AU2020103492A4 - An external medicine for promoting hair regeneration and its preparation - Google Patents
An external medicine for promoting hair regeneration and its preparation Download PDFInfo
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- AU2020103492A4 AU2020103492A4 AU2020103492A AU2020103492A AU2020103492A4 AU 2020103492 A4 AU2020103492 A4 AU 2020103492A4 AU 2020103492 A AU2020103492 A AU 2020103492A AU 2020103492 A AU2020103492 A AU 2020103492A AU 2020103492 A4 AU2020103492 A4 AU 2020103492A4
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- hair follicle
- follicle stem
- hair
- stem cell
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- 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
- C12N5/0628—Hair stem cells; Hair progenitors
-
- 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
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/2292—Thymosin; Related peptides
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/09—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells
- C12N2506/092—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells from hair cells
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Abstract
The purpose of the present invention is to study the related mechanism of hair follicle
stem cells promoting hair regeneration and explore the apply of allogeneic hair follicle stem
cells as drugs for treating pathological alopecia. The topical medicine for promoting hair
regeneration comprises supernatant of hair follicle stem cell culture medium and thymosin. The
external medicinal composition provided by the invention is mainly suitable for alopecia
symptoms such as androgyny, it can effectively stimulate hair follicles and promote hair
regeneration of alopecia patients by combining supernatant of hair follicle stem cell culture
medium with thymosin. The medicine has the advantages of convenience, safety, low cost,
remarkable effect, simple treatment process and high patient acceptance.
Description
PATENTS ACT 1990
An external medicine for promoting hair regeneration and its preparation
The invention is described in the following statement:-
An external medicine for promoting hair regeneration and its preparation
The invention relates to the field of genetic engineering technology and biological
medicine, in particular to an external drug for promoting hair regeneration and its
preparation method.
Alopecia is a common disease in dermatology, which can be divided into non
cicatricial alopecia and cicatricial alopecia. Androgenic alopecia, alopecia areata and
trichotilla belong to the former, while lichen planus, folliculitis alopecia and discoid lupus
erythematosus alopecia belong to the latter. Androgen alopecia is the most common disease,
which is characterized by progressive reduction of hair density, which is an androgen
dependent autosomal dominant polymorphosis.
At present, there are several treatment options for alopecia patients, such as oral or
topical drugs, or surgical treatment. However, these treatments have limitations. Drug
treatment can only relieve symptoms for a short time, and when patients stop taking drugs,
their hair will start to fall off again. For example, oral administration of finasteride has
sexual side effects and can lead to deformity. After stopping taking the drug, the hair will
fall off quickly. Autologous single hair follicle and follicular unit transplantation is a
reliable surgical treatment, but the number of hair follicle donors is limited.
Therefore,novel treatments are urgently needed for alopecia.
Hair follicle stem cells (HFSCs) are a group of multipotential stem cells which exist
in the bulge of outer root sheath of hair follicle, it can self-renew and proliferate. They can
differentiate into hair follicle, sebaceous gland and epidermis, and play an important role
in hair regeneration. Studies have shown that human hair follicle stem cells express various
cell surface molecular markers such as CD200, keratin 15 (KI5), keratin 19 (K19), integrin
06, integrin P 1, Nestin, etc.. But they do not express endothelial cell marker molecule CD31. In addition, CD34, a marker molecule of mouse hair follicle stem cells, is not expressed
either. Adopting these positive and negative marker molecules, researchers can better
isolate and purify hair follicle stem cells, which accelerates the research process of hair
follicle stem cells. Compared with embryonic stem cells and other adult stem cells, hair
follicle stem cells have the advantages of abundant sources, convenient materials, no harm
to the body and no ethical problems. It has been reported in the literature that hair can grow
in nude mice by transplanting hair follicle stem cells, which proves that transplanting
healthy hair follicle stem cells has the potential to repair damaged hair follicles. If
allogeneic hair follicle stem cells can be used as donors, it will greatly expand the
application scope of hair follicle stem cells. In view of this, the present invention studies
the related mechanism of hair follicle stem cells promoting hair regeneration, and explores
the application of allogeneic hair follicle stem cells as drugs for treating pathological
alopecia.
The purpose of the present invention is to study the related mechanism of hair follicle
stem cells promoting hair regeneration and explore the adoption of allogeneic hair follicle stem cells as drugs for treating pathological alopecia.
The external medicine for promoting hair regeneration comprises a hair follicle stem
cell culture medium supernatant and thymosin in combination.
Further, the mass fraction of the thymosin is 0.01-0.1%.
Further, the supernatant of hair follicle stem cell culture medium is subpackaged and
stored at 4°C, which is mixed with thymosin with the mass fraction of 0.01-0.1%.
Furthermore, the supernatant of the hair follicle stem cell culture medium is
subpackaged and stored at 4°C, and thymosin is administered intravenously or
intraperitoneally.
The culture method of the supernatant of the hair follicle stem cell culture medium
comprises the following steps:
(1) Taking healthy hair follicles and washing them repeatedly with penicillin
streptomycin PBS solution;
(2) Under the dissecting microscope, cutting off the bulge of the outer root sheath of
the hair follicle by microsurgery, which is placed in a petri dish; Adding hair follicle stem
cell culture medium, which is incubated in the incubator of 5%CO2 at 37°C;
(3) Supplementing the same amount of medium 1.5mL the next day, observing under
an inverted microscope whether there is spindle-shaped cells grow in the outer root sheath
of the hair follicle or not, and carrying out subculturing after the cells have reached 80%
fusion;
(4) Digesting the hair follicle stem cells, preparing a hair follicle stem cell suspension of 1.Ox105 cells /mL with the hair follicle stem cell culture solution, and counting;
(5) After mixing 1.2% agarose and DMEM/F12 culture medium according to the mass
ratio of 1:1, taking 2ml of the mixed solution which is injected into a 35cm2 culture dish,
and putting it into an incubator for later use after cooling and solidification;
(6) After mixing 0.7% agarose and hair follicle stem cell culture medium in the ratio
of 1:1 in a sterile test tube, adding 1/10 volume of hair follicle stem cell suspension to the
tube, which are mixed well. Then injecting medium I into the petri dish, the amount added
is equal to medium I;
(7) Replacing the culture medium once every 2 days for a total of 10 days.
In addition, the hair follicle stem cell culture solution contains DMEM/F12 containing
% FBS, 100U/mL streptomycin double antibody and 2ng/ml bFGF.
Additional, the culture medium I is mixed with 1.2% agarose and DMEM/F12 culture
medium according to the ratio of 1:1, and then 2ml of the mixed solution is injected into a
cm2 culture dish, which is put into an incubator for later use after being cooled and
solidified.
Furthermore, the digestive solution digested by hair follicle stem cells contains 0.1%
trypsin and 0.008% EDTA phosphate buffer solution.
Further, the healthy hair follicle is taken from the occipital part of the head.
Further, the digestion condition is 37 °C for 3-5 minutes.
The external medicinal composition provided by the invention is mainly suitable for
alopecia symptoms such as androgyny, it can effectively stimulate hair follicles and promote hair regeneration of alopecia patients by combining supernatant of hair follicle stem cell culture medium with thymosin. It has the advantages of convenience, safety, low cost, remarkable effect, simple treatment process and high patient acceptance.
Figure 1 shows the skin HE of nude mice in each group after injection for 5 weeks.
Figure 2 is the hair growth status after subcutaneous injection of HFSC.
In order to explain the technical scheme and purpose of the present invention, the
following specific embodiments will further introduce the present invention.
1 Drug screening
1. Sample collection and grouping: recruiting 20 healthy volunteers and 20
pathologically alopecia volunteers. 30 intact hair follicles were extracted from the superior
donor area (posterior occipital region) and forehead area of each volunteer. According to
different sources, the hair follicle samples were divided into three groups: healthy dominant
donor group, healthy forehead group, alopecia dominant donor group and alopecia area
group.
2. Identification of the number of hair follicle stem cells in hair follicle samples by
immunofluorescence double staining: 5 hair follicle samples were taken from each group,
which were made into frozen sections of 5 m thick.They are solidified with 4%
paraformaldehyde for 10min. After cleaning with PBS for 3 times, treating the sections with 10% goat serum blocking solution containing 0.25% Triton-100 for 20min minutes, respectively adding appropriately diluted primary antibodies (K19, integrin P 1), and incubating the groups at 4°C overnight. The next day, after washing with PBS for three times, the fluorescent secondary antibody corresponding to the species of the primary antibody was added to react at 37°C for 30min; After cleaning with PBS and re-staining with DAPI, the film was observed and photographed under fluorescence microscope. The number of hair follicle stem cells in each group was counted and compared.
3. Gene expression profile of hair follicle samples in each group was analyzed by gene
chip: 15 hair follicle samples were taken from each group, and the gene expression profile
data of hair follicle samples were obtained by Affymetrix Human Gene 2.0 ST Array gene
chip analysis.
4. Bioinformatics analysis of gene expression profile data: the gene expression profile
data obtained in step 4 was imported into NGF model to obtain the gene importance score
in gene expression profile data. The data of NGF top 3000 genes and their IS scores (as
initial heat) were input into HotNet2 model, and the key regulatory subnetworks were
identified by HotNet2 method. 10 hair follicle samples were taken from each group to
extract mRNA. The expression levels of these key genes in hair follicle samples were
verified by real-time fluorescent quantitative PCR.
5. Systematic pharmacology method to screen candidate drugs: the sub-network genes
identified by HotNet2 and the expression levels of these genes were used as tag information,
which were input into the Connectivity Map (cMap) database for query (website:
http://www.broadinstitute.org/cmap/), sorting the returned results by P value, and selecting the top three small molecules for subsequent experimental verification.
2 Further screening drug molecules
1) Preparing low-melting-point agarose liquids with 1.2% and 0.7% concentrations
with triply distilled water. After sterilization under high pressure, it was maintained at 40°C
to keep dissolved.
2) Preparing 2xDMEM/F12 complete medium (2xDMEM/F12 medium containing
% FBS and 200U/mL streptomycin double antibody), which is filtered with 0.1 m
disposable sterile filter, and stored at 37°C.
3) After mixing 1.2% agarose and 2xDMEM/F12 culture medium in the ratio of 1:1,
taking 2ml of the mixed solution and inject it into a 35cm2 culture dish. After cooling and
solidifying, putting it into the incubator for later use.
4) 0.7% agarose and 2xDMEM/F12 culture medium were mixed in a sterile test tube
according to the ratio of 1: 1, and then injected into a Petri dish paved with 1.2% agarose,
with the amount of 2ml.
5) Collecting 40 complete hair follicles from the hair loss area of 20 volunteers with
pathological hair loss, which were randomly divided into control group, No.1 drug group,
No.3 drug group and No.3 drug group, with 10 hair follicles in each group; Washing
repeatedly the follicles for 3 times with PBS solution containing high concentration of
cyanine and streptomycin. When the upper agarose is in a semi-solidified state, inserting
the upper agarose and putting it into the incubator for co-culture for 3 days.
6) During the culture period, dripping 1OuL of PBS or corresponding drugs into the root of each hair follicle in each experimental group, once every 2 days, and the culture period was 10 days in total. Observing the hair growth of damaged hair follicles.
7) 5 hair follicle samples were taken from each group for fluorescence staining to
identify the number of hair follicle stem cells in hair follicle samples; Five other hair follicle
samples were used to detect the expression level of the key genes of hair regeneration in
damaged hair follicles after drug treatment by real-time fluorescence quantitative PCR.
8) According to the experimental results, the drug molecule with the best therapeutic
effect is thymosin.
3 The culture method of the supernatant of hair follicle stem cell culture medium
includes the following steps:
(1) Taking healthy hair follicles which is washed repeatedly with PBS solution of
cyanine-streptomycin;
(2) Under the dissecting microscope, the bulge of the outer root sheath of the hair
follicle was cut by microsurgery and placed in a petri dish; Adding hair follicle stem cell
culture medium, which is incubated in the incubator of 5%CO2 at 37°C;
(3) The next day, adding 1.5mL of the same amount of culture medium. Observing
whether the spindle-shaped cells grew out of the bulge of the outer root sheath of the hair
follicle under the inverted microscope, and then subcultured after the cell fusion reached
(4) Digesting the hair follicle stem cells, preparing a hair follicle stem cell suspension
of 1.Ox105 cells /mL with the hair follicle stem cell culture solution, and counting.
(5) After mixing 1.2% agarose and DMEM/F12 culture medium according to the ratio
of 1:1, taking 2ml of the mixed solution and inject it into a 35cm2 culture dish. After being
cooled and solidified, putting it into the incubator for later use.
(6) Mixing 0.7% agarose with hair follicle stem cell culture medium in the sterile test
tube according to the ratio of 1:1, then adding 1/10 volume of hair follicle stem cell
suspension into the tube, which were fully mixed evenly, and injected into the culture dish
of culture medium I, while the addition amount is equal to that of culture medium I.
(7) Changing the culture medium once every 2 days, and culturing for 10 days in total
to obtain the supernatant of the hair follicle stem cell culture medium.
4 Animal experiment
1.1 Establishment of androgen-induced alopecia rat model: selecting male SD rats
aged 6 weeks, which at treated with depilatory cream to remove their back hair, then
gavaging them 1 mL of (1.5 mg) methyltestosterone (60 tablets of methyltestosterone, 0.5
mg for each pill, ground into fine powder, which is mixed with 20 m L of distilled water
and stirred evenly) and 0.2 mL of lard once a day for 60 days continuously to establish
androgen alopecia rat model.
1.2 Animal grouping and hair follicle stem cell transplantation: the successfully
modeled SD rats are randomly divided into control group, hair follicle stem cell group, and
hair follicle stem cell+drug group, with six rats in each group. After digestion, hair follicle
stem cells are collected and washed twice with sterile saline, and then resuspended to
1.0x105 cells/mL cell suspension. In the hair follicle stem cell group, the cell suspension
is injected subcutaneously at multiple points, 100 L at each point, for a total of 500 [L;
Hair follicle stem cells are injected every 2 days for a total of 3 injections. The control
group is injected with the same volume of saline in the same manner. In addition to hair
follicular stem cell transplantation, the hair follicular stem cell+drug group is given
intraperitoneal injection of appropriate amount of drug twice a week.
Control Experimen Experiment Experiment Experimenta Experimental Experimental group 6 group tal group 1 al group 2 al grou p 4 group 5 Physiological Saline Saline Saline Culture Culture Culture supernatant saline +HFSC(1x +HFSC(2x +HFSC(4x supernatant supernatant +HFSC(1x106)+HU 106) 106) 106) +HFSC(1x106) VSMC(1x106)
1.3 Observing the hair growth of the depilated part of rats every week and making
records. After 8 weeks, the rats are killed and the skin tissue of the depilated part of the
back is made into paraffin tissue specimens.
Experim Experim Experim Experim Experime Control Experim Crol ental ental ental ental ental ntal group group group 1 group 2 group 3 group 4 group 5 6
Ow 0 0 0 0 0 0 0
1w 0 0 0 0 0 0 0
2w 0 0 2 2 0 1 2
3w 0 2 3 2 3 2 3
4w 0 2 3 3 2 3 3
5w 0 2 3 3 3 3 3
1.4 HE staining of hair follicles: The paraffin blocks of skin tissue are cut into paraffin
sections with the thickness of 5[m, followed by xylene dewaxing and gradient alcohol
hydration. After washing with tap water for 3 times, soaking and staining the tissues with
Harris hematoxylin staining solution for 3-5min, then washing them with tap water;
Differentiating the material above with 1% hydrochloric acid alcohol for 3-5s, and washing
it with tap water; Soaking the tissue in 0.2% ammonia solution for 1 minute, then it turns
blue, and washing the tissues with tap water; Soaking the tissues with eosin staining solution for 30 s; Dehydrating with 95% alcohol and absolute ethanol, and sealing with xylene transparent and neutral gum; Observing the samples and taking pictures under the microscope, as shown in Figure 1. Comparing the pathological differences between the experimental groups and the control group.
1.5 Immunofluorescence staining of hair follicles: paraffin sections of skin tissue were
dewaxed with xylene and hydrated with gradient alcohol, washed with distilled water for
3-5 times, treated with 10% goat serum blocking solution containing 0.25% Triton-100 for
minutes, and incubated at 4°C overnight with appropriately diluted primary antibodies
(K19, CD200 and integrin P1). The next day, after washing with PBS for three times, the
fluorescent secondary antibody corresponding to the species of the primary antibody was
added to react at 37°C for 30min; Cleaning with PBS, re-staining with DAPI, sealing,
observing under fluorescence microscope and taking pictures. Comparing the difference of
hair follicle stem cells between the experimental group and the control group.
2. Isolation and culture of hair follicle stem cells
(1) Isolation and culture of human hair follicle stem cells: 10 healthy volunteers are
recruited. 8-10 intact hair follicles are extracted from the posterior occipital region of the
brain of each volunteer and washed three times with PBS solution containing high
concentrations of penicillin and streptomycin. Under the dissecting microscope, hair
follicles are cut off from the bulge of the outer root sheath with microsurgical scissors and
placed in a petri dish of 35cm2 ; Adding 1.5ml of hair follicle medium (DMEM/F12 medium
containing 10% FBS, 100U/mL penicillin and 2ng/ml bFGF), which are incubated in a 5%
C02 incubator at 37°C. On the next day, adding 1.5mL of medium and the cells are observed under the inverted microscope to see if there are any shuttle-shaped cells grow out of the hair follicle outer root sheath projections. Cells subcultured to the third generation is for the subsequent experiments.
2) Identification of the expression of surface marker molecules and negative marker
molecules of hair follicle stem cells by flow cytometry: the 3rd generation hair follicle stem
cells are digested with 0.25% trypsin, washed once with PBS, centrifuged and collected,
and resuspended with PBS solution containing 1% BSA respectively. Adding the
fluorescent antibodies of K15, K19, CD200, integrin 1, CD31 and CD34 respectively, and
the cells are labeled by incubation at room temperature for 30 min in the dark. After
washing with PBS, the expression of these labeled molecules is detected by flow cytometry.
2. Identification of immunogenicity of hair follicle stem cells
1) Detecting the expression of MHC-II molecules in hair follicle stem cells by WB:
collecting hair follicle stem cells, adding a cell total protein extraction reagent to extract
and quantify the total protein. Adding 15kg protein sample into 2xSDS gel loading buffer,
which are boiled for 5min, centrifuged at 6000rpm for 3min. Taking the supernatant which
is transferred to the protein on gel to PVDF membrane after electrophoresis. Sealing the
membrane at room temperature for 2 h after transferring, putting PVDF membrane into the
hybridization bag, and adding appropriately diluted primary antibodies (HLA-DPA1,
HLA-DQA1, HLA-DRA1 and internal reference GAPDH) 4 respectively. Adding HRP
labeled secondary antibody corresponding to the primary antibody species, which are
incubate at room temperature with shaking for 1 h. Rinsing the filter membrane, adding
developer for color rendering, and putting it into a gel imager for exposure imaging. The optical density value of the target zone is analyzed by Quantity One software processing system.
2) Detection of MHC-II molecule expression in hair follicle stem cells by real-time
fluorescence quantitative PCR: collecting hair follicle stem cells, extracting cell total
mRNA by Trizol method, identifying and quantifying by ultraviolet spectrophotometer.
Total RNA is reverse transcribed into cDNA by adopting reverse transcription reaction kit
(prime script rt reagent kit with gDNA eraser, TAKARA). According to Genebank
sequence, HLA-DPA1, HLA-DQA1, HLA-DRA1 and internal reference GAPDH primers
are designed by Primer 5.0 software and synthesized by our company. The qRT-PCR
reaction is performed according to the instructions of the kit (SYBR Premix ExTaqTM II,
TAKARA). HLA-DPA1, HLA-DQA1, HLA-DRA1 and internal reference GAPDH
primers are shown in the following table:
Table 1. Primer sequences of HLA-dpal, HLA-DQA1, HLA-DRA1 and internal
reference GAPDH
Gene name Forward primer Reverse primer
HLA-DPA1 CTGGACAAGAAGGAGACCGT TCAATGTGGCAGATGAGGGT
HLA-DQA1 AACGCTACAACTCTACCGCT TCTGTGACTGACTGCCCATT
The above shows and describes the basic principles and main features of the present
invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments. The above embodiments and descriptions only illustrate the principles of the present invention.
Without departing from the spirit and scope of the present invention, there will be various
changes and improvements in the present invention, all of which fall within the scope of
the claimed invention. The claim scope of that present invention is defined by the append
claims and their equivalents.
Claims (9)
1. The external medicine for promoting hair regeneration, which is characterized in
that it comprises a hair follicle stem cell culture medium supernatant and thymosin in
combination.
2. The external medicine for promoting hair regeneration according to claim 1,
characterized in that the mass fraction of the thymosin is 0.01-0.1%.
3. The external medicine for promoting hair regeneration according to claim 1, which
is characterized in that the supernatant of hair follicle stem cell culture medium is
subpackaged and stored at 4°C, and thymosin is administered intravenously or
intraperitoneally.
4. The preparation method of an external drug for promoting hair regeneration, which
is characterized in that: the supernatant of the hair follicle stem cell culture medium is
subpackaged and stored at 4°C, which is mixed with thymosin with the mass fraction of
.0 1 - 0 .1%. The culture method of the supernatant of the hair follicle stem cell culture
medium comprises the following steps:
(1) Taking healthy hair follicles and washing them repeatedly with penicillin
streptomycin PBS solution;
(2) Under the dissecting microscope, cutting off the bulge of the outer root sheath of
the hair follicle by microsurgery, which is placed in a petri dish; Adding hair follicle stem
cell culture medium, which is incubated in the incubator of 5%CO2 at 37°C;
(3) Supplementing the same amount of medium 1.5mL the next day, observing under an inverted microscope whether there is spindle-shaped cells grow in the outer root sheath of the hair follicle or not, and carrying out subculturing after the cells have reached 80% fusion;
(4) Digesting the hair follicle stem cells, preparing a hair follicle stem cell suspension
of 1.0x105 cells /mL with the hair follicle stem cell culture solution, and counting;
(5) After mixing 1.2% agarose and DMEM/F12 culture medium according to the mass
ratio of 1:1, taking 2ml of the mixed solution which is injected into a 35cm2 culture dish,
and putting it into an incubator for later use after cooling and solidification;
(6) After mixing 0.7% agarose and hair follicle stem cell culture medium in the ratio
of 1:1 in a sterile test tube, adding 1/10 volume of hair follicle stem cell suspension to the
tube, which are mixed well. Then injecting medium I into the petri dish, the amount added
is equal to medium I;
(7) Replacing the culture medium once every 2 days for a total of 10 days.
5. The method for preparing an external medicine for promoting hair regeneration
according to claim 4, which is characterized in that the hair follicle stem cell culture
solution contains DMEM/F12 containing 10% FBS, 100U/mL streptomycin double
antibody and 2ng/ml bFGF.
6. The method for preparing an external medicine for promoting hair regeneration
according to claim 4, which is characterized in that the culture medium I is mixed with 1.2%
agarose and DMEM/F12 culture medium according to the ratio of 1:1, and then 2ml of the
mixed solution is injected into a 35cm2 culture dish, which is put into an incubator for later
use after being cooled and solidified.
7. The preparation method of an external drug for promoting hair regeneration
according to claim 4, which is characterized in that the digestive solution digested by hair
follicle stem cells contains 0.1% trypsin and 0.008% EDTA phosphate buffer solution.
8. The preparation method of external medicine for promoting hair regeneration
according to claim 4, which is characterized in that the healthy hair follicle is taken from
the occipital part of the head.
9. The preparation method of external medicine for promoting hair regeneration
according to claim 7, which is characterized in that the digestion condition is 37 °C for 3
minutes.
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