；
It is molten to obtain the immunocyte culture secretion that total protein concentration is 6mg/ml respectively for 0.22um filter membrane aseptic filtration
Liquid, immunocyte lysate solution and mescenchymal stem cell culture secretion solution extract sample and carry out Quality Control.
High bioactivity skin injury reparative factor composition is mixed with:
Immunocyte culture secretion solution, immunocyte lysate solution and mescenchymal stem cell training after will be quantitative
Nutrient secretion solution is combined according to the ratio of volume ratio 1:3:3, obtains skin injury reparative factor composition of the invention.It should
Skin injury reparative factor composition carry out biochemistry detection, detection project include bacterium, fungi, mycoplasma, Chlamydia, concentration,
Endotoxin, specific detection quality standard are as follows:
Bacterium: negative；
Fungi: negative；
Mycoplasma: negative；
Chlamydia: negative；
Endotoxin: less than 0.25EU/ml；
Concentration: 6mg/ml.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of skin injury reparative factor composition, which is characterized in that the skin injury reparative factor composition, which contains, exempts from
Epidemic disease cell culture secretion and immunocyte lysate.
2. skin injury reparative factor composition according to claim 1, which is characterized in that the skin injury repair because
Sub-portfolio object also contains mescenchymal stem cell culture secretion.
3. skin injury reparative factor composition according to claim 2, which is characterized in that the skin injury repair because
The albumen of immunocyte culture secretion, immunocyte lysate and mescenchymal stem cell culture secretion contains in sub-portfolio object
Amount mass ratio is 2:(1~3): (1~3).
4. skin injury reparative factor composition according to claim 2, which is characterized in that the mescenchymal stem cell training
Nutrient secretion produces the culture supernatant of mescenchymal stem cell from the working cardial cell strain that algebra is 3~10.
5. skin injury reparative factor composition according to claim 2, which is characterized in that the immunocyte derives from
Any one or a few in healthy human peripheral blood, marrow and umbilical cord blood.
6. skin injury reparative factor composition according to claim 2, which is characterized in that the mescenchymal stem cell comes
Derived from the blood of Healthy People, fat, skin and newborn with any one or a few tissue in tissue.
7. according to claim 1 to skin injury reparative factor composition described in 6 any one, which is characterized in that the skin
The total protein concentration of skin trauma reduction factors composition is 2mg/ml~6mg/ml.
8. a kind of preparation method of skin injury reparative factor composition as claimed in any one of claims 1 to 7, feature
It is, the preparation method comprises the following steps:
Immunocyte is subjected to induction stress stimulation culture, collects the culture supernatant in the induction stress stimulation incubation
The culture supernatant is obtained the immunocyte culture secretion through separating treatment by liquid；
Immunocyte after the induction stress stimulation culture is subjected to 2 times~4 times multigelations, is exempted from described in separating treatment acquisition
Epidemic disease cell lysate；
The immunocyte culture secretion and the immunocyte lysate are mixed obtain the skin injury repair because
Sub-portfolio object.
9. preparation method according to claim 8, which is characterized in that by immunocyte carry out induction stress stimulation culture,
Collect the culture supernatant in the induction stress stimulation incubation, comprising the following steps:
Fiber differentiation for the first time, by the immunocyte of collection be inoculated in Fiber differentiation 4d in the culture medium containing inducing cytokine~
12d collects culture supernatant；
Lipopolysaccharides stress stimulation, by the immunocyte after Fiber differentiation for the first time in the training containing inducing cytokine and lipopolysaccharides
It supports and cultivates 10.0h~12.0h in base；
Continue Fiber differentiation, the immunocyte after lipopolysaccharides stress stimulation is continued in the culture medium containing inducing cytokine
Fiber differentiation 5d~13d collects culture supernatant；
Hydrogen peroxide treatment, by the immunocyte after continuation Fiber differentiation in the culture containing inducing cytokine and hydrogen peroxide
0.5h~1.0h is handled in base；
Heat stress impact culture, by hydrogen peroxide treated immunocyte in 38 DEG C in the culture medium containing inducing cytokine
Heat stress impact culture 4.0h~5.0h, collects culture supernatant under~40 DEG C of cultivation temperature.
10. preparation method according to claim 9, which is characterized in that the inducing cytokine includes interleukins
IL-2, interleukins IL-1a and interferon.
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