CN105079783B - Pharmaceutical composition and its preparation method and application - Google Patents
Pharmaceutical composition and its preparation method and application Download PDFInfo
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- CN105079783B CN105079783B CN201410220064.2A CN201410220064A CN105079783B CN 105079783 B CN105079783 B CN 105079783B CN 201410220064 A CN201410220064 A CN 201410220064A CN 105079783 B CN105079783 B CN 105079783B
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Abstract
The present invention provides pharmaceutical composition for skin healing and hair follicle regeneration and its preparation method and application, wherein, which includes：Self assembly polypeptide hydrogel, the self assembly polypeptide hydrogel are by least one being formed of RADA16 I and PRG；And Skin Cell, the Skin Cell are set to inside the self assembly polypeptide hydrogel.Inventor has found, using the pharmaceutical composition of the present invention, can effectively facilitate skin healing and hair follicle regeneration.In addition, the self assembly polypeptide hydrogel in the pharmaceutical composition of the present invention is entirely artificial synthesized, zoonosis is not propagated, with good tissue specificity, be conducive to the structure of tissue specifc stem cells differentiation microenvironment, and material source is more stablized, quality standard is easily controllable.
Description
Technical field
The present invention relates to regenerative medicine, biomaterial and stem cell biology field, more particularly to pharmaceutical composition
And its preparation method and application.
Background technology
Tissue of the skin as human body maximum is the barrier contacted with external environment, when due to environmental damage or disease etc.
When factor causes defect of skin, the loss of surface of a wound moisture, electrolyte and protein is often resulted in, the open surface of a wound also adds sense
The probability of dye.In early days, the generation of above-mentioned complication can be reduced by being effectively sealing off the surface of a wound, and clinically the most commonly used is petrolatum gauzes
With tomography/full thickness skin graft, wherein self split-thickness skin graft transplanting be the recognized standard therapy.But suffer from large skin defect
Person, there are still insufficient and the defects of cause body new damage for area.
Self assembly polypeptide hydrogel material is the research field that last decade has just been risen, and Preliminary Applications result has manifested
Its great potential in terms of organizational project and regeneration and restoration.The self assembly polypeptide with bionic function of synthesis
RADA16-I (Ac-RADARADARADARADA-CONH2) is the representative of such polypeptide, and the small peptide is by 16 hydrophobic and hydrophilic ammonia
Base acid is alternately arranged composition, forms stable β-pleated sheet, and the Nanowire of aligned orderly is self-assembly of in the aqueous solution of appropriate pH
Dimension.Its aqueous solution water absorption is up to more than 99.5%, and what nanofiber intersected to form tool mesh skeleton is similar to extracellular matrix
Three-dimensional hydrogel structure, to cell rise supporting function.The material has good cell and histocompatbility, nontoxic, implantation
It can gradually be absorbed after in vivo, be ideal tissue engineering material.
At present, self assembly polypeptide hydrogel is had not been reported for the wound healing of skin and hair follicle regeneration.
Invention content
The present invention is directed to solve at least some of the technical problems in related technologies.For this purpose, the present invention
One purpose be to propose it is a kind of can be effective for the means of skin healing and hair follicle regeneration.
In one aspect of the invention, the present invention provides a kind of pharmaceutical composition for skin healing and hair follicle regeneration.
According to an embodiment of the invention, which includes：Self assembly polypeptide hydrogel, the self assembly polypeptide hydrogel be by
RADA16-I and PRG (Ac- (RADA) 4GPRGDSGYRGDS-CONH2) at least one formation；And Skin Cell, it is described
Skin Cell is set to inside the self assembly polypeptide hydrogel.Inventor has found, utilizes the pharmaceutical composition of the present invention, energy
Enough effectively facilitate skin healing and hair follicle regeneration.In addition, the full people of self assembly polypeptide hydrogel in the pharmaceutical composition of the present invention
Work synthesizes, and does not propagate zoonosis, can be according to the characteristics of different tissues and purposes flexibly adds the functional sheet of different molecular
Section, is allowed to more tissue specificity, is conducive to the structure of tissue specifc stem cells differentiation microenvironment, and material source is more
Stablize, quality standard is easily controllable.
Pharmaceutical composition according to embodiments of the present invention can also have following additional technical feature：
According to an embodiment of the invention, when the hydrogel is collectively formed by RADA16-I and PRG, in the self assembly
In polypeptide hydrogel, the mass ratio of the RADA16-I and the PRG are 1：1.
According to an embodiment of the invention, in the self assembly polypeptide hydrogel, the content of the Skin Cell is 104-
108A/mL.
According to an embodiment of the invention, the Skin Cell is at least one selected from dermal cell and epidermal cell.
According to an embodiment of the invention, the dermal cell is the dermal cell that newly detaches or cultivates the corium in 0-30 generations to do
Cell.
According to an embodiment of the invention, the epidermal cell is the epidermal cell newly detached or the epidermal stem for cultivating 0-30 generations
Cell.
According to an embodiment of the invention, described pharmaceutical composition further comprises：Be conducive to cell survival, the cell grown
Active factors and/or extracellular matrix molecule.According to an embodiment of the invention, the cell active factor is selected from bFGF (alkali
Property fibroblast growth factor), at least one of EGF (epithelical cell growth factor), the extracellular matrix molecule is choosing
At least one of self-induced transparency matter acid, fibronectin splicing variants.Thereby, it is possible to effectively facilitate the survival of Skin Cell and growth, and then
The effect for promoting skin healing and hair follicle regeneration is preferable.
In another aspect of this invention, the present invention provides a kind of methods for preparing foregoing pharmaceutical composition.Root
According to the embodiment of the present invention, this method includes：(1) RADA16-I solution and PRG solution are provided, wherein, the RADA16-I is molten
A concentration of 0.1-1g/100ml of liquid, a concentration of 0.1-1g/100ml of the PRG solution；(2) by the RADA16-I solution
It is mixed in equal volume with the PRG solution, to obtain mixing hydrogel solution；(3) in the RADA16-I solution, the PRG
Skin Cell is inoculated with, and incubated under conditions of suitable for the dermal cell growth in solution or the mixing hydrogel solution
It educates, to obtain described pharmaceutical composition.Inventor has found, front can be fast and effeciently prepared using this method of the present invention
The pharmaceutical composition, and it is simple, convenient it is quick, be easy to control, be easily achieved large-scale production.
According to an embodiment of the invention, before step (2), in advance by the RADA16-I solution and the PRG solution
Carry out 0.22 μm of filtration sterilization processing.
According to an embodiment of the invention, the inoculating cell further comprises：With 10% sterile sucrose solution by the skin
Skin cell is made into a concentration of 104-108The cell suspension of a/mL, by it is described mixing hydrogel solution and the cell suspension according to
Volume ratio is 5：1 ratio mixing.
According to an embodiment of the invention, the incubation further comprises：Every 30 minutes, the culture medium in orifice plate is removed,
It adds in isometric fresh culture to orifice plate, repeats 3-5 times.According to an embodiment of the invention, include in the culture medium
It is according to an embodiment of the invention, described thin conducive to cell survival, the cell active factor and/or extracellular matrix molecule of growth
The cytoactive factor is at least one selected from bFGF, EGF, and the extracellular matrix molecule is selected from hyaluronic acid, fiber adhesion
At least one of albumen.Thereby, it is possible to effectively facilitate the survival of Skin Cell and growth, and then promote skin healing and hair follicle again
Raw effect is preferable.
According to an embodiment of the invention, a concentration of 1g/100mL of the RADA16-I solution.
According to an embodiment of the invention, a concentration of 1g/100mL of the PRG solution.
According to an embodiment of the invention, the Skin Cell is at least one selected from epidermal cell and dermal cell.
According to an embodiment of the invention, the dermal cell is the dermal cell that newly detaches or cultivates the corium in 0-30 generations to do
Cell.
According to an embodiment of the invention, the epidermal cell is the epidermal cell newly detached or the epidermal stem for cultivating 0-30 generations
Cell.
In still another aspect of the invention, the use the present invention provides foregoing pharmaceutical composition in medicine preparation
On the way, the drug is used for skin healing and hair follicle regeneration.
In another aspect of the invention, the present invention provides a kind of drugs for being used for skin healing and hair follicle regeneration.According to
The embodiment of the present invention, the drug include foregoing pharmaceutical composition.Inventor has found that drug of the invention can be effective
For skin healing and hair follicle regeneration.
Description of the drawings
Fig. 1 shows according to an embodiment of the invention, the mode of appearance figure of pharmaceutical composition of the invention；
Fig. 2 is shown according to an embodiment of the invention, and when cultivating 1 day, the copolymerization of corium stem cell is burnt in pharmaceutical composition
Microscope photo；
Fig. 3 is shown according to an embodiment of the invention, and when cultivating 3 days, the copolymerization of corium stem cell is burnt in pharmaceutical composition
Microscope photo；
Fig. 4 shows according to an embodiment of the invention, the bis- colored graphs of the AO/PI of corium stem cell in pharmaceutical composition；
Fig. 5 shows according to an embodiment of the invention, the alkaline phosphatase (AP) of corium stem cell in pharmaceutical composition
Colored graph；
Fig. 6 shows that according to an embodiment of the invention transplant medicine composition is after 3 weeks, and nude mice skin healing and hair follicle are again
Raw situation result figure；
Fig. 7 shows that according to an embodiment of the invention, after cultivating 1 day, MSC is in the mixing hydrogel solution by various concentration
Inverted microscope photo in the hydrogel of formation, wherein,
The inversion that Fig. 7 A are MSC in being the hydrogel that is formed of mixing hydrogel solution of 0.1g/100mL by peptide concentration
Microscope photo,
The inversion that Fig. 7 B are MSC in being the hydrogel that is formed of mixing hydrogel solution of 0.5g/100mL by peptide concentration
Microscope photo,
It is being that inversion in the hydrogel that is formed of mixing hydrogel solution of 1g/100mL is shown by peptide concentration that Fig. 7 C, which are MSC,
Micro mirror photo；
Fig. 8 shows that according to an embodiment of the invention, after cultivating 3 days, corium stem cell is in RADA16-I self assembly polypeptides
Growing state in hydrogel.
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.Particular technique or condition are not specified in embodiment, according to text in the art
It offers described technology or condition or is carried out according to product description.Reagents or instruments used without specified manufacturer,
For can be with conventional products that are commercially available.
In one aspect of the invention, the present invention provides a kind of pharmaceutical composition for skin healing and hair follicle regeneration.
According to an embodiment of the invention, which includes：Self assembly polypeptide hydrogel, the self assembly polypeptide hydrogel be by
At least one formation of RADA16-I and PRG；And Skin Cell, the Skin Cell are set to the self assembly polypeptide water
Inside gel.Inventor has found, using the pharmaceutical composition of the present invention, can effectively facilitate skin healing and hair follicle regeneration.
In addition, the self assembly polypeptide hydrogel in the pharmaceutical composition of the present invention is entirely artificial synthesized, zoonosis is not propagated, it can root
According to the characteristics of different tissues and purposes flexibly adds the function fragment of different molecular, it is allowed to, with more tissue specificity, be conducive to
The structure of tissue specifc stem cells differentiation microenvironment, and material source is more stablized, quality standard is easily controllable.
According to an embodiment of the invention, the self assembly polypeptide hydrogel can be independent by one kind of RADA16-I and PRG
It is formed, can also be formed by the mixture of RADA16-I and PRG, when the hydrogel is collectively formed by RADA16-I and PRG,
In the self assembly polypeptide hydrogel, the mass ratio of the RADA16-I and the PRG are 1：1.Self assembly polypeptide water as a result,
The performance of gel is preferable, can effectively support growth and the proliferation of Skin Cell.
According to an embodiment of the invention, in the self assembly polypeptide hydrogel, the content of the Skin Cell is not by spy
It does not limit, those skilled in the art can flexibly select according to actual conditions.According to some embodiments of the present invention, Skin Cell
Content can be 104-108A/mL.As a result, pharmaceutical composition of the invention promote the effect of skin healing and hair follicle regeneration compared with
It is good.
According to an embodiment of the invention, the type of the Skin Cell is not particularly limited.Some realities according to the present invention
Example is applied, Skin Cell can be at least one selected from dermal cell and epidermal cell.Thereby, it is possible to effectively facilitate skin healing
And hair follicle regeneration.
According to an embodiment of the invention, the type of the dermal cell is not particularly limited.Some realities according to the present invention
Example is applied, dermal cell can be the dermal cell newly detached or the corium stem cell for cultivating 0-30 generations.The dermal cell as a result,
Growth conditions in self assembly polypeptide hydrogel are good, can Effective multiplication, and then can effectively play promotion skin healing
And the effect of hair follicle regeneration.
According to an embodiment of the invention, the type of the epidermal cell is not particularly limited.Some realities according to the present invention
Example is applied, epidermal cell can be the epidermal cell newly detached or the epidermal stem cells for cultivating 0-30 generations.The epidermal cell as a result,
Growth conditions in self assembly polypeptide hydrogel are good, can Effective multiplication, and then can effectively play promotion skin healing
And the effect of hair follicle regeneration.
According to an embodiment of the invention, described pharmaceutical composition can further include：Be conducive to cell survival, growth
Cell active factor and/or extracellular matrix molecule.According to an embodiment of the invention, the type of the cell active factor is not
It is particularly limited, as long as growth and the proliferation of Skin Cell can be effectively facilitated.A specific example according to the present invention,
Cell active factor can be selected from bFGF (basic fibroblast growth factor), EGF (epithelical cell growth factor) extremely
Few one kind.According to an embodiment of the invention, the type of the extracellular matrix molecule is not particularly limited.According to the present invention one
A little embodiments, extracellular matrix molecule can be at least one selected from hyaluronic acid, fibronectin splicing variants.Thereby, it is possible to have
Effect promotes the survival and growth of Skin Cell, and then promotes the effect of skin healing and hair follicle regeneration preferable.
In another aspect of this invention, the present invention provides a kind of methods for preparing foregoing pharmaceutical composition.Root
According to the embodiment of the present invention, this method includes the following steps：
(1) RADA16-I solution and PRG solution are provided, wherein, a concentration of 0.1-1g/ of the RADA16-I solution
100ml, a concentration of 0.1-1g/100ml of the PRG solution.
According to an embodiment of the invention, a concentration of 1g/100mL of the RADA16-I solution.It is obtained from group as a result,
The performance for filling polypeptide hydrogel is preferable, and the effect of skin healing and hair follicle regeneration is preferable.
According to an embodiment of the invention, a concentration of 1g/100mL of the PRG solution.Obtained self assembly is more as a result,
The performance of peptide hydrogel is preferable, and the effect of skin healing and hair follicle regeneration is preferable.
According to an embodiment of the invention, before subsequent step is carried out, in advance by the RADA16-I solution and the PRG
Solution carries out 0.22 μm of filtration sterilization processing.Thereby, it is possible to effectively carry out degerming to RADA16-I solution and PRG solution, obtain
Sterile RADA16-I solution and PRG solution, convenient for the operation of subsequent step.
(2) the RADA16-I solution and the PRG solution are mixed in equal volume, to obtain mixing hydrogel solution.
It according to an embodiment of the invention, can be in advance by RADA16-I before RADA16-I solution and PRG solution are mixed
Solution and PRG solution are 30 minutes ultrasonic respectively.
(3) Skin Cell is inoculated in the RADA16-I solution, the PRG solution or the mixing hydrogel solution,
And be incubated under conditions of suitable for the dermal cell growth, to obtain described pharmaceutical composition.
According to an embodiment of the invention, the inoculation Skin Cell further comprises：With 10% sterile sucrose solution by institute
It states Skin Cell and is made into a concentration of 104-108The cell suspension of a/mL, by the mixing hydrogel solution and the cell suspension
It is 5 according to volume ratio：1 ratio mixing.As a result, under the conditions of appropriate pH, RADA16-I and PRG in hydrogel solution are mixed
Nanofiber can be spontaneously assemble into, and is further crosslinked, forms the hydrogel of porous network structure, in effectively obtaining
Portion is provided with the self assembly polypeptide hydrogel of Skin Cell, i.e. pharmaceutical composition of the invention.
According to an embodiment of the invention, the type of the Skin Cell is not particularly limited.Some realities according to the present invention
Example is applied, Skin Cell can be at least one selected from epidermal cell and dermal cell.Promote skin healing and hair follicle again as a result,
Raw effect is preferable.
According to an embodiment of the invention, the type of the dermal cell is not particularly limited.Some realities according to the present invention
Example is applied, dermal cell can be the dermal cell newly detached or the corium stem cell for cultivating 0-30 generations.The dermal cell as a result,
Growth conditions in self assembly polypeptide hydrogel are good, can Effective multiplication, and then can effectively play promotion skin healing
And the effect of hair follicle regeneration.
According to an embodiment of the invention, the type of the epidermal cell is not particularly limited.Some realities according to the present invention
Example is applied, epidermal cell can be the epidermal cell newly detached or the epidermal stem cells for cultivating 0-30 generations.The epidermal cell as a result,
Growth conditions in self assembly polypeptide hydrogel are good, can Effective multiplication, and then can effectively play promotion skin healing
And the effect of hair follicle regeneration.
According to an embodiment of the invention, the incubation further comprises：Every 30 minutes, the culture medium in orifice plate is removed,
It adds in isometric fresh culture to orifice plate, repeats 3-5 times.Thereby, it is possible to effectively adjust the pH of self assembly polypeptide hydrogel
It is worth the growth so as to fit Skin Cell and proliferation, so as to effective for skin healing and hair follicle regeneration.Reality according to the present invention
Example is applied, the cell active factor and/or extracellular matrix molecule for being conducive to cell survival, growth, root are included in the culture medium
According to the embodiment of the present invention, the cell active factor be at least one selected from bFGF, EGF, the extracellular matrix molecule
To be selected from at least one of hyaluronic acid, fibronectin splicing variants.Thereby, it is possible to effectively facilitate the survival of Skin Cell and growth,
And then promote the effect of skin healing and hair follicle regeneration preferable.
Inventor has found, foregoing pharmaceutical composition can be fast and effeciently prepared using this method of the present invention,
And it is simple, convenient it is quick, be easy to control, be easily achieved large-scale production.
In still another aspect of the invention, the use the present invention provides foregoing pharmaceutical composition in medicine preparation
On the way, the drug is used for skin healing and hair follicle regeneration.Inventor has found that in the pharmaceutical composition of the present invention, self assembly is more
Peptide hydrogel has good cell and histocompatbility, nontoxic, can effectively support growth and the proliferation of Skin Cell, is implanted into
It can gradually be absorbed after in vivo, Skin Cell effectively can grow and be proliferated in self assembly polypeptide hydrogel, more in self assembly
Under the supporting function of peptide hydrogel, skin healing and hair follicle regeneration can be effectively facilitated.
In another aspect of the invention, the present invention provides a kind of drugs for being used for skin healing and hair follicle regeneration.According to
The embodiment of the present invention, the drug include foregoing pharmaceutical composition.Inventor's discovery, the pharmaceutical composition of the invention
It can be effective for skin healing and hair follicle regeneration.
The embodiment of the present invention is described below in detail, unless otherwise specified, in following embodiments isolated skin stem cell from
The skin histology of C57 mouse is detached and is cultivated；The separation method of Skin Cell is：Mouse skin is cut into small pieces and uses dispase
II (sigma), 37 DEG C digest 2 hours, remove corium and epidermis, are newly detached with type i collagen enzyme (sigma) digestion respectively
Corium and epidermal cell；Carbonic ester film (transwell) is purchased from Corning companies, catalog number 3470；Cell culture
Base composition is DMEM/F12 (3:1) it (buys in Invitrogen companies), (is bought containing 2%B27 in Invitrogen companies),
40ng/ μ L bFGF (Peprotech) and 20ng/ μ L EGF (Peprotech), in addition to embodiment 1, other embodiment culture is true
The culture medium of skin stem cell culture medium thus；3M films are TegadermTMFilm；Culture dish is suspension cell culture ware, is purchased from
In Guangzhou Ztel's biofiltration Products Co., Ltd, article No. MCD-000-090.Experimental animal is purchased from Guangdong Province's medicine reality
Test animal center.
Embodiment 1
The cell compatibility of self assembly polypeptide hydrogel is measured using people source mescenchymal stem cell (MSC), it is specific as follows：
(1) Peptide systhesis is carried out using solid phase synthesis process, passes through Peptide synthesizer synthesis polypeptide molecule, the N-terminal of peptide chain
It is modified with C-terminal by acetylation and amidation, prevents it from degrading rapidly.Polypeptide RADA16-I (Ac- after synthesis
RADARADARADARADA-CONH2) and PRG (Ac- (RADA)4GPRGDSGYRGDS-CONH2) carried out after purification with HPLC, it is cold
It is lyophilized dry, respectively obtains RADA16-I and PRG polypeptide freeze-dried powders.
(2) RADA16-I the and PRG polypeptides freeze-dried powder prepared in step (1) is obtained respectively with deionized water dissolving
It is the RADA16-I solution of 1g/100mL, 0.5g/100mL, 0.1g/100mL and PRG solution to peptide concentration, then, with 0.22
μm membrane filtration degerming, obtain the sterile RADA16-I solution of various concentration and PRG solution.
(3) it is the sterile RADA16-I solution and PRG solution of various concentration is 30 minutes ultrasonic respectively.
(4) the RADA16-I solution by the same concentrations being ultrasonically treated and PRG solution are mixed in equal volume, in particular
Each 50 μ L obtain the mixing hydrogel solution of three kinds of concentration.
(5) it after the quick mixing of hydrogel solution will be mixed, adds in 24 hole transwell, then along culture plate hole wall
It is slowly added to culture medium (DMEM, Corning cellgro, containing 10% fetal calf serum).
(6) in 5%CO2, stand 30min in 37 DEG C of incubators after, slowly siphon away culture medium with pipette tips, then add again new
In fresh culture medium to orifice plate.
(7) 30min is stood again, then the repeatedly operation of step (6).
(8) 30min is stood again, then the repeatedly operation of step (6).
(9) 30min or longer time are stood again, then the repeatedly operation of step (6).
(10) by MSC kinds to self assembly polypeptide hydrogel surface, and culture medium is added, is subsequently placed in 37 DEG C, 5%CO2Training
It supports in case, quiescent culture.The adherency situation of cell and self assembly polypeptide hydrogel is observed under inverted microscope.After culture 1 day
Its aspect graph is shown in Fig. 7, wherein, Fig. 7 A are the water that MSC is formed in the mixing hydrogel solution for being 0.1g/100mL by peptide concentration
Inverted microscope photo in gel, Fig. 7 B are that MSC is formed in the mixing hydrogel solution for being 0.5g/100mL by peptide concentration
Hydrogel in inverted microscope photo, Fig. 7 C are MSC in the mixing hydrogel solution shape for being 1g/100mL by peptide concentration
Into hydrogel in inverted microscope photo.As seen from Figure 7, the self assembly polypeptide hydrogel of three kinds of concentration is all to cell adherence
There is certain supporting function, and cell can preferably stretch in the hydrogel of 1g/100mL, to cell growth and be stained with more
Good supporting function.
Embodiment 2
(1) Peptide systhesis is carried out using solid phase synthesis process, passes through Peptide synthesizer synthesis polypeptide molecule, the N-terminal of peptide chain
It is modified with C-terminal by acetylation and amidation, prevents it from degrading rapidly.Polypeptide RADA16-I (Ac- after synthesis
RADARADARADARADA-CONH2) and PRG (Ac- (RADA)4GPRGDSGYRGDS-CONH2) carried out after purification with HPLC, it is cold
It is lyophilized dry, respectively obtains RADA16-I and PRG polypeptide freeze-dried powders.
(2) RADA16-I the and PRG polypeptides freeze-dried powder prepared in step (1) is obtained respectively with deionized water dissolving
It is the RADA16-I solution of 1g/100mL and PRG solution to peptide concentration, then, with 0.22 μm of membrane filtration degerming, obtains
To sterile RADA16-I solution and PRG solution.
(3) it is sterile RADA16-I solution and PRG solution is 30 minutes ultrasonic respectively.
(4) the RADA16-I solution by being ultrasonically treated and PRG solution are mixed in equal volume, in particular each 50 μ L are obtained
To mixing hydrogel solution.
(5) by the 3rd generation mouse skin corium stem cell (SKP) 10 of culture that suspends4A, centrifugation removal culture medium is used
The sterile sucrose solution washing cells of 1mL10%, are then centrifuged for, remove supernatant, add the sterile sucrose solutions of 20 μ L10% and prepare
Into cell suspension.
(6) after monoploid product cell suspension being mixed the quick mixing of hydrogel solution with pentaploid product, 24 holes are added in
In transwell, culture medium is slowly added to then along culture plate hole wall.
(7) in 5%CO2, stand 30min in 37 DEG C of incubators after, slowly siphon away culture medium with pipette tips, then add again new
In fresh culture medium to 24 hole transwell and orifice plate, incubator is put back to.
(8) 30min is stood again, then the repeatedly operation of step (7).
(9) 30min is stood again, then the repeatedly operation of step (7).
(10) 30min is stood again, then the repeatedly operation of step (7).
(11) obtained pharmaceutical composition is placed in 37 DEG C, 5%CO2In incubator, quiescent culture obtains pharmaceutical composition
Object, mode of appearance figure are shown in Fig. 1.
After culture 1 or 3 day, corium stem cell is observed in self assembly polypeptide water-setting by Laser Scanning Confocal Microscope (OLYMPUS)
Growing state in glue, Laser Scanning Confocal Microscope photo are shown in Fig. 2 and Fig. 3, wherein, Fig. 2 is cultivates 1 day, the copolymerization of corium stem cell
Focusing microscope photo, Fig. 3 is cultivates 3 days, the Laser Scanning Confocal Microscope photo of corium stem cell.It is more it can be seen from Fig. 2 and Fig. 3
Number corium stem cell starts to stretch, and shows that the growth conditions of corium stem cell are good.
With AO/PI double-stainings identification of cell anyway：The bis- colored graphs of AO/PI are shown in Fig. 4, wherein, green is living cells, red
For dead cell.Red dead cell it can be seen that, is hardly visible by Fig. 4, is illustrated in the pharmaceutical composition of the present invention, corium
Stem cell has preferable survival rate.
With the dryness of BCIP/NBT alkaline phosphatases (AP) colour reagent box (the green skies) detection corium stem cell.Alkalinity
Phosphate (AP) colored graph is shown in Fig. 5.The result shows that skin progenitor cell maintains preferable do in self assembly polypeptide hydrogel
Property.
Embodiment 3
(1) Peptide systhesis is carried out using solid phase synthesis process, passes through Peptide synthesizer synthesis polypeptide molecule, the N-terminal of peptide chain
It is modified with C-terminal by acetylation and amidation, prevents it from degrading rapidly.Polypeptide RADA16-I (Ac- after synthesis
RADARADARADARADA-CONH2) and PRG (Ac- (RADA)4GPRGDSGYRGDS-CONH2) carried out after purification with HPLC, it is cold
It is lyophilized dry, respectively obtains RADA16-I and PRG polypeptide freeze-dried powders.
(2) RADA16-I the and PRG polypeptides freeze-dried powder prepared in step (1) is obtained respectively with deionized water dissolving
It is the RADA16-I solution of 1g/100mL and PRG solution to peptide concentration, then, with 0.22 μm of membrane filtration degerming, obtains
Sterile RADA16-I solution and PRG solution.
(3) it is sterile RADA16-I solution and PRG solution is 30 minutes ultrasonic respectively.
(4) the RADA16-I solution by being ultrasonically treated and PRG solution are mixed in equal volume, in particular each 50 μ L are obtained
To mixing hydrogel solution.
(5) by the 3rd generation mouse skin corium stem cell (SKP) (or the newborn rat dermal cell newly detached) of culture that suspends
106It is a, with the newborn rat epidermal cell 10 newly detached6A mixing, centrifugation removal culture medium, is washed with the sterile sucrose solutions of 1mL10%
Cell is washed, is then centrifuged for, supernatant is removed, adds the 20 sterile sucrose solutions of μ L10% and be configured to cell suspension.
(6) after monoploid product cell suspension being mixed the quick mixing of hydrogel solution with pentaploid product, 24 holes are added in
In transwell, culture medium is slowly added to then along culture plate hole wall.
(7) in 5%CO2, stand 30min in 37 DEG C of incubators after, slowly siphon away culture medium with pipette tips, then add again new
In fresh culture medium to transwell and orifice plate, incubator is put back to.
(8) 30min is stood again, then the repeatedly operation of step (7).
(9) 30min is stood again, then the repeatedly operation of step (7).
(10) 30min is stood again, then the repeatedly operation of step (7).
(11) obtained pharmaceutical composition is placed in 37 DEG C, 5%CO2In incubator, quiescent culture obtains pharmaceutical composition
Object.
Embodiment 4
According to following steps, detect the pharmaceutical composition prepared in embodiment 3 and promote skin healing and hair follicle regeneration
Effect, it is specific as follows：
BALB/c nude mices with 1% yellow Jackets are anaesthetized, area is manufactured about on its skin of back with card punch
7mm2Wound, then, the pharmaceutical composition prepared in embodiment 2 is transplanted to nude mice skin wound surface, and use 3M
Film covers nude mice wound surface, then with bandage by nude mice wound dressing.Normal raising nude mice, strip the bandage off removal 3M after 3 weeks
Film, the healing of observation nude mice skin and hair follicle regeneration situation.
Experimental result is as shown in Figure 6.As seen from Figure 6, nude mice skin wound heals completely, and grows hair, table
The pharmaceutical composition of the bright present invention can be effective for skin healing and hair follicle regeneration.
Embodiment 5
(1) Peptide systhesis is carried out using solid phase synthesis process, passes through Peptide synthesizer synthesis polypeptide molecule, the N-terminal of peptide chain
It is modified with C-terminal by acetylation and amidation, prevents it from degrading rapidly.Polypeptide RADA16-I (Ac- after synthesis
RADARADARADARADA-CONH2), carried out after purification with HPLC, freeze-drying obtains RADA16-I polypeptide freeze-dried powders.
(2) the RADA16-I polypeptides freeze-dried powder prepared in step (1) is obtained into polypeptide respectively with deionized water dissolving
Then with 0.22 μm of membrane filtration degerming, it is molten to obtain sterile RADA16-I for the RADA16-I solution of a concentration of 1g/100mL
Liquid.
(3) it is sterile RADA16-I solution is 30 minutes ultrasonic.
(4) by the 3rd generation mouse skin corium stem cell (SKP) 10 of culture that suspends4A, centrifugation removal culture medium is used
The sterile sucrose solution washing cells of 1mL10%, are then centrifuged for, remove supernatant, add the sterile sucrose solutions of 20 μ L10% and prepare
Into cell suspension.
(5) by after the quick mixing of RADA16-I solution after 20 μ L cell suspensions and 100 μ L ultrasounds, 24 holes are added in
In transwell, culture medium is slowly added to then along culture plate hole wall.
(6) in 5%CO2, stand 30min in 37 DEG C of incubators after, slowly siphon away culture medium with pipette tips, then add again new
In fresh culture medium to 24 hole transwell and orifice plate, incubator is put back to.
(7) 30min, the then repeatedly operation of step (6) are stood.
(8) 30min is stood again, then the repeatedly operation of step (6).
(9) 30min is stood again, then the repeatedly operation of step (6)
(10) obtained pharmaceutical composition is placed in 37 DEG C, 5%CO2In incubator, quiescent culture obtains pharmaceutical composition
Object.
By pharmaceutical composition obtained above at 37 DEG C, 5%CO2After being cultivated 3 days in incubator, pass through inverted microscope
(Nikon) growing state of the observation corium stem cell in self assembly polypeptide hydrogel, microscope photo are shown in Fig. 8.It can be with from Fig. 8
Find out, most corium stem cells can preferably grow in RADA16-I self assembly polypeptide hydrogels.
In the description of this specification, reference term " one embodiment ", " example ", " is specifically shown " some embodiments "
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 are 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 or examples described in this specification and the feature of different embodiments or examples
It closes and combines.
Although the embodiments 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 (18)
1. a kind of pharmaceutical composition for skin healing and hair follicle regeneration, which is characterized in that including：
Self assembly polypeptide hydrogel, the self assembly polypeptide hydrogel be by mass ratio be 1：The 1 common shape of RADA16-I and PRG
Into；And
Skin Cell, the Skin Cell is set to inside the self assembly polypeptide hydrogel, and the content of the Skin Cell
It is 104-108A/mL.
2. pharmaceutical composition according to claim 1, which is characterized in that the Skin Cell is selected from dermal cell and table
At least one of chrotoplast.
3. pharmaceutical composition according to claim 2, which is characterized in that the dermal cell is the dermal cell newly detached
Or the corium stem cell in culture 0-30 generations.
4. pharmaceutical composition according to claim 2, which is characterized in that the epidermal cell is the epidermal cell newly detached
Or the epidermal stem cells in culture 0-30 generations.
5. pharmaceutical composition according to claim 1, which is characterized in that further comprise：
Be conducive to cell survival, the cell active factor and/or extracellular matrix molecule that grow.
6. pharmaceutical composition according to claim 5, which is characterized in that the cell active factor is selected from bFGF, EGF
At least one, the extracellular matrix molecule be at least one selected from hyaluronic acid, fibronectin splicing variants.
A kind of 7. method for preparing claim 1-6 any one of them pharmaceutical compositions, which is characterized in that including：
(1) RADA16-I solution and PRG solution are provided, wherein, a concentration of 0.1-1g/100ml of the RADA16-I solution, institute
State a concentration of 0.1-1g/100ml of PRG solution；
(2) the RADA16-I solution and the PRG solution are mixed in equal volume, to obtain mixing hydrogel solution；
(3) it is inoculated with Skin Cell in the RADA16-I solution, the PRG solution or the mixing hydrogel solution, and
Suitable for being incubated under conditions of the dermal cell growth, to obtain described pharmaceutical composition.
8. the method according to the description of claim 7 is characterized in that before step (2), in advance by the RADA16-I solution
0.22 μm of filtration sterilization processing is carried out with the PRG solution.
9. according to the method described in claim 8, it is characterized in that, the inoculating cell further comprises：
The Skin Cell is made into a concentration of 10 with 10% sterile sucrose solution4-108The cell suspension of a/mL, will be described mixed
Heshui gel solution and the cell suspension are 5 according to volume ratio：1 ratio mixing.
10. according to the method described in claim 8, it is characterized in that, the incubation further comprises：
Every 30 minutes, the culture medium in orifice plate is removed, is added in isometric fresh culture to orifice plate, repeated 3-5 times.
11. according to the method described in claim 10, be conducive to cell survival, life it is characterized in that, being included in the culture medium
Long cell active factor and/or extracellular matrix molecule.
12. according to the method for claim 11, which is characterized in that the cell active factor be selected from bFGF, EGF extremely
Few one kind, the extracellular matrix molecule are at least one selected from hyaluronic acid, fibronectin splicing variants.
13. according to the method described in claim 8, it is characterized in that, a concentration of 1g/100mL of the RADA16-I solution, institute
State a concentration of 1g/100mL of PRG solution.
14. according to the method for claim 13, which is characterized in that the Skin Cell is thin selected from epidermal cell and corium
At least one of born of the same parents.
15. according to the method for claim 14, which is characterized in that the dermal cell is the dermal cell or training newly detached
Support the corium stem cell in 0-30 generations.
16. according to the method for claim 14, which is characterized in that the epidermal cell is the epidermal cell or training newly detached
Support the epidermal stem cells in 0-30 generations.
17. the purposes of claim 1-6 any one of them pharmaceutical composition in medicine preparation, the drug are cured for skin
Conjunction and hair follicle regeneration.
It is 18. a kind of for skin healing and the drug of hair follicle regeneration, which is characterized in that comprising described in claim any one of 1-6
Pharmaceutical composition.
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| CN107174653B (en) * | 2016-03-10 | 2020-11-17 | 深圳培元生物科技有限公司 | A method for promoting hair follicle regeneration |
| CN106492269A (en) * | 2016-12-09 | 2017-03-15 | 广东颜值科技有限公司 | A kind of temperature-sensitive hydrogel and preparation method thereof |
| CN107126556B (en) * | 2017-05-17 | 2021-01-26 | 沈阳何氏眼产业集团有限公司 | Stem cell extract, preparation method thereof and application thereof in preparation of skin wound repair preparation |
| CN108014328A (en) * | 2017-12-19 | 2018-05-11 | 吴广印 | A kind of pre-Anti-hair loss, the formula for promoting hair tonic material and preparation method thereof |
| CN111885999A (en) * | 2018-03-15 | 2020-11-03 | 艾得佩索拉公司 | Gel-forming polypeptides |
| CN109771694A (en) * | 2019-03-20 | 2019-05-21 | 江苏瑞思坦生物科技有限公司 | The preparation method and application of self assembly polypeptide nano fiber water gel scaffold material |
| CN110804580B (en) * | 2019-11-21 | 2021-09-28 | 山东大学 | Culture method for forming early micro hair follicle in vitro and application thereof |
| IL296772B2 (en) | 2020-03-31 | 2023-12-01 | 3 D Matrix Ltd | Sterilization of self-assembling peptides by irradiation |
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