CN108066748A - New application of the excretion body in skin injury - Google Patents
New application of the excretion body in skin injury Download PDFInfo
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- CN108066748A CN108066748A CN201610983610.7A CN201610983610A CN108066748A CN 108066748 A CN108066748 A CN 108066748A CN 201610983610 A CN201610983610 A CN 201610983610A CN 108066748 A CN108066748 A CN 108066748A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
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Abstract
The invention discloses the purposes of kit in medicine preparation.For treating epidermis injury, the kit includes the drug：Excretion body and cell factor.The drug prepared using the kit of the present invention can effectively promote skin histology mesocuticle cell Proliferation, accelerate the reparation and healing of the damage of the epidermis surface of a wound.
Description
Technical field
The present invention relates to biomedicine fields.In particular it relates to new application of the excretion body in skin injury.
Background technology
Largest organ of the skin as human body has the function of barrier, protection, adjusts body temperature and sensation.Due to inflammation, burst
Defect of skin caused by the factors such as ulcer, scald, burn, it is serious can threat to life.
However, the therapy and drug currently for skin injury still have it is to be developed.
The content of the invention
It is contemplated that one of technical problem in the prior art is solved at least to a certain extent.
It should be noted that the present invention is the following discovery based on inventor and completes：
The clinic self flap of generally use or dermatoplastic method treatment Wound Defect at present, but it is new to face skin donor site
The problems such as wound defect and the deficiency in skin donor site source.In addition there are color, matter for the skin that graft closes on transplantation site
Ground, difference functionally.
The study found that stem cell can generate a kind of imitated vesicle structure for being referred to as excretion body.Excretion body diameter is between 30-
It is a kind of cell excretion vesica of small volume between 150nm, and with double-layer of lipoid membrane structure.Excretion body is containing there are many thin
Born of the same parents' specific proteins, lipid material and nucleic acid substances release signal molecule and can pass it to other cells, so as to change
Its physiological function.
In view of this, excretion body is acted on damaged skin by inventor, it is found that the epidermal cell proliferation for the skin that treated
Speed is substantially accelerated, and so as to accelerate the reparation of damaged skin, skin physiology function makes moderate progress.Further, inventor is unexpected
Ground is found, by excretion body and cell factor collective effect in damaged skin, the growth rate of epidermal cell and repairing for damaged skin
Complex velocity is significantly faster than that excretion body independent role, and physiological function is obviously improved.
For this purpose, the present invention proposes a kind of purposes of kit in medicine preparation.According to an embodiment of the invention, it is described
For treating epidermis injury, the kit includes drug：Excretion body and cell factor.
Excretion body is acted on impaired epidermis by inventor, it is found that the reparation speed of impaired epidermis is accelerated, physiological function is
Improve.Inventor is it was unexpectedly observed that by excretion body and cell factor collective effect in impaired epidermis, the reparation speed of impaired epidermis
It is significantly faster than that excretion body independent role, and physiological function is obviously improved.Inventor by further investigation find, excretion body can by by
Epidermal cell at damage epidermis is swallowed, and inclusion in excretion body, such as the substances such as protein, nucleic acid can be discharged into intracellular performance
Its regulating and controlling effect, meanwhile, stimulus signal is transferred into the cell by cell factor by being combined with cell surface receptor, can be with
The collaboration of excretion body repairs and reconstructs the regeneration microenvironment of damaged tissues, and regulating cell Proliferation, Differentiation simultaneously promotes regeneration.As a result,
Drug prepared by kit according to embodiments of the present invention can effectively promote surface of a wound epidermal cell proliferation, accelerate epidermis injury
Reparation and healing.
Known in those skilled in the art, one kind that " cell factor " refers to be synthesized, secreted by cell has raw extensively
The small molecular weight protein or polypeptides matter of object activity.
According to an embodiment of the invention, the purposes of the kit in medicine preparation can also have following supplementary technology
Feature：
According to an embodiment of the invention, the kit includes at least one following：Basic Fibroblast Growth Factor；And
Epidermal growth factor.Inventor obtains the more excellent combination of above-mentioned cell factor by many experiments, can be risen between each cell factor
To preferable synergistic effect, repair and reconstruct the regeneration microenvironment of damaged tissues, regulating cell Proliferation, Differentiation.As a result, according to this
Drug prepared by the kit of inventive embodiments can effectively promote surface of a wound epidermal cell proliferation, accelerate the reparation of epidermis injury
And healing.
According to an embodiment of the invention, the kit includes：The excretion body of 2~100 μ g/ml；The alkali of 10~50ng/ml
Property fibroblast growth factor；And the epidermal growth factor of 10~50ng/ml.In some embodiments, the kit includes：
The excretion body of 2~70 μ g/ml；The Basic Fibroblast Growth Factor of 10~40ng/ml；And the epidermal growth of 10~40ng/ml
The factor.In some embodiments, the kit includes：The excretion body of 2~50 μ g/ml；The basic fibroblast of 10~30ng/ml
Growth factor；And the epidermal growth factor of 10~30ng/ml.In some embodiments, the kit includes：2~20 μ g/
The excretion body of ml；The Basic Fibroblast Growth Factor of 10~20ng/ml；And the epidermal growth factor of 10~20ng/ml.Invention
People has found, enables to each cell factor and excretion body synergistic action effect preferable with this condition, can be quickly and efficiently
It repairs and reconstructs the regeneration microenvironment of damaged tissues, regulating cell Proliferation, Differentiation.Kit according to embodiments of the present invention as a result,
The drug of preparation can effectively promote surface of a wound epidermal cell proliferation, accelerate the reparation and healing of epidermis injury.
According to an embodiment of the invention, the kit includes：5 μ g/ml excretion bodies；The basic fibroblast life of 10ng/ml
The long factor；And the epidermal growth factor of 20ng/ml.Inventor has found, enables to each cell factor and outer with this condition
It secretes that body synergistic action effect is preferable, can quickly and efficiently repair and reconstruct the regeneration microenvironment of damaged tissues, regulating cell
Proliferation, Differentiation.Kit according to embodiments of the present invention can effectively promote surface of a wound epidermal cell proliferation as a result, accelerate epidermis
The reparation and healing of damage.
It should be noted that do not make considered critical for the acquisition source of excretion body.According to an embodiment of the invention, excretion
Body derives from mescenchymal stem cell.Inventor has found that the composition difference of inclusion is that different tissues or cell is caused in excretion body
Source excretion body possesses the main reason for various physiological functions.And then inventor has found by many experiments, mescenchymal stem cell comes
The excretion body in source is stronger for the repair ability of damaged tissues.Kit according to embodiments of the present invention can be effectively as a result,
Promote surface of a wound epidermal cell proliferation, accelerate the reparation and healing of epidermis injury.
According to an embodiment of the invention, the excretion body is obtained through the following steps：Mescenchymal stem cell is connect
Kind is cultivated to the mescenchymal stem cell in the Tissue Culture Dish containing cell growth medium and is in growth logarithmic phase
And cell confluency degree reaches more than 90%, cell growth medium is changed to excretion body collects culture medium, additive amount 6ml/
Ware when culture cell 48 is small under 37 DEG C, 5% carbon dioxide condition of culture, collects culture solution；By the culture solution in 4 DEG C,
300g is centrifuged 10 minutes, collects the first supernatant；By first supernatant in 4 DEG C, 2000g is centrifuged 10 minutes, collects second
Supernatant；By second supernatant in 4 DEG C, 10000g is centrifuged 30 minutes, collects the 3rd supernatant；By the 3rd supernatant
In 4 DEG C, when 110000g centrifugations 2 are small, the first sediment is collected；First sediment is resuspended with 35ml PBS buffer solution, and in
4 DEG C, when 110000g centrifugations 2 are small, collect the second sediment；And it is resuspended described second with 100~200 μ l PBS buffer solution and sinks
Starch, to obtain the excretion body.According to a particular embodiment of the invention, the excretion body collection culture medium contains α MEM trainings
Support base, 1% nonessential amino acid, 1% glutamine and 1% Insulin-Transferrin-selenium compound.Inventor unexpectedly sends out
Existing, obtained excretion body can be repaired preferably and reconstruct the regeneration microenvironment of damaged tissues with this condition, so as to effectively
Regulate and control multiplication and the differentiation of damaged tissues epidermal cell.Kit according to embodiments of the present invention can effectively promote as a result,
Surface of a wound epidermal cell proliferation accelerates the reparation and healing of epidermis injury.
According to an embodiment of the invention, the excretion body and cell factor are provided in the form of mixed solution.As a result,
In order to which excretion body and cell factor collaboration are repaired and reconstruct the regeneration microenvironment of damaged tissues, regulating cell Proliferation, Differentiation.By
This, kit according to embodiments of the present invention can effectively promote surface of a wound epidermal cell proliferation, accelerate the reparation of epidermis injury
And healing.
According to an embodiment of the invention, the solvent of the mixed solution is physiological saline.The solvent can effectively dissolve outer
Secrete body and cell factor, and to skin wound is non-stimulated or other side effects.
According to an embodiment of the invention, the excretion body and cell factor are sealed in spray bottle.It is convenient for medication as a result, and gives
Pharmaceutical quantities control.
The administration frequency and dosage of the drug of the present invention can be determined by multiple correlative factors, which includes will quilt
Disease type, administration route, patient age, gender, weight and the severity of disease for the treatment of and as active component
Drug type.According to some embodiments of the present invention, daily dose can be divided into 1 dose, 2 doses or multi-agent of suitable form, with entire
With 1 time, 2 times or multiple dosing in period, as long as reaching therapeutically effective amount.
Term " treatment " obtains desired pharmacology and/or physiologic effect for referring to.The effect is with regard to complete or partial
It can be preventative for prevention disease or its symptom and/or just partially or completely cure caused by disease and/or disease not
Can be curative for good action." treatment " used herein covers the disease of mammal, particularly people, including：(a)
In easy diseased prevention disease (such as preventing skin injury) or illness generation in still not yet making a definite diagnosis the individual fallen ill；(b) press down
Disease processed, such as retardance disease development；Or (c) alleviates disease, such as mitigate and the relevant symptom of disease.It is used herein " to control
Treat " cover and give drug or compound to individual to treat, cure, alleviate, improve, mitigate or inhibit any of the disease of individual
Medication including but not limited to gives the drug containing kit described herein to individual in need.
According to an embodiment of the invention, drug of the invention can be used in combination with conventional treatments and/or therapy or
Person can be used separately with conventional treatments and/or therapy.When the drug of the present invention is using the conjoint therapy with other medicines
During middle administration, they can sequentially or simultaneously give individual.Alternatively, the present invention drug can include the present invention kit,
Pharmaceutically acceptable carrier or pharmaceutically acceptable excipient and other medicines known in the art or preventive medicine
Combination.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
It obtains substantially or is recognized by the practice of the present invention.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination accompanying drawings below to embodiment
Apparent and strong understanding, wherein：
Fig. 1 shows the microphoto of the mescenchymal stem cell of 40 times of amplification according to an embodiment of the invention；
Fig. 2 shows mescenchymal stem cell surface marker protein expression analysis figure according to an embodiment of the invention；
Fig. 3 shows the transmission electron microscope photo of excretion body according to an embodiment of the invention；
Fig. 4 shows the micro- photograph of the epidermal cell system HaCaT cells of 100 times of amplification according to an embodiment of the invention
Piece；
Fig. 5 shows that thermal damage according to an embodiment of the invention amplifies the micro- of 40 times of HaCaT cells after handling
Photo；And
Fig. 6 shows that one embodiment amplifies HaCaT cell Ki67 fluorescence after 100 times of thermal damages repair according to the present invention and contaminates
The microphoto of color.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
It should be noted that term " first ", " second " are only used for description purpose, and it is not intended that instruction or hint phase
To importance or the implicit quantity for indicating indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the present invention, unless otherwise saying
Bright, " multiple " are meant that two or more.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it is carried out according to the described technology of document in the art or condition or according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
The acquisition and identification of 1 excretion body of embodiment
1st, the acquisition of excretion body
(1) mescenchymal stem cell is provided
By mescenchymal stem cell (form is as shown in Figure 1) be incubated at containing serum free medium (purchased from three favourable companies, in
State) 10cm Tissue Culture Dish in, after covering with plate, suction abandon culture medium and use PBS buffer solution rinse cell, then addition
TrypLe Express enzymic digestion cells, when cell rounding and when coming off from culture plate in time with the α containing 10% hyclone
MEM culture mediums terminate digestion, collect cell suspension into 15ml centrifuge tubes, 1200rpm is centrifuged 5 minutes.Suction uses PBS after abandoning supernatant
Once, and cell then is resuspended with 100 μ l/ pipes PBS buffer solution in buffer solution washing cell, adds in 2 μ l correspondences into each pipe respectively
Streaming antibody (HLA-CR, CD14, CD34, CD45, CD19, CD105, CD73, CD90), 4 DEG C are protected from light incubated cell 40 minutes,
And then cell is washed with PBS buffer solution twice, and most progress streaming point is resuspended into 300 μ l PBS buffer solution in cell at last
Analysis, the results are shown in Figure 2, wherein (A)~(H) be respectively HLA-CR, CD14, CD19, CD105, CD34, CD45, CD73,
CD90 antibody.It can determine that obtained cell is mescenchymal stem cell by table 1.
1 flow cytomery of table
| Cell phenotype | Detected value | Normal reference value |
| CD90 | 100% | >95% |
| CD73 | 99.95% | >95% |
| CD105 | 99.66% | >95% |
| CD34 | 0.90% | <2% |
| CD45 | 0.02% | <2% |
| CD14 | 0% | <2% |
| CD19 | 0.06% | <2% |
| HLA-DR | 0% | <2% |
(2) amplification of mescenchymal stem cell：
By mescenchymal stem cell (form is as shown in Figure 1) be incubated at containing serum free medium (purchased from three favourable companies, in
State) 10cm Tissue Culture Dish in.When cell growth and when reaching more than 90% degree of converging, suction is buffered after abandoning culture medium with PBS
Liquid rinse cell once, adds in TrypLe Express enzymic digestion cells, when cell rounding and while coming off from culture plate it is timely
Digestion is terminated with the α MEM culture mediums containing 10% hyclone, collects cell suspension into 15ml centrifuge tubes, 1200rpm centrifugations 5
Minute.Suction abandons after supernatant and cell is resuspended with fresh serum-free media, by 1:5 passage ratio is by cell inoculation to new culture
In ware, 7~10 generation of secondary culture.
(3) when the growth of mesenchymal stem cells in last generation to be amplified reaches more than 90% degree of converging, culture medium is abandoned in suction,
It is changed to excretion body and collects culture medium, additive amount is 6ml/ wares.Excretion body collects the formula of culture medium as α MEM (being purchased from Gibco)
+ 1% Insulin-Transferrin-selenium compound (being purchased from Gibco)+1% nonessential amino acid (being purchased from Gibco)+1% glutamy
Amine (is purchased from Gibco).Continue culture 48 it is small when after collect culture solution, carry out centrifugally operated in the steps below successively：4 DEG C, 300g
Centrifugation 10 minutes, collects supernatant；4 DEG C, 2000g is centrifuged 10 minutes, is collected supernatant；4 DEG C, 10000g is centrifuged 30 minutes, is received
Collect supernatant；It 4 DEG C, when 110000g centrifugations 2 are small, collect sediment and is resuspended with 35ml PBS buffer solution and precipitated；4 DEG C,
When 110000g centrifugations 2 are small, collect sediment and be resuspended with 100~200 μ l PBS buffer solution and precipitated, to obtain excretion body.
2nd, the identification of excretion body
Using the morphological appearance of excretion body of the transmission electron microscope observing after fixation and dyeing, amplify at 15000-25000 times
Sample is observed under multiplying power, the results are shown in Figure 3.Excretion is seen in vitro in the central disc of nick or the spheroid shape of one-sided notch,
Diameter is between 50~150nm.
2 excretion body of embodiment is used to promote the research of epidermal cell proliferation with cell factor
1st, cultured epidermal cell：
Epidermal cell system HaCaT cells (form is as shown in Figure 4) is incubated in the MEM culture mediums containing 10% hyclone,
When cell growth is converged and reaches more than 90%, culture medium is abandoned in suction, and containing for preheating is once added in afterwards with PBS buffer solution rinse cell
0.03%EDTANa20.25% trypsase, digest 10 minutes, used when cell rounding and while departing from culture dish bottom containing 10%
The MEM culture mediums of hyclone terminate digestion, collect cell suspension into centrifuge tube, and 1200rpm is centrifuged 5 minutes, and supernatant is abandoned in suction
Cell is resuspended with fresh culture medium afterwards, cell is added in 96 orifice plates by the inoculum concentration of 3000 cells/wells after cell count and is trained
Support 8 it is small when, HaCaT cells are completely adherent.
2nd, HaCaT cells thermal damage processing and culture：
Orifice plate containing adherent HaCaT cells completely is placed in 45 DEG C of incubators and handles that (cellular morphology is such as 90 minutes
Shown in Fig. 5), it then inhales and abandons culture medium, and add in 0.15mL by following group and handle culture medium accordingly, in 37 DEG C, 5% dioxy
Change carbon incubator in continue culture 24 it is small when.
Processing group
A. control group (+10% hyclone of MEM culture mediums)；
B. experimental group 1 (+10% hyclone+20ng/ml epidermal growth factor of MEM culture mediums)；
C. experimental group 2 (+10% hyclone+10ng/ml Basic Fibroblast Growth Factors of MEM culture mediums)；
D. experimental group 3 (1 obtained excretion body of+5 μ g/ml embodiments of+10% hyclone of MEM culture mediums)；
E. (+10% hyclone+20ng/ml epidermal growth factor+10ng/ml alkalescence of MEM culture mediums is into fibre for experimental group 4
Tie up growth factor)；
F. (+5 μ g/ml of+10% hyclone+10ng/ml Basic Fibroblast Growth Factors of MEM culture mediums are implemented experimental group 5
1 obtained excretion body of example)；
G. (1 institute of+5 μ g/ml embodiments of+10% hyclone+20ng/ml epidermal growth factor of MEM culture mediums of experimental group 6
Obtained excretion body)；
H. (+10% hyclone+10ng/ml Basic Fibroblast Growth Factor+20ng/ml tables of MEM culture mediums of experimental group 7
1 obtained excretion body of+5 μ g/ml embodiments of skin growth factor).
3rd, cellular immunofluorescence detects：
After culture, culture medium is abandoned in suction, with PBS buffer solution rinse once, is added in 4% paraformaldehyde solution room temperature and is fixed
Cell 15 minutes.With PBS buffer solution rinse cell three times, add in the PBS buffer solution of the X100 containing 0.25%Triton to cell into
Row wears film process 15 minutes (room temperature).With the PBS buffer solution (confining liquid) containing 10% donkey serum, 0.3%Triton X100 to thin
When born of the same parents' progress antigen blockade 1 is small (room temperature).1 is pressed with confining liquid:400 volume ratios dilution rabbit-anti people Ki67 antibody (CST companies), and
By obtained antibody diluent in incubated cell overnight (4 DEG C).Ki67 antibody diluents and thin with PBS buffer solution rinse are abandoned in suction
Born of the same parents three times, 5 minutes every time.1 is pressed with confining liquid:400 volume ratios dilution donkey anti-rabbit fluorescence secondary antibody Alexa Fluor 488IgG (H+
L) (Invitrogen companies), and with obtained antibody diluent room temperature be protected from light incubated cell 1 it is small when.Secondary antibody dilution is abandoned in suction
And with PBS buffer solution rinse cell three times, 5 minutes every time.1 is pressed with PBS buffer solution：300 volume ratios dilute DAPI, and use gained
To DAPI dilution room temperatures be protected from light incubated cell 3 minutes.Suction is placed on twice after abandoning dilution with PBS buffer solution rinse cell
The staining conditions of cell are observed under inverted fluorescence microscope, and three visuals field of random counter calculate the stained positive rate of Ki67.Dye
The results are shown in Figure 6 for color, wherein, (1), (3), (5), (7), (9), (11), (13) and (15) is respectively control group, experimental group 1
~7 Ki67 protein fluorescence coloration results, white point represent Ki67 protein positives；(2)、(4)、(6)、(8)、(10)、(12)、(14)
(16) be respectively control group, experimental group 1~7 nucleus dyestuff DAPI coloration result, white point represents nucleus.
Ki67 albumen is a kind of and relevant nuclear antigen of cell Proliferation, participates in regulating cell mitosis process, is expressed in
All cell cycle phases outside the G0 phases are the marker proteins for marking proliferative activity.HaCaT cells are after thermal damage is received
Cell state is remarkably decreased, and cell Proliferation is stagnated.Compared with the control group, epidermal growth factor or Basic Fibroblast Growth Factor with
And after excretion body is jointly processed by, Ki67 positive cells ratio significantly improves in nucleus, shows that more cells enter cell week
Phase is simultaneously in vegetative state, and after the processing of experimental group 4~7, the positive rate of Ki67 is above experimental group 1~3, by experiment
After 7 processing of group, the positive rate highest of Ki67 further demonstrates that excretion body, Basic Fibroblast Growth Factor and epidermal growth factor
Synergistic effect, can play preferably repairing effect.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment of the present invention or example.In the present specification, schematic expression of the above terms is not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It is combined in an appropriate manner in one or more embodiments or example.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the different embodiments described in this specification or example and different embodiments or exemplary feature
It closes and combines.
Although the embodiment of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, those of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (9)
1. a kind of purposes of kit in medicine preparation, which is characterized in that the drug is used to treat epidermis injury,
The kit includes：Excretion body and cell factor.
2. purposes according to claim 1, which is characterized in that the cell factor includes at least one following：
Basic Fibroblast Growth Factor；And
Epidermal growth factor.
3. purposes according to claim 2, which is characterized in that the kit includes：
The excretion body of 2~100 μ g/ml；
The Basic Fibroblast Growth Factor of 10~50ng/ml；And
The epidermal growth factor of 10~50ng/ml.
4. purposes according to claim 2, which is characterized in that the kit includes：
The excretion body of 2~50 μ g/ml；
The Basic Fibroblast Growth Factor of 10~30ng/ml；And
The epidermal growth factor of 10~30ng/ml.
5. purposes according to claim 2, which is characterized in that the kit includes：
5 μ g/ml excretion bodies；
The Basic Fibroblast Growth Factor of 10ng/ml；And
The epidermal growth factor of 20ng/ml.
6. purposes according to claim 1, which is characterized in that the excretion body is obtained through the following steps：
Mescenchymal stem cell is inoculated in the Tissue Culture Dish containing cell growth medium, is cultivated to the mesenchyma
Stem cell is in growth logarithmic phase and cell confluency degree reaches more than 90%, and cell growth medium is changed to excretion body collects
Culture medium, additive amount are 6ml/ wares, when culture cell 48 is small under 37 DEG C, 5% carbon dioxide condition of culture, collect culture solution；
By the culture solution in 4 DEG C, 300g is centrifuged 10 minutes, collects the first supernatant；
By first supernatant in 4 DEG C, 2000g is centrifuged 10 minutes, collects the second supernatant；
By second supernatant in 4 DEG C, 10000g is centrifuged 30 minutes, collects the 3rd supernatant；
By the 3rd supernatant in 4 DEG C, when 110000g centrifugations 2 are small, the first sediment is collected；
First sediment is resuspended with 35ml PBS buffer solution, and in 4 DEG C, when 110000g centrifugations 2 are small, collects the second precipitation
Object；And
Second sediment is resuspended with 100~200 μ l PBS buffer solution, to obtain the excretion body.
7. purposes according to claim 1, which is characterized in that the excretion body and cell factor are the shapes with mixed solution
What formula provided.
8. purposes according to claim 7, which is characterized in that the solvent of the mixed solution is physiological saline.
9. purposes according to claim 1, which is characterized in that the excretion body and cell factor are sealed in spray bottle.
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| CN201610983610.7A CN108066748A (en) | 2016-11-08 | 2016-11-08 | New application of the excretion body in skin injury |
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| CN201610983610.7A CN108066748A (en) | 2016-11-08 | 2016-11-08 | New application of the excretion body in skin injury |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109172859A (en) * | 2018-09-06 | 2019-01-11 | 上海长海医院 | Human stem cell source excretion bluk recombination exogenous hyaluronic acid is preparing the application in skin wound defect repair drug or material |
| CN110339147A (en) * | 2018-11-27 | 2019-10-18 | 浙江梵泊细胞工程有限公司 | A kind of composition for external application and application thereof |
| CN111110699A (en) * | 2020-04-01 | 2020-05-08 | 北京岳昊科技发展有限公司 | Application of combination of polypeptide conjugate and epidermal stem cell exosome in medicines and cosmetics |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104490931A (en) * | 2014-12-17 | 2015-04-08 | 江苏大学 | Application of exosome secreted by human umbilical cord mesenchymal stem cells to treating skin injury |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109172859A (en) * | 2018-09-06 | 2019-01-11 | 上海长海医院 | Human stem cell source excretion bluk recombination exogenous hyaluronic acid is preparing the application in skin wound defect repair drug or material |
| CN110339147A (en) * | 2018-11-27 | 2019-10-18 | 浙江梵泊细胞工程有限公司 | A kind of composition for external application and application thereof |
| CN111110699A (en) * | 2020-04-01 | 2020-05-08 | 北京岳昊科技发展有限公司 | Application of combination of polypeptide conjugate and epidermal stem cell exosome in medicines and cosmetics |
| CN111110699B (en) * | 2020-04-01 | 2020-07-17 | 广州市新纪元化妆品有限公司 | Application of combination of polypeptide conjugate and epidermal stem cell exosome in medicines and cosmetics |
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