CN105079783A - Pharmaceutical composition and preparation method and application thereof - Google Patents
Pharmaceutical composition and preparation method and application thereof Download PDFInfo
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- CN105079783A CN105079783A CN201410220064.2A CN201410220064A CN105079783A CN 105079783 A CN105079783 A CN 105079783A CN 201410220064 A CN201410220064 A CN 201410220064A CN 105079783 A CN105079783 A CN 105079783A
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Abstract
The invention provides a pharmaceutical composition used for healing skin and regenerating hair follicles and a preparation method and application thereof. The pharmaceutical composition comprises self-assembled polypeptide hydrogel and skin cells; the self-assembled polypeptide hydrogel is made from at least one of RADA16-I and PRG, the skin cells are disposed in the self-assembled polypeptide hydrogel. The inventor discovers that the use of the pharmaceutical composition is effective in promoting skin healing and hair follicle regeneration; in addition, the self-assembled polypeptide hydrogel in the pharmaceutical composition is fully artificially synthesized, never spreads animal-origin diseases, has good tissue specificity and is good for constructing a tissue specific stem cell differentiation microenvironment, material resources are more stable, and quality standards are easy to control.
Description
Technical field
The present invention relates to regenerative medicine, biomaterial and stem cell biology field, relate to pharmaceutical composition and its production and use particularly.
Background technology
Skin, as the maximum tissue of human body, is the barrier contacted with external environment, when causing skin injury due to the factor such as environmental damage or disease, usually causes the loss of wound surface moisture, electrolyte and protein, and open wound surface also add the probability of infection.In early days, effectively wound closure can reduce the generation of above-mentioned complication, conventional is kpetrolatum gauze and tomography/full thickness skin graft clinically, and wherein autologous split-thickness skin graft is transplanted is the recognized standard therapy.But large skin defect patient, still there is the defects such as and that cause body new damage not enough for district.
Self assembly polypeptide hydrogel material is the research field of just having risen for nearly ten years, and Preliminary Applications result has manifested its great potential in organizational project and regeneration and restoration.The self assembly polypeptide RADA16-I (Ac-RADARADARADARADA-CONH2) with bionic function of synthesis is the representative of such polypeptide, this small peptide is alternately arranged by 16 hydrophobic and hydrophilic amino acids and forms, form stable β-pleated sheet, in the aqueous solution of suitable pH, self assembly forms the orderly nanofiber of arrangement.Its aqueous solution water absorption is up to more than 99.5%, and nanofiber intersects to form the three-dimensional hydrogel structure being similar to extracellular matrix of tool mesh skeleton, plays supporting function to cell.This material has the good biological cells and tissues compatibility, nontoxic, can progressively be absorbed after implanting, and is desirable tissue engineering material.
At present, the wound healing and the hair follicle regeneration that self assembly polypeptide hydrogel are used for skin not yet have report.
Summary of the invention
The present invention is intended to solve one of technical problem in correlation technique at least to a certain extent.For this reason, one object of the present invention is to propose a kind of means that can be effective to skin healing and hair follicle regeneration.
In one aspect of the invention, the invention provides a kind of pharmaceutical composition for skin healing and hair follicle regeneration.According to embodiments of the invention, this pharmaceutical composition comprises: self assembly polypeptide hydrogel, and described self assembly polypeptide hydrogel is by RADA16-I and PRG (Ac-(RADA) 4GPRGDSGYRGDS-CONH
2) at least one formed; And Skin Cell, it is inner that described Skin Cell is arranged at described self assembly polypeptide hydrogel.Inventor finds, utilizes this pharmaceutical composition of the present invention, can effectively promote skin healing and hair follicle regeneration.In addition, the full synthetic of self assembly polypeptide hydrogel in pharmaceutical composition of the present invention, do not propagate zoonosis, the function fragment of different molecular can be added flexibly according to the features and applications of different tissues, make it to have more tissue specificity, be conducive to the structure of tissue specifc stem cells differentiation microenvironment, and material source is more stable, quality standard is easy to control.
According to the pharmaceutical composition of the embodiment of the present invention, following additional technical feature can also be had:
According to embodiments of the invention, when described hydrogel is formed jointly by RADA16-I and PRG, in described self assembly polypeptide hydrogel, the mass ratio of described RADA16-I and described PRG is 1:1.
According to embodiments of the invention, in described self assembly polypeptide hydrogel, the content of described Skin Cell is 10
4-10
8individual/mL.
According to embodiments of the invention, described Skin Cell is at least one being selected from hypodermal cell and epidermis cell.
According to embodiments of the invention, described hypodermal cell is the new hypodermal cell be separated or the corium stem cell cultivating 0-30 generation.
According to embodiments of the invention, described epidermis cell is the new epidermis cell be separated or the epidermal stem cells cultivating 0-30 generation.
According to embodiments of the invention, described pharmaceutical composition comprises further: be conducive to cell survival, the cell active factor of growth and/or extracellular matrix molecule.According to embodiments of the invention, described cell active factor is for being selected from least one of bFGF (basic fibroblast growth factor), EGF (epithelical cell growth factor), and described extracellular matrix molecule is at least one being selected from hyaluronic acid, fibronectin splicing variants.Thereby, it is possible to effectively promote the survival and growth of Skin Cell, and then promote that the effect of skin healing and hair follicle regeneration is better.
In another aspect of this invention, the invention provides a kind of method preparing foregoing pharmaceutical composition.According to embodiments of the invention, the method comprises: (1) provides RADA16-I solution and PRG solution, and wherein, the concentration of described RADA16-I solution is 0.1-1g/100ml, and the concentration of described PRG solution is 0.1-1g/100ml; (2) by described RADA16-I solution and the mixing of described PRG solution equal-volume, to obtain mixing water gel solution; (3) in described RADA16-I solution, described PRG solution or described mixing water gel solution, inoculate Skin Cell, and hatch under the condition being suitable for described dermal cell growth, to obtain described pharmaceutical composition.Inventor finds, utilizes the method for the present invention can fast and effeciently prepare foregoing pharmaceutical composition, and simple, conveniently fast, easily to control, be easy to accomplish scale production.
According to embodiments of the invention, before step (2), in advance described RADA16-I solution and described PRG solution are carried out 0.22 μm of filtration sterilization process.
According to embodiments of the invention, described inoculating cell comprises further: with 10% aseptic sucrose solution, described Skin Cell being made into concentration is 10
4-10
8the cell suspension of individual/mL, mixes described mixing water gel solution and described cell suspension according to the ratio that volume ratio is 5:1.
According to embodiments of the invention, described in hatch and comprise further: every 30 minutes, remove the culture medium in orifice plate, add equal-volume fresh culture in orifice plate, repeat 3-5 time.According to embodiments of the invention, include in described culture medium and be beneficial to cell survival, the cell active factor of growth and/or extracellular matrix molecule, according to embodiments of the invention, described cell active factor is at least one being selected from bFGF, EGF, and described extracellular matrix molecule is at least one being selected from hyaluronic acid, fibronectin splicing variants.Thereby, it is possible to effectively promote the survival and growth of Skin Cell, and then promote that the effect of skin healing and hair follicle regeneration is better.
According to embodiments of the invention, the concentration of described RADA16-I solution is 1g/100mL.
According to embodiments of the invention, the concentration of described PRG solution is 1g/100mL.
According to embodiments of the invention, described Skin Cell is at least one being selected from epidermis cell and hypodermal cell.
According to embodiments of the invention, described hypodermal cell is the new hypodermal cell be separated or the corium stem cell cultivating 0-30 generation.
According to embodiments of the invention, described epidermis cell is the new epidermis cell be separated or the epidermal stem cells cultivating 0-30 generation.
In still another aspect of the invention, the invention provides foregoing pharmaceutical composition and preparing the purposes in medicine, described medicine is used for skin healing and hair follicle regeneration.
In another aspect of the invention, the invention provides a kind of medicine for skin healing and hair follicle regeneration.According to embodiments of the invention, this pharmaceutical pack is containing foregoing pharmaceutical composition.Inventor finds, medicine of the present invention can be effective to skin healing and hair follicle regeneration.
Accompanying drawing explanation
Fig. 1 shows according to embodiments of the invention, the mode of appearance figure of pharmaceutical composition of the present invention;
Fig. 2 shows according to embodiments of the invention, when cultivating 1 day, and the Laser Scanning Confocal Microscope photo of corium stem cell in pharmaceutical composition;
Fig. 3 shows according to embodiments of the invention, when cultivating 3 days, and the Laser Scanning Confocal Microscope photo of corium stem cell in pharmaceutical composition;
Fig. 4 shows according to embodiments of the invention, the two colored graph of the AO/PI of corium stem cell in pharmaceutical composition;
Fig. 5 shows according to embodiments of the invention, alkaline phosphatase (AP) colored graph of corium stem cell in pharmaceutical composition;
Fig. 6 shows according to embodiments of the invention, and transplant medicine compositions is after 3 weeks, and nude mice skin heals and hair follicle regeneration situation result figure;
Fig. 7 shows according to embodiments of the invention, cultivates after 1 day, the inverted microscope photo in the hydrogel that MSC is formed at the mixing water gel solution by variable concentrations, wherein,
Inverted microscope photo in Fig. 7 A hydrogel that to be MSC formed at the mixing water gel solution by peptide concentration being 0.1g/100mL,
Inverted microscope photo in Fig. 7 B hydrogel that to be MSC formed at the mixing water gel solution by peptide concentration being 0.5g/100mL,
Inverted microscope photo in Fig. 7 C hydrogel that to be MSC formed at the mixing water gel solution by peptide concentration being 1g/100mL;
Fig. 8 shows according to embodiments of the invention, cultivates after 3 days, the growing state of corium stem cell in RADA16-I self assembly polypeptide hydrogel.
Detailed description of the invention
Embodiments of the invention are described below in detail.Embodiment described below is exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.Unreceipted concrete technology or condition in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
In one aspect of the invention, the invention provides a kind of pharmaceutical composition for skin healing and hair follicle regeneration.According to embodiments of the invention, this pharmaceutical composition comprises: self assembly polypeptide hydrogel, and described self assembly polypeptide hydrogel is formed by least one of RADA16-I and PRG; And Skin Cell, it is inner that described Skin Cell is arranged at described self assembly polypeptide hydrogel.Inventor finds, utilizes this pharmaceutical composition of the present invention, can effectively promote skin healing and hair follicle regeneration.In addition, the full synthetic of self assembly polypeptide hydrogel in pharmaceutical composition of the present invention, do not propagate zoonosis, the function fragment of different molecular can be added flexibly according to the features and applications of different tissues, make it to have more tissue specificity, be conducive to the structure of tissue specifc stem cells differentiation microenvironment, and material source is more stable, quality standard is easy to control.
According to embodiments of the invention, described self assembly polypeptide hydrogel can be formed separately by the one of RADA16-I and PRG, also can be formed by the mixture of RADA16-I and PRG, when described hydrogel is formed jointly by RADA16-I and PRG, in described self assembly polypeptide hydrogel, the mass ratio of described RADA16-I and described PRG is 1:1.Thus, the performance of self assembly polypeptide hydrogel is better, effectively can support growth and the propagation of Skin Cell.
According to embodiments of the invention, in described self assembly polypeptide hydrogel, the content of described Skin Cell is not particularly limited, and those skilled in the art can select flexibly according to practical situation.According to some embodiments of the present invention, the content of Skin Cell can be 10
4-10
8individual/mL.Thus, pharmaceutical composition of the present invention promotes that the effect of skin healing and hair follicle regeneration is better.
According to embodiments of the invention, the kind of described Skin Cell is not particularly limited.According to some embodiments of the present invention, Skin Cell can for being selected from least one of hypodermal cell and epidermis cell.Thereby, it is possible to effectively promote skin healing and hair follicle regeneration.
According to embodiments of the invention, the kind of described hypodermal cell is not particularly limited.According to some embodiments of the present invention, hypodermal cell can be the new hypodermal cell be separated or the corium stem cell cultivating 0-30 generation.Thus, the growth conditions of described hypodermal cell in self assembly polypeptide hydrogel is good, can Effective multiplication, and then effectively can play the effect promoting skin healing and hair follicle regeneration.
According to embodiments of the invention, the kind of described epidermis cell is not particularly limited.According to some embodiments of the present invention, epidermis cell can be the new epidermis cell be separated or the epidermal stem cells cultivating 0-30 generation.Thus, the growth conditions of described epidermis cell in self assembly polypeptide hydrogel is good, can Effective multiplication, and then effectively can play the effect promoting skin healing and hair follicle regeneration.
According to embodiments of the invention, described pharmaceutical composition can further include: be conducive to cell survival, the cell active factor of growth and/or extracellular matrix molecule.According to embodiments of the invention, the kind of described cell active factor is not particularly limited, as long as can effectively promote the growth of Skin Cell and breed.According to a concrete example of the present invention, cell active factor can for being selected from least one of bFGF (basic fibroblast growth factor), EGF (epithelical cell growth factor).According to embodiments of the invention, the kind of described extracellular matrix molecule is not particularly limited.According to some embodiments of the present invention, extracellular matrix molecule can for being selected from least one of hyaluronic acid, fibronectin splicing variants.Thereby, it is possible to effectively promote the survival and growth of Skin Cell, and then promote that the effect of skin healing and hair follicle regeneration is better.
In another aspect of this invention, the invention provides a kind of method preparing foregoing pharmaceutical composition.According to embodiments of the invention, the method comprises the following steps:
(1) provide RADA16-I solution and PRG solution, wherein, the concentration of described RADA16-I solution is 0.1-1g/100ml, and the concentration of described PRG solution is 0.1-1g/100ml.
According to embodiments of the invention, the concentration of described RADA16-I solution is 1g/100mL.Thus, the performance of the self assembly polypeptide hydrogel obtained is better, and the effect of skin healing and hair follicle regeneration is better.
According to embodiments of the invention, the concentration of described PRG solution is 1g/100mL.Thus, the performance of the self assembly polypeptide hydrogel obtained is better, and the effect of skin healing and hair follicle regeneration is better.
According to embodiments of the invention, before carrying out subsequent step, in advance described RADA16-I solution and described PRG solution are carried out 0.22 μm of filtration sterilization process.Thereby, it is possible to effectively carry out degerming to RADA16-I solution and PRG solution, obtain aseptic RADA16-I solution and PRG solution, be convenient to the operation of subsequent step.
(2) by described RADA16-I solution and the mixing of described PRG solution equal-volume, to obtain mixing water gel solution.
According to embodiments of the invention, before by RADA16-I solution and the mixing of PRG solution, in advance RADA16-I solution and PRG solution can be distinguished ultrasonic 30 minutes.
(3) in described RADA16-I solution, described PRG solution or described mixing water gel solution, inoculate Skin Cell, and hatch under the condition being suitable for described dermal cell growth, to obtain described pharmaceutical composition.
According to embodiments of the invention, described inoculation Skin Cell comprises further: with 10% aseptic sucrose solution, described Skin Cell being made into concentration is 10
4-10
8the cell suspension of individual/mL, mixes described mixing water gel solution and described cell suspension according to the ratio that volume ratio is 5:1.Thus, under suitable pH condition, RADA16-I and PRG in mixing water gel solution spontaneously can be assembled into nanofiber, and occur crosslinked further, form the hydrogel of porous network structure, thus effectively obtain the inner self assembly polypeptide hydrogel being provided with Skin Cell, i.e. pharmaceutical composition of the present invention.
According to embodiments of the invention, the kind of described Skin Cell is not particularly limited.According to some embodiments of the present invention, Skin Cell can for being selected from least one of epidermis cell and hypodermal cell.Thus, promote that the effect of skin healing and hair follicle regeneration is better.
According to embodiments of the invention, the kind of described hypodermal cell is not particularly limited.According to some embodiments of the present invention, hypodermal cell can be the new hypodermal cell be separated or the corium stem cell cultivating 0-30 generation.Thus, the growth conditions of described hypodermal cell in self assembly polypeptide hydrogel is good, can Effective multiplication, and then effectively can play the effect promoting skin healing and hair follicle regeneration.
According to embodiments of the invention, the kind of described epidermis cell is not particularly limited.According to some embodiments of the present invention, epidermis cell can be the new epidermis cell be separated or the epidermal stem cells cultivating 0-30 generation.Thus, the growth conditions of described epidermis cell in self assembly polypeptide hydrogel is good, can Effective multiplication, and then effectively can play the effect promoting skin healing and hair follicle regeneration.
According to embodiments of the invention, described in hatch and comprise further: every 30 minutes, remove the culture medium in orifice plate, add equal-volume fresh culture in orifice plate, repeat 3-5 time.Thereby, it is possible to effectively regulate the pH value of self assembly polypeptide hydrogel, make growth and the propagation of its applicable Skin Cell, thus be effective to skin healing and hair follicle regeneration.According to embodiments of the invention, include in described culture medium and be beneficial to cell survival, the cell active factor of growth and/or extracellular matrix molecule, according to embodiments of the invention, described cell active factor is at least one being selected from bFGF, EGF, and described extracellular matrix molecule is at least one being selected from hyaluronic acid, fibronectin splicing variants.Thereby, it is possible to effectively promote the survival and growth of Skin Cell, and then promote that the effect of skin healing and hair follicle regeneration is better.
Inventor finds, utilizes the method for the present invention can fast and effeciently prepare foregoing pharmaceutical composition, and simple, conveniently fast, easily to control, be easy to accomplish scale production.
In still another aspect of the invention, the invention provides foregoing pharmaceutical composition and preparing the purposes in medicine, described medicine is used for skin healing and hair follicle regeneration.Inventor finds, in pharmaceutical composition of the present invention, self assembly polypeptide hydrogel has the good biological cells and tissues compatibility, nontoxic, effectively can support growth and the propagation of Skin Cell, can progressively be absorbed after implanting, Skin Cell can effectively grow and propagation in self assembly polypeptide hydrogel, under the supporting function of self assembly polypeptide hydrogel, can effectively promote skin healing and hair follicle regeneration.
In another aspect of the invention, the invention provides a kind of medicine for skin healing and hair follicle regeneration.According to embodiments of the invention, this pharmaceutical pack is containing foregoing pharmaceutical composition.Inventor finds, this pharmaceutical composition of the present invention can be effective to skin healing and hair follicle regeneration.
Be described below in detail embodiments of the invention, if no special instructions, in following embodiment, isolated skin stem cell is separated from the skin histology of C57 mice and cultivates; The separation method of Skin Cell is: be cut into small pieces by mouse skin with dispaseII (sigma), 37 DEG C digest 2 hours, peel off corium and epidermis, use type i collagen enzyme (sigma) digestion to obtain corium and the epidermis cell of new separation respectively; Carbonic ester film (transwell) is purchased from Corning company, and catalog number is 3470; Cell culture medium consists of DMEM/F12 (3:1) (buying in Invitrogen company), containing 2%B27 (buying in Invitrogen company), 40ng/ μ LbFGF (Peprotech) and 20ng/ μ LEGF (Peprotech), except embodiment 1, other embodiments cultivate the culture medium culture medium all for this reason of corium stem cell; 3M film is TegadermTMFilm; Culture dish is suspension cell culture ware, and purchased from Guangzhou Ztel's biofiltration Products Co., Ltd, article No. is MCD-000-090.Laboratory animal is all purchased from Guangdong Medical Lab Animal Center.
Embodiment 1
People source mescenchymal stem cell (MSC) is utilized to measure the cell compatibility of self assembly polypeptide hydrogel, specific as follows:
(1) adopt solid phase synthesis process to carry out Peptide systhesis, by Peptide synthesizer improvement on synthesis molecule, N end and the C end of peptide chain are modified by acetylation and amidatioon, prevent it from degrading rapidly.Polypeptide RADA16-I (Ac-RADARADARADARADA-CONH after synthesis
2) and PRG (Ac-(RADA)
4gPRGDSGYRGDS-CONH
2) carry out purification with HPLC after, lyophilization, obtains RADA16-I and PRG polypeptide lyophilized powder respectively.
(2) RADA16-I and the PRG polypeptide lyophilized powder prepared in step (1) is used deionized water dissolving respectively, obtain RADA16-I solution that peptide concentration is 1g/100mL, 0.5g/100mL, 0.1g/100mL and PRG solution, then, degerming with the membrane filtration of 0.22 μm, obtain aseptic RADA16-I solution and the PRG solution of variable concentrations.
(3) the aseptic RADA16-I solution of variable concentrations and PRG solution are distinguished ultrasonic 30 minutes.
(4) by the RADA16-I solution of the same concentrations through supersound process and the mixing of PRG solution equal-volume, be in particular each 50 μ L, obtain the mixing water gel solution of three kinds of concentration.
(5) after mixing water gel solution being mixed fast, add in 24 hole transwell, then slowly add culture medium (DMEM, Corningcellgro, containing 10% hyclone) along culture plate hole wall.
(6) at 5%CO
2, leave standstill 30min in 37 DEG C of incubators after, slowly siphon away culture medium with rifle head, more again add fresh culture medium in orifice plate.
(7) leave standstill 30min again, then repeat the operation of step (6).
(8) leave standstill 30min again, then repeat the operation of step (6).
(9) leave standstill 30min or longer time again, then repeat the operation of step (6).
(10) MSC kind is surperficial to self assembly polypeptide hydrogel, and add culture medium, be then placed in 37 DEG C, 5%CO
2in incubator, quiescent culture.The adhesion situation of observation of cell and self assembly polypeptide hydrogel under inverted microscope.Cultivate its aspect graph after 1 day and see Fig. 7, wherein, inverted microscope photo in Fig. 7 A hydrogel that to be MSC formed at the mixing water gel solution by peptide concentration being 0.1g/100mL, inverted microscope photo in Fig. 7 B hydrogel that to be MSC formed at the mixing water gel solution by peptide concentration being 0.5g/100mL, the inverted microscope photo in Fig. 7 C hydrogel that to be MSC formed at the mixing water gel solution by peptide concentration being 1g/100mL.As seen from Figure 7, the self assembly polypeptide hydrogel of three kinds of concentration all has certain supporting function to cell adhesion, and in the hydrogel of 1g/100mL, cell can stretch better, cell growth and be stained with better supporting function.
Embodiment 2
(1) adopt solid phase synthesis process to carry out Peptide systhesis, by Peptide synthesizer improvement on synthesis molecule, N end and the C end of peptide chain are modified by acetylation and amidatioon, prevent it from degrading rapidly.Polypeptide RADA16-I (Ac-RADARADARADARADA-CONH after synthesis
2) and PRG (Ac-(RADA)
4gPRGDSGYRGDS-CONH
2) carry out purification with HPLC after, lyophilization, obtains RADA16-I and PRG polypeptide lyophilized powder respectively.
(2) RADA16-I and the PRG polypeptide lyophilized powder prepared in step (1) is used deionized water dissolving respectively, obtain RADA16-I solution and PRG solution that peptide concentration is 1g/100mL, then, degerming with the membrane filtration of 0.22 μm, obtain aseptic RADA16-I solution and PRG solution.
(3) aseptic RADA16-I solution and PRG solution are distinguished ultrasonic 30 minutes.
(4) by through the RADA16-I solution of supersound process and the mixing of PRG solution equal-volume, be in particular each 50 μ L, obtain mixing water gel solution.
(5) by the 3rd generation mouse skin corium stem cell (SKP) 10 of suspension culture
4individual, centrifugal segregation culture medium, with the aseptic sucrose solution washed cell of 1mL10%, then centrifugal, remove supernatant, then add the aseptic sucrose solution of 20 μ L10% and be mixed with cell suspension.
(6) monoploid being amassed cell suspension and pentaploid amasss after mixing water gel solution mixes fast, adds in 24 hole transwell, then slowly adds culture medium along culture plate hole wall.
(7) at 5%CO
2, leave standstill 30min in 37 DEG C of incubators after, slowly siphon away culture medium with rifle head, more again add fresh culture medium in 24 hole transwell and orifice plate, put back to incubator.
(8) leave standstill 30min again, then repeat the operation of step (7).
(9) leave standstill 30min again, then repeat the operation of step (7).
(10) leave standstill 30min again, then repeat the operation of step (7).
(11) obtained pharmaceutical composition is placed in 37 DEG C, 5%CO
2in incubator, quiescent culture, obtains pharmaceutical composition, and its mode of appearance figure is shown in Fig. 1.
Cultivate after 1 or 3 day, the growing state of corium stem cell in self assembly polypeptide hydrogel is observed by Laser Scanning Confocal Microscope (OLYMPUS), Fig. 2 and Fig. 3 be shown in by Laser Scanning Confocal Microscope photo, wherein, Fig. 2 is cultivation 1 day, the Laser Scanning Confocal Microscope photo of corium stem cell, Fig. 3 is the Laser Scanning Confocal Microscope photo of cultivation 3 days, corium stem cell.As can be seen from Fig. 2 and Fig. 3, most corium stem cell starts to stretch, and shows that the growth conditions of corium stem cell is good.
See Fig. 4 with the two colored graph of AO/PI double-staining identification of cell life or death: AO/PI, wherein, green is living cells, and redness is dead cell.Can be seen by Fig. 4, almost can't see red dead cell, illustrate that, in pharmaceutical composition of the present invention, corium stem cell has good survival rate.
The dryness of corium stem cell is detected with BCIP/NBT alkaline phosphatase (AP) colour reagent box (the green skies).Alkaline phosphatase (AP) colored graph is shown in Fig. 5.Result shows that skin progenitor cell maintains good dryness in self assembly polypeptide hydrogel.
Embodiment 3
(1) adopt solid phase synthesis process to carry out Peptide systhesis, by Peptide synthesizer improvement on synthesis molecule, N end and the C end of peptide chain are modified by acetylation and amidatioon, prevent it from degrading rapidly.Polypeptide RADA16-I (Ac-RADARADARADARADA-CONH after synthesis
2) and PRG (Ac-(RADA)
4gPRGDSGYRGDS-CONH
2) carry out purification with HPLC after, lyophilization, obtains RADA16-I and PRG polypeptide lyophilized powder respectively.
(2) RADA16-I and the PRG polypeptide lyophilized powder prepared in step (1) is used deionized water dissolving respectively, obtain RADA16-I solution that peptide concentration is 1g/100mL and PRG solution, then, degerming with the membrane filtration of 0.22 μm, obtain aseptic RADA16-I solution and PRG solution.
(3) aseptic RADA16-I solution and PRG solution are distinguished ultrasonic 30 minutes.
(4) by through the RADA16-I solution of supersound process and the mixing of PRG solution equal-volume, be in particular each 50 μ L, obtain mixing water gel solution.
(5) by the 3rd generation mouse skin corium stem cell (SKP) (or new newborn rat hypodermal cell be separated) 10 of suspension culture
6individual, with the newborn rat epidermis cell 10 be newly separated
6individual mixing, centrifugal segregation culture medium, with the aseptic sucrose solution washed cell of 1mL10%, then centrifugal, remove supernatant, then add the aseptic sucrose solution of 20 μ L10% and be mixed with cell suspension.
(6) monoploid being amassed cell suspension and pentaploid amasss after mixing water gel solution mixes fast, adds in 24 hole transwell, then slowly adds culture medium along culture plate hole wall.
(7) at 5%CO
2, leave standstill 30min in 37 DEG C of incubators after, slowly siphon away culture medium with rifle head, more again add fresh culture medium in transwell and orifice plate, put back to incubator.
(8) leave standstill 30min again, then repeat the operation of step (7).
(9) leave standstill 30min again, then repeat the operation of step (7).
(10) leave standstill 30min again, then repeat the operation of step (7).
(11) obtained pharmaceutical composition is placed in 37 DEG C, 5%CO
2in incubator, quiescent culture, obtains pharmaceutical composition.
Embodiment 4
According to following steps, detect the effect that the pharmaceutical composition prepared in embodiment 3 promotes skin healing and hair follicle regeneration, specific as follows:
By the pentobarbital sodium anesthesia of 1% of BALB/c nude mice, on its skin of back, manufacture area with card punch and be about 7mm
2wound, then, the pharmaceutical composition prepared in embodiment 2 is transplanted to nude mice skin wound surface, and covers nude mice wound surface with 3M film, then with binder by nude mice wound dressing.Normal raising nude mice, strips the bandage off after 3 weeks and removes 3M film, observes nude mice skin healing and hair follicle regeneration situation.
Experimental result as shown in Figure 6.As seen from Figure 6, nude mice skin wound heals completely, and grows hair, shows that pharmaceutical composition of the present invention can be effective to skin healing and hair follicle regeneration.
Embodiment 5
(1) adopt solid phase synthesis process to carry out Peptide systhesis, by Peptide synthesizer improvement on synthesis molecule, N end and the C end of peptide chain are modified by acetylation and amidatioon, prevent it from degrading rapidly.Polypeptide RADA16-I (Ac-RADARADARADARADA-CONH2) after synthesis, after carrying out purification with HPLC, lyophilization, obtains RADA16-I polypeptide lyophilized powder.
(2) the RADA16-I polypeptide lyophilized powder prepared in step (1) is used deionized water dissolving respectively, obtain the RADA16-I solution that peptide concentration is 1g/100mL, then use the membrane filtration of 0.22 μm degerming, obtain aseptic RADA16-I solution.
(3) by ultrasonic for aseptic RADA16-I solution 30 minutes.
(4) by the 3rd generation mouse skin corium stem cell (SKP) 10 of suspension culture
4individual, centrifugal segregation culture medium, with the aseptic sucrose solution washed cell of 1mL10%, then centrifugal, remove supernatant, then add the aseptic sucrose solution of 20 μ L10% and be mixed with cell suspension.
(5) by 20 μ L cell suspension and 100 μ L ultrasonic after RADA16-I solution mix fast after, add in 24 hole transwell, then slowly add culture medium along culture plate hole wall.
(6) at 5%CO
2, leave standstill 30min in 37 DEG C of incubators after, slowly siphon away culture medium with rifle head, more again add fresh culture medium in 24 hole transwell and orifice plate, put back to incubator.
(7) leave standstill 30min, then repeat the operation of step (6).
(8) leave standstill 30min again, then repeat the operation of step (6).
(9) leave standstill 30min again, then repeat the operation of step (6)
(10) obtained pharmaceutical composition is placed in 37 DEG C, 5%CO
2in incubator, quiescent culture, obtains pharmaceutical composition.
By pharmaceutical composition obtained above at 37 DEG C, 5%CO
2cultivate after 3 days in incubator, observe the growing state of corium stem cell in self assembly polypeptide hydrogel by inverted microscope (Nikon), microphotograph is shown in Fig. 8.As can be seen from Figure 8, most corium stem cell can grow preferably in RADA16-I self assembly polypeptide hydrogel.
In the description of this description, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not must for be identical embodiment or example.And the specific features of description, structure, material or feature can combine in one or more embodiment in office or example in an appropriate manner.In addition, when not conflicting, the feature of the different embodiment described in this description or example and different embodiment or example can carry out combining and combining by those skilled in the art.
Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, and those of ordinary skill in the art can change above-described embodiment within the scope of the invention, revises, replace and modification.
Claims (10)
1., for a pharmaceutical composition for skin healing and hair follicle regeneration, it is characterized in that, comprising:
Self assembly polypeptide hydrogel, described self assembly polypeptide hydrogel is formed by least one of RADA16-I and PRG; And
Skin Cell, it is inner that described Skin Cell is arranged at described self assembly polypeptide hydrogel.
2. pharmaceutical composition according to claim 1, is characterized in that, when described hydrogel is formed jointly by RADA16-I and PRG, in described self assembly polypeptide hydrogel, the mass ratio of described RADA16-I and described PRG is 1:1.
3. pharmaceutical composition according to claim 1, is characterized in that, in described self assembly polypeptide hydrogel, the content of described Skin Cell is 10
4-10
8individual/mL.
4. pharmaceutical composition according to claim 3, is characterized in that, described Skin Cell is at least one being selected from hypodermal cell and epidermis cell,
Optionally, described hypodermal cell is the new hypodermal cell be separated or the corium stem cell cultivating 0-30 generation,
Optionally, described epidermis cell is the new epidermis cell be separated or the epidermal stem cells cultivating 0-30 generation.
5. pharmaceutical composition according to claim 1, is characterized in that, comprises further:
Be conducive to cell survival, the cell active factor of growth and/or extracellular matrix molecule,
Optionally, described cell active factor is at least one being selected from bFGF, EGF, and described extracellular matrix molecule is at least one being selected from hyaluronic acid, fibronectin splicing variants.
6. prepare a method for the pharmaceutical composition described in any one of claim 1-5, it is characterized in that, comprising:
(1) provide RADA16-I solution and PRG solution, wherein, the concentration of described RADA16-I solution is 0.1-1g/100ml, and the concentration of described PRG solution is 0.1-1g/100ml;
(2) by described RADA16-I solution and the mixing of described PRG solution equal-volume, to obtain mixing water gel solution;
(3) in described RADA16-I solution, described PRG solution or described mixing water gel solution, inoculate Skin Cell, and hatch under the condition being suitable for described dermal cell growth, to obtain described pharmaceutical composition.
7. method according to claim 6, is characterized in that, before step (2), in advance described RADA16-I solution and described PRG solution is carried out 0.22 μm of filtration sterilization process,
Optionally, described inoculating cell comprises further:
With 10% aseptic sucrose solution, described Skin Cell being made into concentration is 10
4-10
8the cell suspension of individual/mL, mixes described mixing water gel solution and described cell suspension according to the ratio that volume ratio is 5:1,
Optionally, hatch described in and comprise further:
Every 30 minutes, remove the culture medium in orifice plate, add equal-volume fresh culture in orifice plate, repeat 3-5 time,
Optionally, include in described culture medium and be beneficial to cell survival, the cell active factor of growth and/or extracellular matrix molecule,
Optionally, described cell active factor is at least one being selected from bFGF, EGF, and described extracellular matrix molecule is at least one being selected from hyaluronic acid, fibronectin splicing variants.
8. method according to claim 6, is characterized in that, the concentration of described RADA16-I solution is 1g/100mL,
Optionally, the concentration of described PRG solution is 1g/100mL,
Optionally, described Skin Cell is at least one being selected from epidermis cell and hypodermal cell,
Optionally, described hypodermal cell is the new hypodermal cell be separated or the corium stem cell cultivating 0-30 generation,
Optionally, described epidermis cell is the new epidermis cell be separated or the epidermal stem cells cultivating 0-30 generation.
9. the pharmaceutical composition described in any one of claim 1-5 is preparing the purposes in medicine, and described medicine is used for skin healing and hair follicle regeneration.
10., for a medicine for skin healing and hair follicle regeneration, it is characterized in that, comprise the pharmaceutical composition described in any one of claim 1-5.
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| 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 |
| CN107126556A (en) * | 2017-05-17 | 2017-09-05 | 何伟 | A kind of stem cell extract and preparation method thereof and the application in skin wound preparation for repairing is prepared |
| CN107174653A (en) * | 2016-03-10 | 2017-09-19 | 深圳培元生物科技有限公司 | One kind promotes skin follicle regeneration method |
| 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 |
| 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 |
| CN110804580A (en) * | 2019-11-21 | 2020-02-18 | 山东大学 | Culture method for forming early micro hair follicle in vitro and application thereof |
| CN111885999A (en) * | 2018-03-15 | 2020-11-03 | 艾得佩索拉公司 | Gel-forming polypeptides |
| EP4125995A4 (en) * | 2020-03-31 | 2024-04-10 | 3 D Matrix Ltd | Sterilization of self-assembling peptides by irradiation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107174653A (en) * | 2016-03-10 | 2017-09-19 | 深圳培元生物科技有限公司 | One kind promotes skin follicle regeneration method |
| 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 |
| CN107126556A (en) * | 2017-05-17 | 2017-09-05 | 何伟 | A kind of stem cell extract and preparation method thereof and the application in skin wound preparation for repairing is prepared |
| 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 |
| CN110804580A (en) * | 2019-11-21 | 2020-02-18 | 山东大学 | Culture method for forming early micro hair follicle in vitro and application thereof |
| 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 |
| EP4125995A4 (en) * | 2020-03-31 | 2024-04-10 | 3 D Matrix Ltd | Sterilization of self-assembling peptides by irradiation |
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