CN108992466B - Preparation method of injection for treating androgenetic alopecia by using hair papilla cell exosomes - Google Patents
Preparation method of injection for treating androgenetic alopecia by using hair papilla cell exosomes Download PDFInfo
- Publication number
- CN108992466B CN108992466B CN201811161456.0A CN201811161456A CN108992466B CN 108992466 B CN108992466 B CN 108992466B CN 201811161456 A CN201811161456 A CN 201811161456A CN 108992466 B CN108992466 B CN 108992466B
- Authority
- CN
- China
- Prior art keywords
- hair
- culture
- hair papilla
- cells
- injection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0627—Hair cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Dermatology (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Virology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Developmental Biology & Embryology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Cosmetics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to an injection for treating androgenetic alopecia by utilizing hair papilla cell exosomes, which is a physiological saline solvent containing the hair papilla cell exosomes. The hair papilla cells obtained by the preparation method have large secretion amount and high content of effective components for promoting hair regeneration, so that the injection has good effect of treating androgenetic alopecia.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of an injection for treating androgenetic alopecia by using hair papilla cell exosomes.
Background
The prevalence of androgenetic alopecia (AGA) increases with age and affects both men and women. Patients diagnosed with AGA may experience a reduction in quality of life. While natural and synthetic ingredients can be used to treat AGA and promote hair growth, they have serious side effects and unknown mechanisms of action, limiting their use. At present, studies have been made to isolate Dermal Papilla Cells (DPC) that play a key role in hair follicle regeneration from the hair follicles of normal patient's own hair, culture and proliferate them in vitro to obtain exosomes, and inject them into bald areas of patients, thereby generating new normal hair. Researchers further find that in the process of promoting hair growth by exosome, Wnt1a protein carried by exosome stimulates hair follicle portion hair papilla (DP) cells of a subject to induce hair circulation so as to promote hair regeneration.
However, the collection of the stem cell exosomes at the present stage is difficult to achieve the treatment requirement, and the differences between the secreted exosome amount and the types of main substances contained in the exosomes are large under different environments, different pH values and different induction conditions, so that troubles are brought to the research and clinical application of the exosomes.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of an injection for treating androgenetic alopecia by utilizing hair papilla cell exosomes, and the amount of the exosomes secreted by the hair papilla cells obtained in the preparation process is large, and the content of effective components for promoting hair regeneration is high, so that the injection has a good effect of treating androgenetic alopecia.
In order to solve the above technical problems, the present invention provides a method for preparing an injection for treating androgenetic alopecia using hair papilla cell exosomes, wherein the injection is a physiological saline solvent containing hair papilla cell exosomes obtained in a serum-free basic culture medium, and the preparation method comprises the following steps:
(1) separating hair papilla cells;
(2) adherent culture of hair papilla cells: carrying out adherent culture on the dermal papilla cells obtained in the step (1);
(3) stimulating and inducing the culture of hair papilla cells: transferring the primary hair papilla cells obtained by adherent culture in the step (2) to a cell culture system when the primary hair papilla cells are transferred to the third generation, culturing in a three-air culture box, reducing the oxygen concentration in the culture box to 0.1-3% when the primary hair papilla cells are cultured to the 7 th day, and simultaneously adding 1-5 x 10% of final concentration−4 Stimulating and inducing the minoxidil of M; after 24 hours of stimulation induction, removing the culture medium for adherent culture in the step (2), and cleaning for 3 times by using a serum-free basal culture medium, wherein the culture medium is changed into the serum-free basal culture medium, and the oxygen content is changed to 5%;
(4) preparing exosome freeze-dried powder: culturing in a serum-free basic culture medium for 24-48 h in the step (3), collecting cell supernatant, and dissolving with PBS (phosphate buffer solution) after low-temperature ultracentrifugation to obtain papilla cell exosomes; counting, subpackaging into ampoule bottles, placing at the temperature of-50 to-80 ℃ for freezing treatment, and then carrying out freeze-drying treatment at the temperature of-50 ℃ to obtain hair papilla cell exosome freeze-dried powder;
(5) preparing an injection: the hair papilla cell exosome freeze-dried powder is dissolved into a physiological saline solvent containing the hair papilla cell exosomes by using physiological saline.
Further, the separation of papilla pili cells in the step (1) comprises the following steps:
a. placing a single hair follicle after operation in a hair follicle collection liquid, preserving the hair follicle in an environment at 4 ℃, and cleaning hair follicle tissues for 2-3 times by using a PBS (phosphate buffered saline) solution containing 1% of double antibodies by mass fraction;
b. separating the hair bulb tissue with a 1ml syringe needle under a microscope, removing residual hair matrix, and transferring the tissue to a new culture dish containing PBS solution with a pipette;
c. washing PBS once, adding 0.2% collagenase IV, digesting at 37 ℃ for 1-2 h, observing the digestion state at intervals of 10min, simultaneously blowing and beating the tissue by using a suction pipe, after the dermal sheath tissue around the papillary hair tissue is completely dissociated, selecting single papillary hair cells by using a pipette under a microscope, and placing the papillary hair cells into a new culture dish.
Further, the papilla pili cells are cultured at 37 ℃ in 5% CO in the step (2)2And (3) carrying out adherent culture in an incubator, replacing the culture medium after the tissues are completely adherent and cells climb out, replacing the culture medium every 3 days, and carrying out subculture when the cells grow and fuse to reach 80%.
Further, the culture medium for adherent culture in the step (2) comprises DMEM/F12, fetal bovine serum, EGF, bFGF and double antibody.
The invention has the advantages that:
1. based on the important function of hair papilla cells and exosomes secreted by the hair papilla cells in the process of regulating and controlling the hair cycle, the exosomes of the hair papilla cells are used for treating androgenetic alopecia and are prepared into injection, so that the treatment is convenient and the effect is good.
2. The minoxidil is adopted to stimulate hair papilla cells, so that the hair papilla cells secrete exosomes with specific alopecia treatment effect, and the alopecia treatment effect is achieved.
3. The stimulation induction culture environment of the hair papilla cells adopts a low-oxygen condition, which is favorable for improving the capability of the hair papilla cells for secreting exosomes, thereby obtaining more exosomes.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and embodiments.
FIG. 1 is a drawing showing the immunohistochemical identification of a hair papilla cell surface marker (. alpha. -SMA), wherein A, B is a drawing showing the immunohistochemical identification of a fibroblast and a third generation hair papilla cell obtained by the method of the present invention, respectively.
FIG. 2 is an electron micrograph of exosomes obtained with the method of the invention (examples 1-3) and with the conventional culture method (without hypoxia and minoxidil stimulation) (example 4).
FIG. 3 shows Western blot analysis of Wnt1a protein level expression (GAPDH as internal reference) in exosomes obtained from the present invention (examples 1-3) and conventional culture (without hypoxia and minoxidil stimulation) (example 4).
FIG. 4 is a graph comparing the experimental results of mice treated with the inventive injection and with the exosome injections obtained from the conventional culture method (without hypoxia and minoxidil stimulation).
FIG. 5 is a graph showing the effect of the injection of the present invention for external application to treat alopecia in human body.
Detailed Description
Example 1
A preparation method of an injection for treating androgenetic alopecia by using hair papilla cell exosomes, wherein the injection is a physiological saline solvent containing the hair papilla cell exosomes obtained in a serum-free basal medium, and the preparation method comprises the following steps:
(1) separation of hair papilla cells:
a. placing a single hair follicle after operation in a hair follicle collection liquid, preserving the hair follicle in an environment at 4 ℃, and cleaning hair follicle tissues for 2-3 times by using a double-resistant PBS solution containing 1% of mass fraction;
b. separating the hair bulb tissue with a 1ml syringe needle under a microscope, removing residual hair matrix, and transferring the tissue to a new culture dish containing PBS solution with a pipette;
c. washing PBS once, adding 0.2% collagenase IV, digesting at 37 ℃ for 1-2 h, observing the digestion state at intervals of 10min, simultaneously blowing and beating the tissue by using a suction pipe, after the dermal sheath tissue around the papillary hair tissue is completely dissociated, selecting single papillary hair cells by using a pipette under a microscope, and placing the papillary hair cells into a new culture dish.
(2) Adherent culture of hair papilla cells: carrying out adherent culture on the dermal papilla cells obtained in the step (1): dermal papilla cells at 37 deg.C with 5% CO2And (3) carrying out adherent culture in an incubator, replacing the culture medium after the tissues are completely adherent and cells climb out, replacing the culture medium every 3 days, and carrying out subculture when the cells grow and fuse to reach 80%. The culture medium for adherent culture contains DMEM/F12, fetal bovine serum, EGF, bFGF and double antibody.
(3) Stimulating and inducing the culture of hair papilla cells: transferring the primary hair papilla cells obtained by adherent culture in the step (2) to a cell culture system when the primary hair papilla cells are transferred to the third generation, culturing in a three-air culture box, reducing the oxygen concentration in the culture box to 0.1% when the primary hair papilla cells are cultured to the 7 th day, and simultaneously adding 1 x 10 of final concentration−4Stimulating and inducing the minoxidil of M; after 24 hours of stimulation induction, removing the culture medium for adherent culture in the step (2), and cleaning for 3 times by using a serum-free basal culture medium, wherein the culture medium is changed into the serum-free basal culture medium, and the oxygen content is changed to 5%;
(4) preparing exosome freeze-dried powder: culturing in a serum-free basic culture medium for 24-48 h in the step (3), collecting cell supernatant, and dissolving with PBS (phosphate buffer solution) after low-temperature ultracentrifugation to obtain papilla cell exosomes; counting, filling into 5ml ampoule bottles, placing at the temperature of-50 to-80 ℃ for freezing treatment, and then carrying out freeze-drying treatment at the temperature of-50 ℃ for 24 hours to obtain hair papilla cell exosome freeze-dried powder;
(5) preparing an injection: the hair papilla cell exosome freeze-dried powder is dissolved by normal saline to form hair papilla cell exosome containing concentration of 1 x 1010Granules/ml of physiological saline solvent.
Example 2
An injection for treating androgenetic alopecia by using hair papilla cell exosomes, which is a physiological saline solvent containing the hair papilla cell exosomes.
The preparation method of the injection for treating androgenetic alopecia by using the hair papilla cell exosomes comprises the following steps:
(1) separation of hair papilla cells:
a. placing a single hair follicle after operation in a hair follicle collection liquid, preserving the hair follicle in an environment at 4 ℃, and cleaning hair follicle tissues for 2-3 times by using a double-resistant PBS solution containing 1% of mass fraction;
b. separating the hair bulb tissue with a 1ml syringe needle under a microscope, removing residual hair matrix, and transferring the tissue to a new culture dish containing PBS solution with a pipette;
c. washing PBS once, adding 0.2% collagenase IV, digesting at 37 ℃ for 1-2 h, observing the digestion state at intervals of 10min, simultaneously blowing and beating the tissue by using a suction pipe, after the dermal sheath tissue around the papillary hair tissue is completely dissociated, selecting single papillary hair cells by using a pipette under a microscope, and placing the papillary hair cells into a new culture dish.
(2) Adherent culture of hair papilla cells: carrying out adherent culture on the dermal papilla cells obtained in the step (1): dermal papilla cells at 37 deg.C with 5% CO2And (3) carrying out adherent culture in an incubator, replacing the culture medium after the tissues are completely adherent and cells climb out, replacing the culture medium every 3 days, and carrying out subculture when the cells grow and fuse to reach 80%. The culture medium for adherent culture contains DMEM/F12, fetal bovine serum, EGF, bFGF and double antibody.
(3) Stimulating and inducing the culture of hair papilla cells: transferring the primary dermal papilla cells obtained by adherent culture in the step (2) into a cell culture system when the primary dermal papilla cells are transferred to the third generation, and culturing in a three-gas incubatorMedium culture, reducing oxygen concentration in incubator to 3% and adding final concentration of 5 x 10 by 7 days−4Stimulating and inducing the minoxidil of M; after 24 hours of stimulation induction, removing the culture medium for adherent culture in the step (2), and cleaning for 3 times by using a serum-free basal culture medium, wherein the culture medium is changed into the serum-free basal culture medium, and the oxygen content is changed to 5%;
(4) preparing exosome freeze-dried powder: culturing in a serum-free basic culture medium for 24-48 h in the step (3), collecting cell supernatant, and dissolving with PBS (phosphate buffer solution) after low-temperature ultracentrifugation to obtain papilla cell exosomes; counting, filling into 5ml ampoule bottles, placing at the temperature of-50 to-80 ℃ for freezing treatment, and then carrying out freeze-drying treatment at the temperature of-50 ℃ for 24 hours to obtain hair papilla cell exosome freeze-dried powder;
(5) preparing an injection: the hair papilla cell exosome freeze-dried powder is dissolved by normal saline to form hair papilla cell exosome containing concentration of 1 x 1010Granules/ml of physiological saline solvent.
Example 3
An injection for treating androgenetic alopecia by using hair papilla cell exosomes, which is a physiological saline solvent containing the hair papilla cell exosomes.
The preparation method of the injection for treating androgenetic alopecia by using the hair papilla cell exosomes comprises the following steps:
(1) separation of hair papilla cells:
a. placing a single hair follicle after operation in a hair follicle collection liquid, preserving the hair follicle in an environment at 4 ℃, and cleaning hair follicle tissues for 2-3 times by using a double-resistant PBS solution containing 1% of mass fraction;
b. separating the hair bulb tissue with a 1ml syringe needle under a microscope, removing residual hair matrix, and transferring the tissue to a new culture dish containing PBS solution with a pipette;
c. washing PBS once, adding 0.2% collagenase IV, digesting at 37 ℃ for 1-2 h, observing the digestion state at intervals of 10min, simultaneously blowing and beating the tissue by using a suction pipe, after the dermal sheath tissue around the papillary hair tissue is completely dissociated, selecting single papillary hair cells by using a pipette under a microscope, and placing the papillary hair cells into a new culture dish.
(2) Adherent culture of hair papilla cells: carrying out adherent culture on the dermal papilla cells obtained in the step (1): adherent culture of hair papilla cells is carried out in a 5% CO2 incubator at 37 ℃, the culture medium is replaced after tissues are completely adherent and cells climb out, the culture medium is replaced every 3 days, and subculture is carried out when the cells grow and fuse to reach 80%. The culture medium for adherent culture contains DMEM/F12, fetal bovine serum, EGF, bFGF and double antibody.
(3) Stimulating and inducing the culture of hair papilla cells: transferring the primary hair papilla cells obtained by adherent culture in the step (2) to a cell culture system when the primary hair papilla cells are transferred to the third generation, culturing in a three-air culture box, reducing the oxygen concentration in the culture box to 1.5% when the primary hair papilla cells are cultured to the 7 th day, and simultaneously adding 3 x 10% of final concentration−4Stimulating and inducing the minoxidil of M; after 24 hours of stimulation induction, removing the culture medium for adherent culture in the step (2), and cleaning for 3 times by using a serum-free basal culture medium, wherein the culture medium is changed into the serum-free basal culture medium, and the oxygen content is changed to 5%;
(4) preparing exosome freeze-dried powder: culturing in a serum-free basic culture medium for 24-48 h in the step (3), collecting cell supernatant, and dissolving with PBS (phosphate buffer solution) after low-temperature ultracentrifugation to obtain papilla cell exosomes; counting, filling into 5ml ampoule bottles, placing at the temperature of-50 to-80 ℃ for freezing treatment, and then carrying out freeze-drying treatment at the temperature of-50 ℃ for 24 hours to obtain hair papilla cell exosome freeze-dried powder;
(5) preparing an injection: the hair papilla cell exosome freeze-dried powder is dissolved by normal saline to form hair papilla cell exosome containing concentration of 1 x 1010Granules/ml of physiological saline solvent.
Example 4
This example is a control experiment, hair papilla cell exosomes were obtained by a conventional culture method (without hypoxia and minoxidil stimulation), and an injection solution containing the hair papilla cell exosomes in a physiological saline solvent was prepared.
The preparation method of the injection comprises the following steps:
(1) separation of hair papilla cells:
a. placing a single hair follicle after operation in a hair follicle collection liquid, preserving the hair follicle in an environment at 4 ℃, and cleaning hair follicle tissues for 2-3 times by using a double-resistant PBS solution containing 1% of mass fraction;
b. separating the hair bulb tissue with a 1ml syringe needle under a microscope, removing residual hair matrix, and transferring the tissue to a new culture dish containing PBS solution with a pipette;
c. washing PBS once, adding 0.2% collagenase IV, digesting at 37 ℃ for 1-2 h, observing the digestion state at intervals of 10min, simultaneously blowing and beating the tissue by using a suction pipe, after the dermal sheath tissue around the papillary hair tissue is completely dissociated, selecting single papillary hair cells by using a pipette under a microscope, and placing the papillary hair cells into a new culture dish.
(2) Adherent culture of hair papilla cells: carrying out adherent culture on the dermal papilla cells obtained in the step (1): dermal papilla cells at 37 deg.C with 5% CO2And (3) carrying out adherent culture in an incubator, replacing the culture medium after the tissues are completely adherent and cells climb out, replacing the culture medium every 3 days, and carrying out subculture when the cells grow and fuse to reach 80%. The culture medium for adherent culture contains DMEM/F12, fetal bovine serum, EGF, bFGF and double antibody.
(3) Stimulating and inducing the culture of hair papilla cells: transferring the primary dermal papilla cells obtained by adherent culture in the step (2) into a cell culture system when the primary dermal papilla cells are transferred to the third generation, culturing in a three-gas culture box, and replacing a new culture medium for adherent culture when the primary dermal papilla cells are cultured to the 7 th day; after 24 hours of culture medium, removing the culture medium for adherent culture in the step (2), cleaning for 3 times by using a serum-free basal culture medium, changing the culture medium into the serum-free basal culture medium, and continuing to culture, wherein the oxygen content is always kept at 5%;
(4) preparing exosome freeze-dried powder: culturing in a serum-free basic culture medium for 24-48 h in the step (3), collecting cell supernatant, and dissolving with PBS (phosphate buffer solution) after low-temperature ultracentrifugation to obtain papilla cell exosomes; counting, filling into 5ml ampoule bottles, placing at the temperature of-50 to-80 ℃ for freezing treatment, and then carrying out freeze-drying treatment at the temperature of-50 ℃ for 24 hours to obtain hair papilla cell exosome freeze-dried powder;
(5) preparing an injection: the hair papilla cell exosome freeze-dried powder is dissolved by normal saline to form hair papilla cell exosome containing concentration of 1 x 1010Granules/ml of physiological saline solvent.
FIG. 1 shows the immunohistochemical characterization of alpha-SMA from fibroblasts and third generation hair papilla cells obtained by the method of the present invention, respectively, at A, B. The observation form under the cytoscope in the B picture is uniform, a large number of brown yellow cells are shown after dyeing, and the characteristic of the hair papilla cells is met. The cells obtained by the method are dermal papilla cells and have uniform cell purity.
As shown in FIG. 2, electron microscope images of the exosomes obtained in examples 1-4 are obtained, in the images, 10ml of exosomes extracted from the supernatant are collected by four methods, and electron microscope results show that after equivalent dilution and uniform mixing operations, the exosome concentration obtained in examples 1-3 is obviously higher than that obtained by the conventional culture method (example 4), while the exosome concentration obtained in examples 1-3 is not different, and the results obtained by counting analysis are consistent with the observation results.
As shown in FIG. 3, the expression of Wnt1a protein level (GAPDH as internal reference) in the exosomes obtained in examples 1-4 was detected by Western blot method. At the same protein concentration, the expression level of Wnt1a in exosomes collected in examples 1-3 was significantly higher than that in example 4 (control, normal method culture) (the difference between the bars with different superscript letters was significant, p < 0.01). Wnt1a has been proved in the existing research, and can stimulate DP cells at the hair follicle part of a subject to induce the hair cycle and further promote the hair regeneration, so that the proportion of effective components for inducing the hair regeneration in exosomes secreted by the DP cells after the stimulation of minoxidil is obviously increased.
Animal experiments are carried out on the injection of the invention, 6 nude mice with 4-5 weeks are selected and randomly divided into control groups (hypoxia and minoxidil puncture are not carried out)Stimulation treatment) and treatment groups (hypoxia and minoxidil stimulation treatment). Treatment group: 1ml of the injection solution of the present invention (containing the exosome particles in an amount of 1 x 10)10/ml) was injected into the mouse depilatory site; control (exosomes obtained without hypoxic and minoxidil stimulation treatments) treatment and dose were injected in the same manner as treatment. Once every 5 days for four weeks, and then continuously photographing for observation. As shown in FIG. 4, both formulations can promote hair regeneration, but the injection of the present invention has significant advantages in terms of hair growth rate and final hair regeneration effect.
The injection of the present invention was subjected to human body external application test (since no clinical approval was obtained, the human body injection test could not be performed) 2 times a week at a dose of 3ml each time (the amount of the particles containing exosome was 1 x 10)10And/ml), uniformly applying on the affected part, massaging the affected part properly, and washing with warm water for one month after 30 minutes. As shown in FIG. 5, the hair loss was significantly increased after the external application of the injection of the present invention to the hair loss site for 3 months.
In conclusion, the method of the invention is based on the important function of hair papilla cells and the exosomes secreted by the hair papilla cells in the process of regulating and controlling the hair cycle, the exosomes of the hair papilla cells are used for treating androgenetic alopecia, and are prepared into injection, so that the treatment is convenient and the effect is good. The minoxidil is adopted to stimulate hair papilla cells, so that the hair papilla cells secrete exosomes with specific alopecia treatment effect, and the alopecia treatment effect is achieved. The stimulation induction culture environment of the hair papilla cells adopts a low-oxygen condition, which is favorable for improving the capability of the hair papilla cells for secreting exosomes, thereby obtaining more exosomes. In the process of stimulating and inducing culture, the culture medium is replaced into a serum-free basic culture medium after 24 hours of stimulating and inducing, so that the hair papilla cells in the early stage can be normally proliferated, a large amount of exogenous exosomes contained in the serum can be eliminated, and the influence on the purity of the exosomes is avoided.
The above description is illustrative and not restrictive. Many modifications and variations of the present invention will be apparent to those skilled in the art in light of the above teachings, which will fall within the spirit and scope of the invention.
Claims (4)
1. A preparation method of an injection for treating androgenetic alopecia by using hair papilla cell exosomes is characterized in that the injection is a physiological saline solvent containing the hair papilla cell exosomes obtained in a serum-free basal medium, and the preparation method comprises the following steps:
(1) separating hair papilla cells;
(2) adherent culture of hair papilla cells: carrying out adherent culture on the dermal papilla cells obtained in the step (1);
(3) stimulating and inducing the culture of hair papilla cells: transferring the primary hair papilla cells obtained by adherent culture in the step (2) to a cell culture system when the primary hair papilla cells are transferred to the third generation, culturing in a three-air culture box, reducing the oxygen concentration in the culture box to 0.1-3% when the primary hair papilla cells are cultured to the 7 th day, and simultaneously adding 1-5 x 10% of final concentration−4 Stimulating and inducing the minoxidil of M; after 24 hours of stimulation induction, removing the culture medium for adherent culture in the step (2), and cleaning for 3 times by using a serum-free basal culture medium, wherein the culture medium is changed into the serum-free basal culture medium, and the oxygen content is changed to 5%;
(4) preparing exosome freeze-dried powder: culturing in a serum-free basic culture medium for 24-48 h in the step (3), collecting cell supernatant, and dissolving with PBS (phosphate buffer solution) after low-temperature ultracentrifugation to obtain papilla cell exosomes; counting, subpackaging into ampoule bottles, placing at the temperature of-50 to-80 ℃ for freezing treatment, and then carrying out freeze-drying treatment at the temperature of-50 ℃ to obtain hair papilla cell exosome freeze-dried powder;
(5) preparing an injection: the hair papilla cell exosome freeze-dried powder is dissolved into a physiological saline solvent containing the hair papilla cell exosomes by using physiological saline.
2. The method for preparing an injection for treating androgenetic alopecia using hair papilla cell exosomes according to claim 1, wherein the separation of hair papilla cells in the step (1) comprises the steps of:
a. placing a single hair follicle after operation in a hair follicle collection liquid, preserving the hair follicle in an environment at 4 ℃, and cleaning hair follicle tissues for 2-3 times by using a double-resistant PBS solution containing 1% of mass fraction;
b. separating the hair bulb tissue with a 1ml syringe needle under a microscope, removing residual hair matrix, and transferring the tissue to a new culture dish containing PBS solution with a pipette;
c. washing PBS once, adding 0.2% collagenase IV, digesting at 37 ℃ for 1-2 h, observing the digestion state at intervals of 10min, simultaneously blowing and beating the tissue by using a suction pipe, after the dermal sheath tissue around the papillary hair tissue is completely dissociated, selecting single papillary hair cells by using a pipette under a microscope, and placing the papillary hair cells into a new culture dish.
3. The method for preparing an injection for treating androgenetic alopecia using hair papilla cell exosomes according to claim 1, wherein the hair papilla cells in the step (2) are treated at 37 ℃ and 5% CO2And (3) carrying out adherent culture in an incubator, replacing the culture medium after the tissues are completely adherent and cells climb out, replacing the culture medium every 3 days, and carrying out subculture when the cells grow and fuse to reach 80%.
4. The method for preparing an injection for treating androgenetic alopecia using hair papilla cell exosomes according to claim 1 or 3, wherein the culture medium for adherent culture in the step (2) comprises DMEM/F12, fetal bovine serum, EGF, bFGF and diabody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811161456.0A CN108992466B (en) | 2018-09-30 | 2018-09-30 | Preparation method of injection for treating androgenetic alopecia by using hair papilla cell exosomes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811161456.0A CN108992466B (en) | 2018-09-30 | 2018-09-30 | Preparation method of injection for treating androgenetic alopecia by using hair papilla cell exosomes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108992466A CN108992466A (en) | 2018-12-14 |
| CN108992466B true CN108992466B (en) | 2021-07-16 |
Family
ID=64589869
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811161456.0A Active CN108992466B (en) | 2018-09-30 | 2018-09-30 | Preparation method of injection for treating androgenetic alopecia by using hair papilla cell exosomes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108992466B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980796A (en) * | 2019-12-17 | 2021-06-18 | 东莞市东阳光生物药研发有限公司 | Liquid medium for reducing oxidation of recombinant protein expressed in CHO cells |
| CN111617237A (en) * | 2020-06-16 | 2020-09-04 | 南京元荟生物科技有限公司 | Hair growth promoting liquid containing hair papilla cell-derived exosome and collagen and preparation method thereof |
| CN111544373A (en) * | 2020-06-16 | 2020-08-18 | 南京元荟生物科技有限公司 | Hair growth promoting liquid containing hair papilla cell-derived exosomes and preparation method thereof |
| CN112618572A (en) * | 2020-10-16 | 2021-04-09 | 中科细胞科技(广州)有限公司 | Composition for treating baldness |
| CN112920988B (en) * | 2021-02-07 | 2022-12-06 | 广州四叶草健康科技有限公司 | Preparation and separation method of hair papilla cell exosome |
| CN113599516B (en) * | 2021-08-16 | 2022-02-18 | 上海蒙彼利生物技术有限公司 | Method for preparing exosome and application of pharmaceutical composition thereof in tissue repair |
| CN113621556B (en) * | 2021-08-25 | 2023-06-13 | 南方医科大学南方医院 | Construction method of androgenetic alopecia cell model |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1824310A (en) * | 2005-12-31 | 2006-08-30 | 罗洋 | Preparation method of medicine for treating pelada |
| CN1850272A (en) * | 2006-02-23 | 2006-10-25 | 罗洋 | Medicine for treating alopecia areata and its clinical use |
| CN101336932A (en) * | 2008-08-09 | 2009-01-07 | 罗洋 | Use of medicine prepared from dermal papilla cells supernatant in treating calvities |
| KR20180015994A (en) * | 2016-08-05 | 2018-02-14 | 한양대학교 에리카산학협력단 | Composition comprising exosomes derived from human adipose stem cell and human dermal papilla cell for promoting hair growth |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016006885A1 (en) * | 2014-07-07 | 2016-01-14 | 메디포스트(주) | Hair growth-promoting function of culture medium of stimulated stem cells and use thereof |
-
2018
- 2018-09-30 CN CN201811161456.0A patent/CN108992466B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1824310A (en) * | 2005-12-31 | 2006-08-30 | 罗洋 | Preparation method of medicine for treating pelada |
| CN1850272A (en) * | 2006-02-23 | 2006-10-25 | 罗洋 | Medicine for treating alopecia areata and its clinical use |
| CN101336932A (en) * | 2008-08-09 | 2009-01-07 | 罗洋 | Use of medicine prepared from dermal papilla cells supernatant in treating calvities |
| KR20180015994A (en) * | 2016-08-05 | 2018-02-14 | 한양대학교 에리카산학협력단 | Composition comprising exosomes derived from human adipose stem cell and human dermal papilla cell for promoting hair growth |
Non-Patent Citations (1)
| Title |
|---|
| Synergistic proliferating effects of electrical stimulation and minoxidil on human dermal papilla cells;Kim Jung-eun等;《Korean Journal of Dermatology》;20161231;第54卷(第5期);第341-349页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108992466A (en) | 2018-12-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108992466B (en) | Preparation method of injection for treating androgenetic alopecia by using hair papilla cell exosomes | |
| RU2396084C1 (en) | Soft tissue filler composition for injection and method for preparing thereof | |
| CN110269833A (en) | A kind of umbilical cord mesenchymal stem cells preparation and its preparation method and application | |
| CN101748096A (en) | Sub totipotential stem cell and preparation method and application thereof | |
| EP1950284A1 (en) | Method for cultivation of hair follicular dermal sheath cell | |
| US20120189585A1 (en) | Method and composition for restoration of age-related tissue loss in the face or selected areas of the body | |
| CN106635973A (en) | Preparation method of cell nutrient solution for promoting hair regeneration | |
| CN105820998A (en) | Isolation extraction and culture method for human adipose-derived stem cells (ADSCs) for clinical back-transfusion grade cell therapy | |
| CN106659741A (en) | Hair growth-promoting function of small-sized stem cells and use thereof | |
| CN108721606A (en) | A kind of striae of pregnancy reparation product and preparation method thereof based on stem cell factor | |
| WO2024120208A1 (en) | Method for preparing umbilical cord mesenchymal stem cell directional secretome for promoting hair growth and use thereof | |
| CN105255822A (en) | Method for screening and culturing extracellular hair follicle stem cell matrix for clinic treatment level cell therapy | |
| CN108066750A (en) | Stem cell and its secretion are used to treat the new application of skin burn | |
| CN111840330B (en) | Anti-hair loss repairing composition and application | |
| CN112891517A (en) | Cytokine composition for promoting scalp hair follicle regeneration and preparation method and application thereof | |
| CN113957040A (en) | Adipose-derived stem cell growth factor extract and preparation method and application thereof | |
| CN102552323A (en) | Medicine for accelerating skin repair and regeneration, preparation method thereof and application thereof | |
| CN105238740A (en) | In-vitro screening and culturing method for tissue engineering skin seed epidermal keratinocyte high-simulation in-vivo cell extracellular matrix attachment | |
| CN102703380A (en) | Sub-totipotent stem cell, preparation method and application thereof | |
| CN113332313A (en) | Stem cell preparation for treating seborrheic alopecia | |
| CN111228311A (en) | Preparation method of stem cell hair growth factor for protecting and inducing hair follicle regeneration | |
| CN112294849A (en) | Acellular matrix-cell composite particle for hair growth and preparation method thereof | |
| CN110592008A (en) | Method for culturing bone marrow mesenchymal stem cells of canine animals | |
| CN109355257A (en) | The mescenchymal stem cell mixed culturing method of different tissue sources | |
| CN116875537B (en) | Method for constructing hair follicle organoids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A preparation method of injection for treating androgenic alopecia with dermal papilla cell exosomes Effective date of registration: 20220125 Granted publication date: 20210716 Pledgee: Cultural and creative sub branch of Bank of Hangzhou Co.,Ltd. Pledgor: ZHEJIANG HEALTHFUTURE BIOMEDICAL TECHNOLOGY CO.,LTD. Registration number: Y2022330000165 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20230504 Granted publication date: 20210716 Pledgee: Cultural and creative sub branch of Bank of Hangzhou Co.,Ltd. Pledgor: ZHEJIANG HEALTHFUTURE BIOMEDICAL TECHNOLOGY CO.,LTD. Registration number: Y2022330000165 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Preparation method of an injection for treating androgenic alopecia using extracellular vesicles of dermal papilla cells Effective date of registration: 20230506 Granted publication date: 20210716 Pledgee: Cultural and creative sub branch of Bank of Hangzhou Co.,Ltd. Pledgor: ZHEJIANG HEALTHFUTURE BIOMEDICAL TECHNOLOGY CO.,LTD. Registration number: Y2023330000848 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |