CN107349220A - A kind of preparation comprising fibroblast excretion body and application thereof - Google Patents
A kind of preparation comprising fibroblast excretion body and application thereof Download PDFInfo
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Abstract
The present invention, into fibroblastic method, Fibroblast cell-culture supernatant is collected after external evoked amplification, is prepared using low temperature ultrafiltration concentration method and obtains excretion body using UC MSC inductions.And using this excretion body as main effective ingredient, it is prepared into spray.Experiments verify that the spray containing fibroblast excretion body can effectively facilitate wound healing.
Description
Technical field
The present invention relates to fibroblast excretion body, its preparation method, and the preparation comprising fibrocyte excretion body and its
Purposes.
Background technology
Skin is the maximum organ of human body, accounts for the 16% of body weight, contains the blood circulation peace treaty of human body about 1/3
1/4 moisture, important defence defencive function is played, be first of the guard wire of human body truly.Normal skin tissue
Inside, the external world, which causes injury, is destroyed skin integrity in the presence of the factor, and the normal function of skin is damaged, so as to form skin
Wound.Slight wound is only limitted to skin epidermis, and slightly severe one has skin and hypodermis fracture, and wound occurs；Serious
Wound can have muscle, tendon, the fracture of nerve and fracture.And wound healing is exactly a function of repairing skin histology in body
And the complex process of integrality division.
In general, wound healing includes four-stage, it is that hemostasis, inflammation, propagation and overline are moulding respectively.These processes
The comprehensive regulation of various cell factors and growth factor is needed, wound is quickly healed within a certain period of time.But some because
It is prolonged refractory that element also results in wound, such as inflammation infection, diabetes foot disease.Also it is exactly that the low crowd of some immunity is for example old
Year, people was waited because contacting between immunocyte and Skin Cell is destroyed, caused no enough cell factor and growth factor
To promote skin fibroblasts fast breeding, secretion collagen to carry out healing of wound.
Fibroblast (fibroblast), also referred to as fibroblast, it is the main cellular of loose connective tissue, by
The mesenchymal cell (mesenchymal cell) of embryonic stage differentiates, and belongs to terminally differentiated cells.Fibroblast born of the same parents
Body is larger, and the weak basophilla of kytoplasm, karyon is larger oval, and the loose coloring of chromatin is shallow, and kernel is obvious.Under Electronic Speculum, its kytoplasm
It can be seen that abundant rough surfaced endoplasmic reticulum (RER), free ribosome and the Golgi complex of prosperity, show that it has synthesis and secretory protein
The function of matter.Fibroblast still synthesis and secretion collagen, elastomer, reticular fibre and organic substrate, thus
Played an important role in repair process after tissue damage.At present, people are mainly obtained by the culture of skin histology etc.
Human fibroblasts, this has certain injury to donor, limits the larger scale clinical application of human fibroblasts Related product.
Human umbilical cord mesenchymal stem cells (Umbilical Cord Mesenchymal Stem Cells, UC-MSC) refer to
A kind of versatile stem cell being present in neonatal umbilical cord tissue, it has higher differentiation potential, can entered to multiple directions
Row differentiation.UC-MSC cell content, multiplication capacity is strong, and immunogenicity is low, and has convenient material drawing, no ethics dispute etc.
Advantage, there is wide clinic in terms of the organizational projects such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium and cardiac muscle
Application prospect, therefore increasingly by the concern of research workers.
On the other hand, excretion body (exosome) is also known as allochthon, is found in the sheep red blood cell (SRBC) supernatant of in vitro culture earliest
It is that the size of cell active secretion is more homogeneous in liquid, a diameter of 40~100 nanometers, 1.10~1.18g/ml of density vesica
Sample corpusculum, the materials such as miRNA, the mRNA and albumen related to cell derived are contained in it.Excretion body both can directly activate by
Body cell, it can also be entered by transporting its bioactive substance included in recipient cell, participate in intercellular information interchange
And many important physiology or pathologic process.The excretion body of different cell secretions has different functions, such as tumor cell secretion
Excretion body there is the function of promoting tumor cell proliferation, reduce immunocyte killing ability, and from the outer of immunocyte
Secrete body function then contrast.Illustrate that excretion body has the part function similar to its derived cell.
Chinese Patent Application No. 201410705636.6 discloses one kind and prepares human amnion mesenchymal stem cell excretion body jelly
The method of dry powder, including cryoprotector is made with trehalose and mixed with human amnion mesenchymal stem cell excretion body, membrane filtration removes
Bacterium, the first cryogenic freezing under certain vacuum degree again after ultralow temperature pre-freeze.
Chinese Patent Application No. 201410705462.3, which discloses human amnion mesenchymal stem cell excretion body, can promote HDF
The growth of cell, and the activity of intracellular SOD enzymes can be improved, reduce because of oxygen radical pair caused by ultraviolet radiation induction
The attack and infringement of cell, the death rate of cell is reduced, show that human amnion mesenchymal stem cell excretion body has anti-light aging
Effect.
The excretion body that Chinese Patent Application No. 201610149852.6 discloses source for mesenchymal stem cells is preparing treatment
Application in the medicine of ankylosing spondylitis.
However, there is presently no openly fibroblast excretion body, and made of fibroblast excretion body for this area
Promote the preparation of wound healing.
More than being based on, UC-MSC is induced into fibroblast we have developed one kind, and extract fibroblast excretion
System into it is a kind of promote wound healing spray.
The content of the invention
It is appreciated that the purpose of terms used herein is only in that description specific embodiment, rather than limited.Enter
One step, unless otherwise defined, all technologies used herein and scientific terminology have ordinary skill of the art
The identical implication that personnel are commonly understood by.By according to the definition that is set forth below, using following term and grammatical variants to describe
With the claimed present invention.
The present invention relates to a kind of method for preparing fibroblast excretion body, and it includes (1) by umbilical cord mesenchymal stem cells
Fibroblast is induced into, (2) cultivate the fibroblast, and (2) extraction fibroblast excretion body.
In one embodiment, the umbilical cord mesenchymal stem cells are the umbilical cord mesenchymal stem cells of mammal.Institute
It is for example, ox, sheep, horse, dog, cat, monkey, and mankind, etc. to state mammal.Preferably, the mammal is the mankind.
In one embodiment, methods described identifies human umbilical cord mesenchymal stem cells before being additionally included in step (1), and
The identified human umbilical cord mesenchymal stem cells of culture.
In another embodiment, human umbilical cord mesenchymal stem cells are induced into fibroblast with conditioned medium,
The conditioned medium includes inactivation Cord Blood Serum, basic fibroblast growth factor, phosphoric acid vitamin C, and proline.It is excellent
Selection of land, the conditioned medium be the inactivation Cord Blood Serum containing volume fraction 10%, 10ng/ml basic fibroblast growths because
Son, 1mmol/L phosphoric acid vitamin C, the α-MEM of 0.04mmo/L proline.
In one embodiment, the invention further relates to the fibroblast excretion body prepared by methods described.
In one embodiment, the present invention relates to a kind of preparation for being used to promote wound healing, it is included into fiber finer
It is extracellular to secrete body and pharmaceutically acceptable carrier.
Brief description of the drawings
By the description to the embodiment of the present invention referring to the drawings, above-mentioned and other purpose of the invention, feature and
Advantage will be apparent from, in the accompanying drawings：
Fig. 1：The immunohistochemical staining picture of human umbilical cord mesenchymal stem cells.As shown in figure 1, human umbilical cord mesenchymal stem cells
Grown in fiber cell attachment, and cellular morphology is homogeneous, in fusiformis, and has certain orientation, swirling growth.
Fig. 2：P6 adds normal incubation medium (inactivation Cord Blood Serums of the α-MEM containing volume fraction 10%) (control for UC-MSC
Group), or add conditioned medium (inactivation Cord Blood Serums of the α-MEM containing volume fraction 10%, 10ng/ml basic fibroblasts
Growth factor, 1mmol/L phosphoric acid vitamin C, 0.04mmo/L proline) induction after cell dyeing picture.It is as shown in Fig. 2 real
Spindle or star-shaped flat structure of group cell into projection are tested, meets fibroblastic morphological feature.
Fig. 3：Control group and the immunohistochemical staining picture of experimental group, as a result show in Fig. 2, the type of cellular control unit I
Collagen expression is negative, and experimental group cell NTx is positive, and illustrates the induction system of the present invention and can make umbilical cord mesenchyma
Stem cell induces to fibroblast.
Fig. 4：Fibroblastic transmission electron microscope observing image (left figure) and Westernblot images (right figure).Transmiting
Observe that fibroblast excretion body is rounded or oval, diameter about 50-180nm, there is complete after birth structure under Electronic Speculum, it is interior
Containing materials of low density, Westernblot confirms that it expresses excretion body surface marker CD9 and CD63.
Embodiment
Definition
When term " such as ", " such as ", " comprising ", "comprising" or its variant be used for this paper when, these terms will not by regarding
For restrictive term, and " but being not limited only to " or " unrestriction " will be construed as.
When be related to measurable magnitude such as measure, temporal duration etc. when, term " about " refers to include designated value ± 20%,
Or in some cases ± 10%, or in some cases ± 5%, or in some cases ± 1%, or in some cases ±
0.1% change, therefore such change is adapted for disclosed method.
When being related to number range, during such as concentration range, the scope includes any value in the range of this, and the scope
Two end points.
The present invention relates to a kind of method for preparing fibroblast excretion body, and it includes (1) by umbilical cord mesenchymal stem cells
Fibroblast is induced into, (2) cultivate the fibroblast, and (2) extraction fibroblast excretion body.Can be with use condition
Culture medium inducing umbilical cord mesenchymal stem is divided into fibroblast.In one embodiment, using α-MEM CMC models
Base (inactivation Cord Blood Serums of the α-MEM containing volume fraction 10%, 10ng/ml basic fibroblast growth factors, 1mmol/L phosphorus
Acid vitamin C, 0.04mmo/L proline) conditioned medium inducing umbilical cord mesenchymal stem is divided into fibroblast.
In one embodiment, the culture of umbilical cord mesenchymal stem cells is also carried out using control medium.By conditioned medium and control
Cell after culture medium is cultivated with umbilical cord mesenchymal stem cells under the conditions of identical accessory respectively is observed under the microscope, and
Compare its morphosis to determine whether umbilical cord mesenchymal stem cells are divided into fibroblast.Alternatively, can also be by exempting from
Cellular morphology is observed after epidemic disease histochemical stain to determine whether umbilical cord mesenchymal stem cells are divided into fibroblast.
Fibroblast described in conventional method culture known in the art can be passed through.Especially, in an embodiment party
In case, fibroblast described in the method culture described in the embodiment of the present application can be used.
Fibrocyte excretion body can be extracted by methods known in the art.Disclosed in this area it is a variety of extraction, it is pure
The outside the pale of civilization method for secreting body.See that (it discloses continuous affine by two for example, Chinese Patent Application No. 201480066123.3
The method that purification step separates excretion body from biofluid)；(it discloses logical for Chinese Patent Application No. 201510915717.3
Cross ultracentrifugation separation excretion body)；Chinese Patent Application No. 201510918998.8 (it discloses by centrifugation, freeze-drying,
The method of buffer extractions obtains excretion body from urine)；(it discloses pass through for Chinese Patent Application No. 201610597323.2
The step such as centrifugation, filtering obtains excretion body).By being incorporated by herein by reference for above-mentioned patent application.
In one embodiment, the umbilical cord mesenchymal stem cells are the umbilical cord mesenchymal stem cells of mammal.Institute
It is for example, ox, sheep, horse, dog, cat, monkey, and mankind, etc. to state mammal.Preferably, the mammal is the mankind.
In one embodiment, methods described identifies human umbilical cord mesenchymal stem cells before being additionally included in step (1), and
The identified human umbilical cord mesenchymal stem cells of culture.
In another embodiment, human umbilical cord mesenchymal stem cells are induced into fibroblast with culture medium.It is preferred that
Ground, use condition culture medium induce human umbilical cord mesenchymal stem cells into fibroblast.It is highly preferred that the conditioned medium
Comprising inactivation Cord Blood Serum, basic fibroblast growth factor, phosphoric acid vitamin C, and proline.Most preferably, the bar
Part culture medium is the inactivation Cord Blood Serum containing volume fraction 10%, 10ng/ml basic fibroblast growth factors, 1mmol/L
α-the MEM of phosphoric acid vitamin C, 0.04mmo/L proline.
In one embodiment, the invention further relates to the fibroblast excretion body prepared by methods described.
In one embodiment, the present invention relates to a kind of preparation for being used to promote wound healing, it is included into fiber finer
It is extracellular to secrete body and pharmaceutically acceptable carrier.The preparation of the present invention can be well known by persons skilled in the art any suitable
Formulation, for example, can be applied herein with other suitable forms in the form of creme, solution, spray, emulsion, dispersion
Described preparation.Preferably, in the form of a spray using preparation as described herein.Described in the preparation outside fibroblast
Secrete body concentration be by weight 1% to 30%, preferably by weight 5% to 20%, more preferably by weight 5%, 10% or
20%.
Embodiment
Below based on embodiment, present invention is described, but the present invention is not restricted to these embodiments.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for those skilled in the art
For, the present invention can have various changes and change.All any modifications made within spirit and principles of the present invention, it is equal
Replace, improve etc., it should be included in the scope of the protection.
Unless otherwise indicated, implementation of the invention will use molecular biology (including recombinant technique), microbiology, cell
Biology and biochemical routine techniques, this is fallen into the range of art technology.Such technology has sufficiently in the literature
Illustrate, such as：《Molecular Cloning:A Laboratory guide》, second edition, Sambrook et al., 1989；Oligonucleotide
Synthesis, MJ Gait are edited, and 1984；Animal Cell Culture, R.I.Freshney are edited, and 1987；Methods
In Enzymology, Academic Press, Inc.；Handbook of Experimental Immunology, the 4th edition,
D.M.Weir＆CC.Blackwell is edited, Blackwell Science Inc., and 1987；GeneTransfer Vectors
For Mammalian Cells, J.M.Miller＆M.P.Calos are edited, and 1987；Current Protocols in
Molecular Biology, F.M.Ausubel et al. are edited, and 1987；And PCR：The Polymerase Chain
Reaction, Mullis et al. are edited, and 1994.
Embodiment 1
Fresh and healthy umbilical cord and bleeding of the umbilicus are taken, takes upper serum standby after inactivating after collecting bleeding of the umbilicus centrifugation.Rinsed well with PBS
Afterwards, umbilical blood vessels is removed with scissors tweezers, strips out the Fahrenheit glue tissue of the inside, gained tissue is fully shredded to 1mm3 sizes, added
Enter α-MEM nutrient solutions and be placed in 37 DEG C, 5% CO2Incubator culture, the umbilical sera containing 10% inactivation in nutrient solution.Umbilical cord tissue
After culture 5-8 days, it is seen that there is part cell to be climbed out of around tissue block, form is in tiny fusiformis, and after one week, cell starts fast
Speed propagation, forms the cell colony to differ in size, after cell covers with, is passed on 0.25% Trypsin Induced.Such as Fig. 1 institutes
Show, human umbilical cord mesenchymal stem cells grow in fiber cell attachment, and cellular morphology is homogeneous, in fusiformis, and has certain orientation
Property, swirling growth.
Embodiment 2
The UC-MSC that P3 and P6 generations are separately cultured, adjustment cell density to 1x10 are taken respectively6/ ml, respectively with FITC-
CD29, PE-CD31, FITC-CD40, PE-CD44, PC5-CD45, PC5-CD90, PE-CD105 and FITC-HLA-DR antibody 4
30min DEG C is incubated altogether, and PBS is washed 3 times, then cell is resuspended with 200 μ l PBS, is detected on flow cytometer.
As shown in table 1, high expression CD29, CD44, CD90 and CD105 of UC-MSC, low expression or do not express CD31, CD40,
CD45 and HLA-DR, meet UC-MSC standard of perfection.
The identification of table 1UC-MSC surface markers
Embodiment 3
Taking P6, adjustment cell density is 1x10 for UC-MSC6/ ml, by every bottle of 2x106Individual cell is seeded to the training of T175 cells
Support and cultivated in bottle.Liquid is changed after 24h and is divided into 2 groups, i.e. experimental group and control group, control group adds normal incubation medium, and (α-MEM contain volume
The inactivation Cord Blood Serum of fraction 10%), experimental group adds conditioned medium (inactivation bleeding of the umbilicus blood of the α-MEM containing volume fraction 10%
Clearly, 10ng/ml basic fibroblast growth factors, 1mmol/L phosphoric acid vitamin C, 0.04mmo/L proline) carry out cell
Induce, morphological observation is carried out to cell respectively after 3d.
As shown in Fig. 2 spindle or star-shaped flat structure of the experimental group cell into projection, meet fibroblastic
Morphological feature.
Embodiment 4
The cover glass of sterilizing is placed in 24 orifice plates, collects two groups of cells in embodiment 3, is resuspended with respective culture medium
After cell, by every hole 2x105Individual cell is inoculated in 24 orifice plates containing cover glass, per hole 0.5ml.Be placed on 37 DEG C, 5%
Continue to cultivate in CO2 cell culture incubator, inhaled after 48h and abandon original fluid, add PBS and wash 3 times.Then with 4% paraformaldehyde
Fixed, room temperature places 20min, and PBS is washed 3 times.0.1%TritonX-100 is added dropwise, room temperature places 10min.PBS is washed 2 times.
Subsequent serum closing 30min, unnecessary serum is wiped with filter paper, adds primary antibody (NTx protein antibodies), 4 DEG C of incubation 2h.PBS
After washing 3 times, 37 DEG C of secondary antibody incubation 30min, the PBS washing 3 times of biotin labeling is added, DAB dyeing, washing terminates.Bush
Essence is redyed, conventional to be dehydrated transparent, mounting, microscopy.
Immunohistochemical staining result (Fig. 3) shows that cellular control unit NTx protein expression is negative, experimental group
Cell NTx is positive, and illustrates the induction system of the present invention and umbilical cord mesenchymal stem cells can be made to be induced to fibroblast.
Embodiment 5
The Fibroblast cell-culture supernatant through induction the 3rd day in embodiment 3 is collected, 2000rpm/min centrifugation 10min, is gone
Except cell fragment.Add 15ml PBS balance Ultra-15 super filter tubes, centrifuge 4000g x 10min, suction abandon super filter tube inner tube and
PBS in collecting pipe.The Fibroblast cell-culture supernatant that 15ml removes cell fragment, centrifugation 4000g x are added into super filter tube
30min, collecting pipe is emptied, add 14mlPBS and carry out buffer exchange.4000g x 30min, withdraw and handle well from concentration tube
Sample (30x concentrations).
Take and isolate and purify the μ l of excretion body 10, add after isometric PBS dilution and be added dropwise on 2mm load sample copper mesh, in room temperature
Surplus liquid is gently sucked with filter paper after standing 1min, with 3% (w/v) sodium phosphotungstate solution (pH6.8) room temperature negative staining 5min,
Room temperature is dried after gently washing one time with distilled water, in transmission electron microscope observation and is taken a picture.Randomly select 20 excretion body examinations
Measure its diameter.It is another to take 15 μ g excretion body suspensions, add SDS-PAGE after adding 75 DEG C of 5 × loadding buffer holdings, 5min
Gel, 150V constant pressures run the expression of transferring film detection CD9 and CD63 after glue 1.5h.
Observe that fibroblast excretion body is rounded or oval, diameter about 50-180nm, has had under transmission electron microscope
Whole after birth structure, includes materials of low density, and Westernblot confirms that it expresses excretion body surface marker CD9 and CD63 (Fig. 4).
Embodiment 6
1ml nitrocellulose solutions are slowly added in 99ml deionized waters, make its final concentration of by weight 1%, adjust
Whole phase pH value is to 7.5.Then add hyaluronic acid, quercitin, vitamin E, make its concentration be respectively by weight 0.01%,
20ng/ μ l and 5ng/ μ l, after above-mentioned solution is mixed, add in embodiment 5 and separate the excretion liquid solution of concentration, make its final concentration
For by weight 20%.It is eventually adding sodium benzoate and menthol.Its final concentration is respectively 4ng/ μ l and 1ng/ μ l.Stand, will
Resulting solution obtains the spray for promoting wound healing with being fitted into after 0.22 μM of membrane filtration in sterile spray bottle.
Embodiment 7
1ml nitrocellulose solutions are slowly added in 99ml deionized waters, make its final concentration of by weight 1%, adjust
Whole phase pH value is to 7.5.Then add hyaluronic acid, quercitin, vitamin E, make its concentration be respectively by weight 0.01%,
20ng/ μ l and 5ng/ μ l, after above-mentioned solution is mixed, add in embodiment 5 and separate the excretion liquid solution of concentration, make its final concentration
For by weight 15%.It is eventually adding sodium benzoate and menthol.Its final concentration is respectively 4ng/ μ l and 1ng/ μ l.Stand, will
Resulting solution obtains the spray for promoting wound healing with being fitted into after 0.22 μM of membrane filtration in sterile spray bottle.
Embodiment 8
1ml nitrocellulose solutions are slowly added in 99ml deionized waters, make its final concentration of by weight 1%, adjust
Whole phase pH value is to 7.5.Then add hyaluronic acid, quercitin, vitamin E, make its concentration be respectively by weight 0.01%,
20ng/ μ l and 5ng/ μ l, after above-mentioned solution is mixed, add in embodiment 5 and separate the excretion liquid solution of concentration, make its final concentration
For by weight 10%.It is eventually adding sodium benzoate and menthol.Its final concentration is respectively 4ng/ μ l and 1ng/ μ l.Stand, will
Resulting solution obtains the spray for promoting wound healing with being fitted into after 0.22 μM of membrane filtration in sterile spray bottle.
Embodiment 9
1ml nitrocellulose solutions are slowly added in 99ml deionized waters, make its final concentration of by weight 1%, adjust
Whole phase pH value is to 7.5.Then add hyaluronic acid, quercitin, vitamin E, make its concentration be respectively by weight 0.01%,
20ng/ μ l and 5ng/ μ l, after above-mentioned solution is mixed, add in embodiment 5 and separate the excretion liquid solution of concentration, make its final concentration
For by weight 5%.It is eventually adding sodium benzoate and menthol.Its final concentration is respectively 4ng/ μ l and 1ng/ μ l.Stand, by institute
Solution obtains the spray for promoting wound healing with being fitted into after 0.22 μM of membrane filtration in sterile spray bottle.
Comparative example 1
1ml nitrocellulose solutions are slowly added in 99ml deionized waters, make its final concentration of by weight 1%, adjust
Whole phase pH value is to 7.5.Then add hyaluronic acid, quercitin, vitamin E, make its concentration be respectively by weight 0.01%,
20ng/ μ l and 5ng/ μ l, after above-mentioned solution is mixed, add sodium benzoate and menthol.Its final concentration be respectively 4ng/ μ l and
1ng/μl.Stand, resulting solution is obtained for promoting wound healing with being fitted into after 0.22 μM of membrane filtration in sterile spray bottle
Spray.
Embodiment 10
Take peptone 10g, beef extract 3g, sodium chloride 5g to be dissolved in 1L distilled water, add 2ml 15% NaOH solution, adjust
PH to 7.2~7.4 is saved, agar 15g is added, dispenses to triangular pyramidal bottle, 121 DEG C of autoclaving 15min.Sterilizing is fallen after terminating
Plate.
Respectively by 1x107CFU large intestine Ai Xi Salmonellas, Staphylococcus aureus and Mycophyta Trichophyton rubrum is inoculated into flat board
In, then each 1ml of embodiment 6-9 spray liquid is tested, first two flat board was observed and made after 37 DEG C of 24-48 hours
Colonial morphology describes and microscopic examination, latter fungi are observed after cultivating 24-48 hours at 28 DEG C and make colonial morphology
Description.
Experimental result is, in three kinds of flat boards containing bacterium after 10% excretion spray body agent liquid is added, cultivates 24-48 hours, nothing
Bacterium colony occurs.As a result show：The spray of the present invention has suppression to Escherichia coli, staphylococcus, Mycophyta Trichophyton rubrum
Effect.
Embodiment 11
KM mouse (6-8 week old) 50 are taken, are divided into 5 groups.Specific test method is by KM mouse back left of spine
One diameter 0.5cm of mark circular incision line at 0.5cm, after iodophor disinfection skin, holostrome is reduced along mark line with scissors
Skin forms circular wound face one by one to deep fascia.Respectively use embodiment 6-9 and comparative example 1 in aerosol therapy, one day
1 time, each 0.5ml, right side only gauze covering wrapping.Every group each 10, Continuous Observation, and record wound area.As a result such as table 1
Shown, speed of wound healing is directly related with excretion bulk concentration, and excretion bulk concentration is higher, and speed of wound healing is faster.
Disclosed herein and claimed all preparations, composition and/or method can be according to the disclosure
Manufacture and implementation, it is not necessary to excessive experiment.Although preparation of the present invention, group are described according to preferred embodiment
Compound and method, but it will be apparent for a person skilled in the art that：Without departing from idea of the invention, spirit and protection model
In the case of enclosing, can into the step of preparation, composition and/or method and method described herein or
Apply a variety of changes in the order of step.All such similar replacements that will be apparent to those skilled in the art and modification are all
It is considered as within the scope of spirit of the present invention, protection domain and the concept that appended claims defines.
Claims (10)
1. a kind of preparation for being used to promote wound healing, it includes fibroblast excretion body and pharmaceutically acceptable carrier.
2. the preparation described in claim 1, wherein the concentration of the fibroblast excretion body is by weight 1% to 30%,
It is preferred that by weight 5% to 20%, more preferably by weight 5%, 10% or 20%.
3. the preparation described in claim any one of 1-2, wherein the preparation also includes hyaluronic acid, quercitin or vitamin
E。
4. the concentration of the preparation described in claim 3, wherein hyaluronic acid is by weight 0.005% to 0.1%, quercitin
Concentration is 10-30ng/ μ l, and the concentration of vitamin E is 1-10ng/ μ l.
5. the preparation described in claim any one of 1-2, wherein the preparation is spray.
6. the preparation described in any one of foregoing claim is being prepared for promoting the purposes in the medicine in wound healing.
7. fibroblast excretion body is being prepared for promoting the purposes in the medicine in wound healing.
8. a kind of method for preparing fibroblast excretion body, it includes (1) by umbilical cord mesenchymal stem cells induction into fiber
The fibroblast, and (2) extraction fibroblast excretion body are cultivated in cell, (2)；Preferably, wherein before step (1)
Human umbilical cord mesenchymal stem cells are identified, and cultivate identified umbilical cord mesenchymal stem cells.
9. the method described in claim 8, wherein induced umbilical cord mesenchymal stem cells into fibroblast with conditioned medium,
The conditioned medium includes inactivation Cord Blood Serum, basic fibroblast growth factor, phosphoric acid vitamin C, and proline；It is excellent
Selection of land, wherein the conditioned medium is the inactivation Cord Blood Serum containing volume fraction 10%, the life of 10ng/ml basic fibroblasts
The long factor, 1mmol/L phosphoric acid vitamin C, the α-MEM of 0.04mmo/L proline.
10. the fibroblast excretion body prepared by any one of claim 8-9 method.
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