CN108057131A - A kind of novel agent box containing stem cell - Google Patents
A kind of novel agent box containing stem cell Download PDFInfo
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- CN108057131A CN108057131A CN201610982785.6A CN201610982785A CN108057131A CN 108057131 A CN108057131 A CN 108057131A CN 201610982785 A CN201610982785 A CN 201610982785A CN 108057131 A CN108057131 A CN 108057131A
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- cell
- culture
- stem cell
- biological support
- excretion body
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
Abstract
The invention discloses kits.The kit includes：First reagent, first reagent include excretion body and cell factor；And second reagent, second reagent include biological support and stem cell.The kit of the present invention can effectively promote the reparation of skin injury and the healing of skin wound.
Description
Technical field
The present invention relates to biomedicine fields.In particular it relates to a kind of novel agent box containing stem cell.
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 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 reparation speed of damaged skin is accelerated, physiology
Function makes moderate progress.Further, inventor it was unexpectedly observed that by excretion body, cell factor and stem cell collective effect in by
Skin is damaged, the reparation speed of damaged skin 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 kit.According to an embodiment of the invention, the kit includes：First examination
Agent, first reagent include excretion body and cell factor；And second reagent, second reagent include biological support and dry
Cell.
Excretion body is acted on damaged skin by inventor, it is found that the reparation speed of damaged skin is accelerated, physiological function is
Improve.Inventor is it was unexpectedly observed that by excretion body and cell factor collective effect in damaged skin, the reparation speed of damaged skin
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
Epidermis and dermal cell at damage tissue are swallowed, and inclusion in excretion body, such as the substances such as protein, nucleic acid can be discharged into cell
Interior its regulating and controlling effect of performance, meanwhile, cell factor is transferred to cell by being combined with cell surface receptor, by stimulus signal
It is interior, it can be cooperateed with excretion body and repair and reconstruct the regeneration microenvironment of damaged tissues, regulating cell Proliferation, Differentiation simultaneously promotes tissue again
It is raw.In addition, the stem cell that kit provides can carry out scale amplification, epidermal cell, fibroblast, blood are efficiently divided into
Various types of skin tissue cells such as endothelial cell, and some promotive factors can be generated in stem cells hyperplasia atomization,
Be conducive to the reparation of damaged tissues.Excretion body, cell factor and stem cell three mutually act synergistically, and will accelerate damaged skin group
The reparation and healing knitted.In addition, biological support plays the role of fixed, support stem cell growth and promotes regeneration.As a result,
Kit according to embodiments of the present invention can effectively promote surface of a wound epidermal cell and dermal cell multiplication, accelerate skin 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 kit can also have following additional technical feature：
According to an embodiment of the invention, the cell factor includes at least one following：Basic Fibroblast Growth Factor；With
And epidermal growth factor.Inventor obtains the more excellent combination of above-mentioned cell factor by many experiments, meeting between each cell factor
Preferable synergistic effect is played, repair and reconstructs the regeneration microenvironment of damaged tissues, regulating cell Proliferation, Differentiation.Basis as a result,
The kit of the embodiment of the present invention can effectively promote surface of a wound epidermal cell and dermal cell multiplication, accelerate repairing for skin injury
Multiple 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；The epidermal growth factor of 10~50ng/ml；Biological support；And mescenchymal stem cell, it is filled between described
Matter stem cell is with 1 × 105~1 × 106A cell/cm2Density be carried on the biological support.In some embodiments, institute
Stating kit includes：The excretion body of 2~70 μ g/ml；The Basic Fibroblast Growth Factor of 10~40ng/ml；10~40ng/ml's
Epidermal growth factor；Biological support；And mescenchymal stem cell, the mescenchymal stem cell is with 1.5 × 105~8 × 105It is a thin
Born of the same parents/cm2Density be carried on the biological support.In some embodiments, the kit includes：2~50 μ g/ml's is outer
Secrete body；The Basic Fibroblast Growth Factor of 10~30ng/ml；The epidermal growth factor of 10~30ng/ml；Biological support；And
Mescenchymal stem cell, the mescenchymal stem cell is with 2 × 105~6 × 105A cell/cm2Density be carried on it is described biology branch
On frame.In some embodiments, the kit includes：The excretion body of 2~20 μ g/ml；The basic fibroblast of 10~20ng/ml
Growth factor；And the epidermal growth factor of 10~20ng/ml, biological support；And mescenchymal stem cell, the mesenchyma are done
Cell is with 2 × 105~6 × 105A cell/cm2Density be carried on the biological support.
Inventor find, enable to this condition each cell factor, excretion body and stem cell synergistic action effect compared with
It is good, it can quickly and efficiently repair and reconstruct the regeneration microenvironment of damaged tissues, regulating cell Proliferation, Differentiation.It is more than stem cell
Proportional load is stated on biological support, can either ensure the normal growth metabolism of stem cell, can also effectively act on it
Undamaged portion carries out Proliferation, Differentiation.If density is excessive, by Reverse transcriptase between cell, influence to be metabolized.It is raw if density is too small
Stem cell population is less on the unit area of object stent, and the cell of Proliferation, Differentiation can not meet demand.It is real according to the present invention as a result,
The kit for applying example can effectively promote surface of a wound epidermal cell and dermal cell multiplication, accelerates the reparation of skin injury and is cured
It closes.
It should be noted that according to an embodiment of the invention, by the way that mescenchymal stem cell is inoculated in and biological support phase
It in the culture medium of contact, and is incubated, so that mescenchymal stem cell is carried on biological support.Above-mentioned load density refers to incubate
The inoculum concentration of mescenchymal stem cell before educating.
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；The epidermal growth factor of 20ng/ml；Biological support；And mescenchymal stem cell, the mescenchymal stem cell with 5 ×
105A cell/cm2Density be carried on the biological support.Enable to this condition each cell factor, excretion body and
Stem cell synergistic action effect is preferable, can quickly and efficiently repair and reconstruct the regeneration microenvironment of damaged tissues, and regulation and control are thin
Born of the same parents' Proliferation, Differentiation.Kit according to embodiments of the present invention can effectively promote surface of a wound epidermal cell and dermal cell as a result,
Multiplication, accelerates the reparation and healing of skin injury.
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 and dermal cell multiplication, accelerate the reparation and healing of skin 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, dermal fibroblast and stem cell.As a result, according to embodiments of the present invention
Kit can effectively promote surface of a wound epidermal cell and dermal cell multiplication, accelerate the reparation and healing of skin 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 and dermal cell multiplication, accelerate skin
The reparation and healing of damage.
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 stem cell is universal stem cell,
The universal stem cell includes：Fat-derived stem cells, bone marrow derived stem cells or umbilical cord derived stem cells, it is excellent
Umbilical cord derived stem cells are selected, the biological support includes：Collagen, fibroin or chitosan, preferably collagen.
Inventor has found that extraction culture preserves universal stem cell, alloimmune originality is relatively low, can secrete with being easy to
A variety of repair cell factors and stromatin, Multidirectional Differentiation ability can efficiently be divided into epidermal cell, into fiber in injury region
Various types of skin tissue cells such as cell, vascular endothelial cell, so as to accelerate the reparation of damaged skin tissue and healing.It is excellent
Umbilical cord derived stem cells are selected, in order to obtain.
According to an embodiment of the invention, second reagent is obtained by comprising the following steps：It will be described dry thin
Born of the same parents are inoculated in the culture medium being in contact with the biological support, are cultivated, to obtain second reagent.
According to an embodiment of the invention, the biological support is obtained by comprising the following steps：By collagen sea
It is continuous to carry out precrosslink with glutaraldehyde solution, it obtains pre-paying co-product；The co-product of pre-paying with acetum is crosslinked, is obtained
To cross-linking products；The cross-linking products are freezed, and obtained lyophilized products are sterilized, obtain sterilizing product；With
And the sterilizing product is soaked in culture solution, the culture solution is replaced, until the pH value of the culture solution is 7.2, is discarded
The culture solution obtains the biological support.Inventor obtains the above-mentioned optimal method for preparing biological support by many experiments,
So as to obtain biological support.
According to an embodiment of the invention, second reagent is obtained by comprising the following steps：(1) by fresh oxtail
It is placed in 75% alcohol and impregnates 10 minutes, then rinsed repeatedly with sterile saline；(2) tail tendon is removed, removes sarolemma and fascia,
It is placed in sterilized petri dishes；(3) tail tendon is shredded, is immersed in 0.05mol/L acetums, when 4 DEG C of shakings 48 are small；(4) move into and crush
It is crushed in machine, places into 4 DEG C of refrigerators and carry out reexpansion；(5) 24 it is small when after, be filtered with 200 mesh screens, collect filtrate,
Obtain collagen solution；(6) in 6 orifice plates, per hole, collagen solution described in addition 5ml, makes it be laid in orifice plate；
(7) by the orifice plate when -30 DEG C of refrigerator pre-freezes 6 are small, then be dried in vacuo 18~24 it is small when, obtain collagen sponge；(8) by described in
Collagen sponge is placed in 10cm culture dishes, is added in the glutaraldehyde solution of 30mL 0.25% and is pre-payed into the culture dish
Connection 10 minutes, obtains pre-paying co-product；(9) addition 30mL0.05mol/L acetums in co-product are pre-payed to described, in 4 DEG C
Be crosslinked 24 it is small when, obtain cross-linking products；(10) freezed after the cross-linking products described in normal saline flushing, and the jelly that will be obtained
Dry product sterilizes, and obtains sterilizing product；(11) 10ml culture solutions are added in into the sterilizing product, is impregnated in 37 DEG C, often
It replaces the culture solution, until the pH value of the culture solution is 7.2, discards the culture solution, obtains the biological support；
(12) biological support is laid in culture dish, 37 DEG C of culture medium is added in into the culture dish, and will be equipped with described
The culture dish of culture medium and biological support is placed in containing 5%CO2Incubator in, stand 30 minutes；And (13) to the standing
According to 5 × 10 in culture medium afterwards5A cell/cm2Inoculum concentration inoculation stem cell, at 37 DEG C, containing 5%CO2Incubator in incubate
Educate 72 it is small when, collect the biological support for loading the stem cell, obtain second reagent.It is dry in culture medium as a result,
Cell can be carried on biological support.
Collagen is natural biologic material, is widely present in the connective tissue, skin and tendon of vertebrate.Collagen
The cell adhesion signal peptide sequence itself included, can specifically be identified with mediated cell, be conducive to sticking and growing for cell.And
And collagen is easy to process, easily obtains, and with the characteristics of antigenic low, catabolite will not trigger adverse reaction.
Inventor has found that collagen solution is freeze-dried, can obtain porous collagen sponge, changes the dense of collagen solution
The size of the controllable collagen sponge hole of degree.Substantial amounts of pore structure in collagen sponge scaffold is conducive to growing into and creating for cell
The infiltration of covering weave.It is degradable due to the poor mechanical property of collagen sponge, due supporting role is easily lost too early, therefore
Some modifications must be often done in application.For example, being chemically crosslinked using glutaraldehyde, can preferably solve mechanical strong
The problem of spending, the collagem membrane after crosslinking have certain tear-resistant ability.
Inventor is utilized ox tendon collagen as timbering material by many experiments, is because beef tendon collagen and people
Collagen homology highest, up to 98%；And the collagen extracted in tendon is mostly Type I collagen, is hardly deposited
In III Collagen Type VI, therefore, the qualitative difference of different batches collagen is less prone to.
According to an embodiment of the invention, first reagent is sealed in spray bottle.It is convenient for medication and dosage control as a result,
System.According to another embodiment of the invention, second reagent is sealed in containing CO2With the sealing container of serum free medium.
The basic metabolism and survival of stem cell are able to maintain that under this condition of culture.According to a particular embodiment of the invention, described first
Reagent is sealed in spray bottle, is placed in -70 DEG C of preservations, and the term of validity is 1 month.According to another specific embodiment of the application, institute
The second reagent is stated to be sealed in containing CO2In the hermetic bag of serum free medium, 4 DEG C of preservations are placed in, when the term of validity is 24 small.
In addition, the present invention proposes the purposes of kit described above in medicine preparation.Reality according to the present invention
Example is applied, the drug is used to treat defect of skin.The drug can effectively promote surface of a wound epidermal cell as a result, and corium is thin
Born of the same parents are proliferated, and accelerate the reparation and healing of skin injury.
According to an embodiment of the invention, the defect of skin is by diabetes, burn and scald, mechanicalness physical damnification or operation
Caused by wound.The drug can effectively promote surface of a wound epidermal cell and dermal cell multiplication as a result, accelerate skin injury
It repairs and heals.
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.
It will be appreciated to those of skill in the art that above for the described feature and advantage of kit, it is equally applicable
In the purposes of the kit in medicine preparation, details are not described herein.
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 statistics according to an embodiment of the invention
Figure；
Fig. 3 shows the transmission electron microscope photo of excretion body according to an embodiment of the invention；
Fig. 4 shows the epidermal cell microphoto of 10 times of amplification according to an embodiment of the invention；
Fig. 5 shows the micro- photograph of epidermal cell immunofluorescence dyeing of 10 times of amplification according to an embodiment of the invention
Piece；
Fig. 6 shows that Wound Contraction rate according to an embodiment of the invention changes over time curve；
Fig. 7 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. 8 shows that thermal damage according to an embodiment of the invention amplifies the aobvious of 40 times of HaCaT cells after handling
Micro- photo；
Fig. 9 shows HaCaT cell Ki67 fluorescence after 100 times of thermal damages' reparations of amplification according to an embodiment of the invention
The microphoto of dyeing；
Figure 10 shows the microscope of the fibroblast BJ cells of 100 times of amplification according to an embodiment of the invention
Photo；
Figure 11 shows 40 times of thermal damage of amplification according to an embodiment of the invention treated fibroblast
The microphotograph of BJ cells；And
Figure 12 shows that one embodiment amplifies BJ cell Ki67 fluorescent stainings after 100 times of thermal damages repair according to the present invention
Microphoto.
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
The forms of umbilical cord mesenchymal stem cells is used as shown in Figure 1, amplification is taken to grow mescenchymal stem cell in good condition
Cell dissociation is unicellular by TrypLEExpress digestive ferments；After then being washed with the PBS containing 1%BSA, be made containing 5 ×
106The single cell suspension of a umbilical cord mesenchymal stem cells, and respectively mark CD90, CD73, CD105, CD34, CD45, CD14,
CD19, HLA-CR streaming antibody (BD companies, the U.S.), often pipe additive amount is that 5 μ l room temperatures are protected from light incubation 20 minutes；With containing 1%BSA
PBS wash 2 times after, cell is resuspended to 400 μ l, and carry out FCM analysis, the results are shown in Figure 2, wherein (A)~
(H) it is respectively HLA-CR, CD14, CD19, CD105, CD34, CD45, CD73, CD90 antibody.It can determine by table 1 obtained
Cell is the high mescenchymal stem cell of purity.
1 flow cytomery of table
(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.
The preparation of 2 second reagent of embodiment
(1) fresh oxtail is placed in 75% alcohol and impregnated 10 minutes, then rinsed repeatedly with sterile saline；
(2) tail tendon is removed, sarolemma and fascia is removed, is placed in sterilized petri dishes；
(3) tail tendon is shredded, is immersed in 0.05mol/L acetums, when 4 DEG C of shakings 48 are small；
(4) move into pulverizer and crush, place into 4 DEG C of refrigerators and carry out reexpansion；
(5) 24 it is small when after, be filtered with 200 mesh screens, collect filtrate, obtain collagen solution；
(6) in 6 orifice plates, 5ml collagen solutions is added in per hole, it is made to be laid in orifice plate；
(7) by orifice plate when -30 DEG C of refrigerator pre-freezes 6 are small, then be dried in vacuo 18~24 it is small when, obtain collagen sponge；
(8) collagen sponge is placed in 10cm culture dishes, the glutaraldehyde that 30mL 0.25% is added in into the culture dish is molten
Precrosslink 10 minutes is carried out in liquid, obtains pre-paying co-product；
(9) to addition 30mL 0.05mol/L acetums in co-product are pre-payed, when 4 DEG C of crosslinkings 24 are small, it is crosslinked
Product；
(10) with being freezed after normal saline flushing cross-linking products, and obtained lyophilized products are sterilized, obtained
Sterilize product；
(11) 10ml culture solutions are added in into sterilizing product, in 37 DEG C of immersions, replaces culture solution daily, until culture solution
PH value is 7.2, discards culture solution, obtains biological support；
(12) biological support is laid in culture dish, 37 DEG C of culture medium is added in into culture dish, and culture will be housed
The culture dish of base and biological support is placed in containing 5%CO2Incubator in, stand 30 minutes；And
(13) into the culture medium after standing according to 5 × 105A cell/cm2Inoculum concentration inoculation stem cell, 37 DEG C, contain
5%CO2Incubator in be incubated 72 it is small when, collect load stem cell biological support, obtain the second reagent.
3 external evoked mescenchymal stem cell of embodiment breaks up to epidermal cell
1st, external evoked mescenchymal stem cell breaks up to epidermal cell
It is covered in advance on 6 orifice plates in matrigel (BD companies, the U.S.), repopulating cell density is 6 × 104A cell/cm2
Umbilical cord mesenchymal stem cells, 2ml is added to contain the low sugar DMEM culture mediums of 10% hyclone, at 37 DEG C, 5%CO per hole2Culture
It is cultivated in case to cell and reaches 50% fusion, used 2ml epidermis inductive condition culture mediums instead and carry out induction 7 days, every other day partly
Amount changes the liquid once.
Epidermis inductive condition culture medium prescription：(DMEM:DF12=1:1 (being purchased from Sigma), 20ng/ml epidermal growths
The factor (being purchased from R＆D), 15ng/ml basic fibroblast growth factors (being purchased from R＆D), 1% Insulin-Transferrin-selenium are multiple
Close object (being purchased from Sigma), 0.1 μM of dexamethasone (being purchased from Sigma), 100U/ml penicillin and 100 μ g/ml streptomysins.
2nd, morphological observation
After being induced using epidermis inductive condition culture medium, the variation of cellular morphology is observed under inverted microscope, such as
Shown in Fig. 4.Occurs more typical epithelioid cell's form in cell.
3rd, immunofluorescence dyeing
To epidermal cell the cell for breaking up 7 days is induced to be added in after PBS buffer solution rinse once mescenchymal stem cell
4% paraformaldehyde room temperature fixes 15 minutes, and the PBS buffer solution of the X100 containing 0.3%Triton carries out cell to wear film process,
Then cell is closed with the PBS buffer solution containing 10% lowlenthal serum, adds in 19 Dan Ke of mouse anti human Cytokeratin
Grand antibody (model C K19, Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) primary antibody working solution (1:50) 4 DEG C of overnight incubations, mark
The FITC fluorescence secondary antibody (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) of mountain sheep anti mouse, 4 DEG C are protected from light incubation 30min, are delayed with PBS
Fliud flushing fully washs cell three times, and in fluorescence microscopy Microscopic observation, the results are shown in Figure 5.Mescenchymal stem cell is lured to epidermal cell
It leads, surface marker CK19 specific to expression epidermal stem cells, the cell under microscope (100 ×) in 10 visuals field of random counter
Sum and the cell number broken up completely, show through statistical analysis, and cell differentiation efficiency is 59.6 ± 4.2%.As can be seen that
Under the conditions of external evoked, mescenchymal stem cell possesses stronger epidermal cell differentiation capability.
4 nude mice model of embodiment is tested
1st, animal packet and modelling
BALB/c nude mices 20 are provided, 20 grams or so of weight is randomly divided into 4 groups, and every group 5, concrete operations are as follows：
With 3.5% chloral hydrate (0.01ml/g) intraperitoneal injection of anesthesia, cut in back center and be deep to the complete of fascia
Layer skin makees the round defect surface of a wound of a diameter of 2cm.Every group is given different reagents, Petroleum gauze is then covered, using clinic
After bolus dressing carries out suture fixation, single cage raising.
Specific embodiment is as follows：
A groups：Without any processing；
B groups：1 obtained 5ug/ml excretions body of embodiment is sprayed on the surface of a wound and then adds collagem membrane and fixation；
C groups：2 obtained second reagent of embodiment is covered on the surface of a wound；
D groups：First 1 obtained excretion body of embodiment is sprayed on the surface of a wound, then covers the examination of embodiment 2 obtained second
Agent.
2nd, Wound healing rate calculates
Animal surface of a wound size is retouched in postoperative 7,14,21 days and is printed on transparent membrane, surface of a wound receipts are carried out using following equation
The calculating of shrinkage, and SAS statistical analysis softwares are utilized, comparison among groups uses variance analysis, compares examined using t two-by-two, P<
0.05 is statistically significant.
Wound Contraction rate (%)=100% × (surface of a wound area after original face area-healing)/original face area
The results are shown in Figure 6.According to the calculating of Wound Contraction rate, D groups (excretion body, stem cells-collagen film) after the transfer 7
It reaches the healing of (58 ± 0.12) %, B groups (simple excretion body) and C groups (stem cells-collagen film) be respectively (35 ±
0.25) % and (40 ± 0.19) %, wound healing effect is suitable, and A groups only reach the healing of (20 ± 0.27) %, the healing of D groups
Effect and B groups, C groups, there are notable difference (P ＜ 0.01) for A groups.Animal implant tests textured 14 days, advantage of the D groups on wound healing
Gradually weaken, to transplantation experiments 21 days, D groups healed completely, and the transplanting surface of a wound is smooth, and elastic good, no clear scar is formed；B groups and C
Group healing effect is only second to D groups.Show that stem cell acts synergistically with excretion body as a result, effectively accelerate the reparation of skin injury
And healing.
5 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 7) 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. 8), 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 9 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.
6 excretion body of embodiment is used for the research that dermal cell is promoted to be proliferated with cell factor
1st, cell culture：
Dermal cell system BJ cells (form is as shown in Figure 10) is incubated in the HDMEM culture mediums containing 10% hyclone,
When cell growth is converged and reaches more than 90%, culture medium is abandoned in suction, and preheating is once added in afterwards with PBS buffer solution rinse cell
TrypLE Express enzymes digest 2 minutes, with containing 10% hyclone when cell rounding and disengaging culture dish bottom
HDMEM culture mediums terminate digestion, collect cell suspension into centrifuge tube, and 1200rpm is centrifuged 5 minutes, and suction is abandoned after supernatant with fresh
HDMEM culture mediums containing 10% hyclone cell is resuspended, cell is added by the inoculum concentration of 3000 cells/wells after cell count
When into 96 orifice plates, culture 8 is small, BJ cells are completely adherent.
2nd, BJ cells thermal damage processing and culture：
Orifice plate containing completely adherent BJ cells is placed in 45 DEG C of incubators and handles 90 minutes (form such as Figure 11 institutes
Show), 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% carbon dioxide
Continue in incubator culture 24 it is small when.
Processing group
A. control group (+10% hyclone of HDMEM culture mediums)；
B. experimental group 1 (+10% hyclone+20ng/ml epidermal growth factor of HDMEM culture mediums)；
C. experimental group 2 (+10% hyclone+10ng/ml Basic Fibroblast Growth Factors of HDMEM culture mediums)；
D. experimental group 3 (1 obtained excretion body of+5 μ g/ml embodiments of+10% hyclone of HDMEM culture mediums)
E. experimental group 4 (+10% hyclone+20ng/ml epidermal growth factor+10ng/ml alkalescence of HDMEM culture mediums into
Fibroblast growth factor)；
F. (+5 μ g/ml of+10% hyclone+10ng/ml Basic Fibroblast Growth Factors of HDMEM culture mediums are real for experimental group 5
Apply 1 obtained excretion body of example)；
G. (+5 μ g/ml embodiments 1 of+10% hyclone+20ng/ml epidermal growth factor of HDMEM culture mediums of experimental group 6
Obtained excretion body)；
H. (+10% hyclone+10ng/ml Basic Fibroblast Growth Factors+20ng/ml of HDMEM culture mediums of experimental group 7
1 obtained excretion body of+5 μ g/ml embodiments of epidermal 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
Obtained dilution is stayed overnight in 4 DEG C of incubated cells.Suction abandon dilution and with PBS buffer solution rinse cell three times, 5 points every time
Clock.1 is pressed with confining liquid:400 volume ratios dilute donkey anti-rabbit fluorescence secondary antibody Alexa Fluor 488IgG (H+L) (Invitrogen
Company), and by obtained dilution room temperature be protected from light incubated cell 1 it is small when.Dilution and thin with PBS buffer solution rinse is abandoned in suction
Born of the same parents three times, 5 minutes every time.1 is pressed with PBS buffer solution:300 volume ratios dilute DAPI, and room temperature is protected from light incubated cell 3 minutes.Suction is abandoned
It is placed on the staining conditions that cell is observed under inverted fluorescence microscope after DAPI solution twice with PBS buffer solution rinse cell, and
Three visuals field of random counter calculate the stained positive rate of Ki67.Coloration result is as shown in figure 12, wherein, (1), (3), (5), (7),
(9), (11), (13) and (15) are respectively control group, the Ki67 protein fluorescence coloration results of experimental group 1~7, and white point represents Ki67
Protein positive；(2), (4), (6), (8), (10), (12), (14) and (16) are respectively control group, the nucleus of experimental group 1~7
The coloration result of dyestuff DAPI, white point represent nucleus.
Compared with the control group, after excretion body, epidermal growth factor or Basic Fibroblast Growth Factor processing, in nucleus
Ki67 positive cell ratios significantly improve, and show that more cells enter the cell cycle and in vegetative state, and pass through experiment
After 4~7 processing of group, the positive rate of Ki67 is above experimental group 1~3, after the processing of experimental group 7, the positive rate highest of Ki67,
It further demonstrates that excretion body, Basic Fibroblast Growth Factor and epidermal growth factor synergistic effect, can play and preferably repair
Effect.
The research of 7 first reagent of embodiment and the second reagent collective effect to wound healing
Nude mice model experiment is carried out according to the method for embodiment 4, wherein, processing group is：
A groups：Without any processing；
B groups：First reagent is sprayed on the surface of a wound, wherein the first reagent contains：10ng/ml Basic Fibroblast Growth Factors+
1 obtained excretion body of+5 μ g/ml embodiments of 20ng/ml epidermal growth factor；
C groups：2 obtained second reagent of embodiment is covered on the surface of a wound；
D groups：First reagent is sprayed on the surface of a wound, wherein the first reagent contains：10ng/ml Basic Fibroblast Growth Factors+
1 obtained excretion body of+5 μ g/ml embodiments of 20ng/ml epidermal growth factor, then covering embodiment 2 obtained second is tried
Agent.
The result shows that the Wound Contraction rate of D groups is significantly raised compared with other each groups, illustrate excretion body, cell factor, stem cell
Collective effect has the surface of a wound preferably reparation and regenerated effect, accelerates healing speed at damaged skin.
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.
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 (10)
1. a kind of kit, which is characterized in that including：
First reagent, first reagent include excretion body and cell factor；And
Second reagent, second reagent include biological support and stem cell.
2. kit 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. kit 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；
The epidermal growth factor of 10~50ng/ml；
Biological support；And
Mescenchymal stem cell, the mescenchymal stem cell is with 1 × 105~1 × 106A cell/cm2Density be carried on the life
On object stent,
Preferably, the kit includes：
The excretion body of 2~50 μ g/ml；
The Basic Fibroblast Growth Factor of 10~30ng/ml；
The epidermal growth factor of 10~30ng/ml；
Biological support；And
Mescenchymal stem cell, the mescenchymal stem cell is with 1.5 × 105~8 × 105A cell/cm2Density be carried on it is described
On biological support,
It is highly preferred that
The kit includes：
5 μ g/ml excretion bodies；
The Basic Fibroblast Growth Factor of 10ng/ml；
The epidermal growth factor of 20ng/ml；
Biological support；And
Mescenchymal stem cell, the mescenchymal stem cell is with 5 × 105A cell/cm2Density be carried on the biological support.
4. kit 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.
5. kit according to claim 1, which is characterized in that the excretion body and cell factor are with mixed solution
What form provided, it is preferable that the solvent of the mixed solution is physiological saline.
6. kit according to claim 1, which is characterized in that the stem cell is universal stem cell,
The universal stem cell includes：Fat-derived stem cells, bone marrow derived stem cells or umbilical cord derived stem cells, preferably navel
Band derived stem cells,
The biological support includes：Collagen, fibroin or chitosan, preferably collagen.
7. kit according to claim 1, which is characterized in that second reagent is as obtained by comprising the following steps
It arrives：
The stem cell is inoculated in the culture medium being in contact with the biological support, is cultivated, to obtain described
Two reagents.
8. kit according to claim 1, which is characterized in that the biological support is by comprising the following steps to obtain
's：
Collagen protein sponge and glutaraldehyde solution are subjected to precrosslink, obtain pre-paying co-product；
The co-product of pre-paying with acetum is crosslinked, obtains cross-linking products；
The cross-linking products are freezed, and obtained lyophilized products are sterilized, obtain sterilizing product；And
The sterilizing product is soaked in culture solution, the culture solution is replaced, until the pH value of the culture solution is 7.2, abandons
The culture solution is removed, obtains the biological support.
9. kit according to claim 1, which is characterized in that second reagent is by comprising the following steps to obtain
's：
(1) fresh oxtail is placed in 75% alcohol and impregnated 10 minutes, then rinsed repeatedly with sterile saline；
(2) tail tendon is removed, sarolemma and fascia is removed, is placed in sterilized petri dishes；
(3) tail tendon is shredded, is immersed in 0.05mol/L acetums, when 4 DEG C of shakings 48 are small；
(4) move into pulverizer and crush, place into 4 DEG C of refrigerators and carry out reexpansion；
(5) 24 it is small when after, be filtered with 200 mesh screens, collect filtrate, obtain collagen solution；
(6) in 6 orifice plates, per hole, collagen solution described in addition 5ml, makes it be laid in orifice plate；
(7) by the orifice plate when -30 DEG C of refrigerator pre-freezes 6 are small, then be dried in vacuo 18~24 it is small when, obtain collagen sponge；
(8) collagen sponge is placed in 10cm culture dishes, the glutaraldehyde that 30mL 0.25% is added in into the culture dish is molten
Precrosslink 10 minutes is carried out in liquid, obtains pre-paying co-product；
(9) addition 30mL 0.05mol/L acetums in co-product are pre-payed to described, when 4 DEG C of crosslinkings 24 are small, is crosslinked
Product；
(10) freezed after the cross-linking products described in normal saline flushing, and obtained lyophilized products are sterilized, obtained
Sterilize product；
(11) 10ml culture solutions are added in into the sterilizing product, in 37 DEG C of immersions, replaces the culture solution daily, until described
The pH value of culture solution is 7.2, discards the culture solution, obtains the biological support；
(12) biological support is laid in culture dish, 37 DEG C of culture medium is added in into the culture dish, and will be equipped with
The culture dish of the culture medium and biological support is placed in containing 5%CO2Incubator in, stand 30 minutes；And
(13) into the culture medium after the standing according to 5 × 105A cell/cm2Inoculum concentration inoculation stem cell, 37 DEG C, contain
5%CO2Incubator in be incubated 72 it is small when, collect the biological support for loading the stem cell, obtain second reagent.
10. kit according to claim 1, which is characterized in that
First reagent is sealed in spray bottle,
Second reagent is sealed in containing CO2With the sealing container of serum free medium.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109106977A (en) * | 2018-08-27 | 2019-01-01 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A kind of self-healing injection aquagel dressing and the preparation method and application thereof for Tissue of Diabetic Wound reparation |
| CN110693912A (en) * | 2019-11-18 | 2020-01-17 | 深圳市人民医院 | Application of stem cell exosome in preparation of product for promoting wound healing |
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| CN101291586A (en) * | 2005-09-02 | 2008-10-22 | 英特菲思生物技术公司 | A method for cell implantation |
| WO2016072821A1 (en) * | 2014-11-07 | 2016-05-12 | 한양대학교 에리카산학협력단 | Composition for differentiation induction of adipocyte containing stem cell-derived exosome, regeneration of adipose tissue, and skin whitening or wrinkle improvement |
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| CN1511593A (en) * | 2002-12-30 | 2004-07-14 | 中国人民解放军军事医学科学院放射医 | Artificial skin containing human bone marrow mesenchymal stem cell and its construction method |
| CN101291586A (en) * | 2005-09-02 | 2008-10-22 | 英特菲思生物技术公司 | A method for cell implantation |
| WO2016072821A1 (en) * | 2014-11-07 | 2016-05-12 | 한양대학교 에리카산학협력단 | Composition for differentiation induction of adipocyte containing stem cell-derived exosome, regeneration of adipose tissue, and skin whitening or wrinkle improvement |
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| CN109106977A (en) * | 2018-08-27 | 2019-01-01 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A kind of self-healing injection aquagel dressing and the preparation method and application thereof for Tissue of Diabetic Wound reparation |
| CN110693912A (en) * | 2019-11-18 | 2020-01-17 | 深圳市人民医院 | Application of stem cell exosome in preparation of product for promoting wound healing |
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Application publication date: 20180522 |