CN113621556B

CN113621556B - Construction method of androgenetic alopecia cell model

Info

Publication number: CN113621556B
Application number: CN202110982838.5A
Authority: CN
Inventors: 苗勇; 胡志奇; 张钰凡; 杜丽娟
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2023-06-13
Anticipated expiration: 2041-08-25
Also published as: CN113621556A

Abstract

The invention discloses a construction method of an androgenetic alopecia cell model, which comprises the following steps: collecting hair follicle bulb part of forehead part of androgenetic alopecia patient, separating hair papilla of hair follicle bulb part, and culturing; taking a carina region of the occipital part of an androgenic alopecia patient, separating outer root sheath cells of the carina region and culturing; 3D culturing the hair papilla cells obtained after the culture treatment to enable the hair papilla cells to gather to form hair papilla cell balls; the hair papilla cell pellet is inoculated in the upper chamber of a transwell culture dish, the outer root sheath cell is inoculated in the lower chamber of a pre-coated transwell culture dish, and dihydrotestosterone is added to obtain the cell model of androgenetic alopecia. The cell model of the invention is more stable, the number of the sheath cells of the outer root is reduced after successful modeling, the change trend of the androgenetic alopecia related marker is consistent with that of the inner body, and the cell model has higher reference value for testing androgenetic alopecia drugs or treatment means.

Description

Construction method of androgenetic alopecia cell model

Technical Field

The invention relates to the technical field of biology, in particular to a construction method of an androgenetic alopecia cell model.

Background

Androgenetic alopecia (Androgen alopecia, AGA), also known as male pattern alopecia. Androgenic alopecia is characterized by androgens inducing premature transition of hair follicles from anagen to catagen, and miniature changes in hair follicles, which are clinically manifested as progressive alopecia (c.a. jahonda, cellular and developmental aspects of androgenetic alopecia, experimental dermatology,7 (1998) 235-248). AGA is a disease with complex etiology and affected by multiple factors, and many unknowns are available in the current etiology research. Previous studies have considered that one of the main causes of male pattern alopecia is a genetic factor. In recent years. With the continuous progress of molecular biology, the research on the pathogenesis of androgenic alopecia is deeper, and the theory that androgens may cause AGA to occur is more mature. The 5-alpha-reductase contained in androgenic alopecia hair papilla cells can lead to the conversion of exocrine Zhou Gaotong to the more active dihydrotestosterone. Dihydrotestosterone binds to androgen receptors in the nucleus and initiates a series of reactions triggering this effect (A.L.Dallob, N.S.Sadick, W.Unger, S.Lipert, L.A.Geissler, S.L.Gregoire, H.H.Nguyen, E.C.Moore, W.K.Tanaka, the effect of finasteride, a 5alpha-reductase inhibitor, on scalp skin testosterone and dihydrotestosterone concentrations in patients with male pattern baldness, the Journal of clinical endocrinology and metabolism,79 (1994) 703-706). In general, in androgenic alopecia hair follicles, hair papilla cells are the target cell type for androgenic action. Androgens drive hair papilla cells in paracrine fashion against hair follicle epidermal cells, resulting in miniaturization of hair follicles and changes in the cycle of hair follicles (V.A.Randall, N.A.Hibberts, M.J.Thornton, A.E.Merrick, K.Hamada, S.Kato, T.J.Jenner, i.de Oliveira, a.g. messenger, do androgens influence hair growth by altering the paracrine factors secreted by dermal papilla cells. Thus, the construction of an androgenic alopecia model is advantageous for further exploring the pathological mechanism of androgenic alopecia and for drug screening.

The current androgenetic alopecia related models are mainly divided into the following two types: animal models and organ models, the former mainly uses mice as experimental bodies, the development cost is high, the construction period is long, huge species differences exist, and the results are difficult to be transformed into clinic. The latter hair follicle micro-organs can only maintain growth in vitro for two weeks, the research window is narrow, and the apoptosis of hair follicle cells induced by the body administration of high androgens cannot completely imitate the hair follicle-related pathological changes in AGA.

Then, in order to elucidate the regulation of hair follicle epidermal cells by dihydrotestosterone-treated hair papilla cells in a more refined androgenic alopecia cell model, itam, S et al set up an androgenic alopecia cell model by placing outer root sheath cells (ORS) in the upper chamber of a Transwell culture dish and hair papilla cells in the lower chamber of the Transwell culture dish, and adding dihydrotestosterone (S.Itami, S.Kurata, T.Sonoda, S.Takayasu, interaction between dermal papilla cells and follicular epithelial cells in vitro: effect of androgen, the British journal of dermatology,132 (1995) 527-532). Subsequently, several scientists have explored the mechanism of androgen-induced hair loss using this co-culture model. Kwack et al have studied to find that DHT mediates secretion of Wnt signaling pathway inhibitor 1 (Dkk 1) by DPC (M.H.Kwack, Y.K.Sung, E.J.Chung, S.U.Im, J.S.Ahn, M.K.Kim, J.C.Kim, dihydrotestosterone-inducible dickkopf 1from balding dermal papilla cells causes apoptosis in follicular keratinocytes,The Journal of investigative dermatology,128 (2008) 262-269). Leirbos et al changed keratinocytes in the lower chamber of the cell model to hair follicle stem cells, found that DHT activated GSK-3β in DPC and inhibited differentiation of hair follicle stem cells by phosphorylating β -catenin (T.Kitagawa, K.Matsuda, S.Inui, H.Takenaka, N.Katoh, S.Itami, S.Kishimoto, M.Kawata, keratinocyte growth inhibition through the modification of Wnt signaling by androgen in balding dermal papilla cells, the Journal of clinical endocrinology and metabolism,94 (2009) 1288-1294). Inui et al studied the role of Androgen-induced hair papilla cell secreted TGF-beta in androgenetic alopecia, which was involved in the process of epidermal cell growth inhibition in a cell model (S.Inui, Y.Fukuzato, T.Nakajima, K.Yoshikawa, S.Itami, androgen-induced TGF-beta 1from balding dermal papilla cells inhibits epithelial cell growth: a clue to understand paradoxical effects of Androgen on human hair growth, FASEB journ: official publication of the Federation of American Societies for Experimental Biology,16 (2002) 1967-1969).

However, the hair papilla cells in the existing cell model are arranged and grown in a two-dimensional plane, so that the communication between the cells and extracellular matrixes is lacking, the two-dimensional growing cells proliferate, migrate, differentiate and in-vivo difference is large (S.Thippabhotla, C.Zhong, M.He,3D cell culture stimulates the secretion of in vivo like extracellular vesicles,Scientific reports,9 (2019) 13012), the form of hair papilla balls in the human body cannot be truly simulated, the characteristics of androgenetic alopecia cannot be truly simulated, and the pathophysiological research, pathogenesis research, in-vitro drug screening, drug evaluation and the like of androgenetic alopecia are greatly limited.

Disclosure of Invention

The invention aims to solve the problems that a 2D androgenetic alopecia model in the prior art lacks communication between cells and extracellular matrixes, and two-dimensional growing cells have large proliferation, migration, differentiation and in-vivo differences and cannot truly simulate the morphology of a hair papilla ball in a human body, and provides a construction method of the androgenetic alopecia cell model.

To achieve the purpose, the invention adopts the following technical scheme:

the method for constructing the androgenetic alopecia cell model comprises the following steps:

s10, taking hair follicle bulb parts of forehead parts of androgenetic alopecia patients, separating hair papilla of the hair follicle bulb parts, and culturing;

s20, taking a carina region of the occipital part of an androgenetic alopecia patient, separating outer root sheath cells of the carina region, and culturing;

s30, culturing the hair papilla cells obtained after the culturing treatment in the step S10 in 3D (three-dimensional) mode, and enabling the hair papilla cells to aggregate to form hair papilla cell balls;

s40, inoculating the hair papilla cell balls into an upper chamber of a transwell culture dish, inoculating the external root sheath cells obtained after the culture treatment in the step S20 into a lower chamber of the pre-coated transwell culture dish, and adding dihydrotestosterone to obtain the cell model of androgenetic alopecia.

Further, the step S10 specifically includes the following steps:

s10a, taking hair follicles of the forehead of an androgenetic alopecia patient and flushing;

s10b, taking the hair bulb part of the washed hair follicle, and adding collagenase for digestion to obtain a hair papilla;

s10c, centrifuging the hair papilla, and discarding supernatant; re-suspending with penicillin-streptomycin and DMEM medium, inoculating into a cell culture flask, and adding penicillin-streptomycin and DMEM medium for culturing;

s10d, when the cells grow to be more than 80% of the bottle bottom area, the adherent papilla cells are digested by trypsin/EDTA and transferred to a new culture dish in a ratio of 1:2, and transferred to the second generation.

Further, the hair follicle in step S10a is taken from the bald area of the forehead of an androgenic alopecia patient.

Further, the concentration of the collagenase is 2mg/ml;

preferably, in step S10c, the centrifugation conditions are 1000rpm for 5min;

preferably, in step S10c, the culture medium is resuspended in 1% (v/v) penicillin-streptomycin and DMEM containing 20% (v/v) fetal bovine serum, and then inoculated into cell cultureIn a flask, 1% (v/v) penicillin-streptomycin and DMEM medium containing 20% (v/v) fetal bovine serum were added at 37℃and 5% CO ₂ Culturing in an incubator.

Further, the step S20 specifically includes the following steps:

s20a, taking hair follicles of occipital areas of patients with androgenetic alopecia who undergo hair transplantation surgery, and rinsing;

s20b, taking a carina region of the hair follicle, adopting neutral enzyme treatment, then separating and removing a dermis sheath, adopting trypsin to treat dermis components and filtering;

s20c, placing the filtered outer root sheath cells into a culture dish pre-coated with 10 mug/mL human fibronectin, and adding human epidermal keratinocyte culture medium (KSFM) into the culture dish.

Further, the hair follicle in step S20a is taken from a non-bald area of the occiput of an androgenic alopecia patient who has undergone a hair transplantation operation.

Further, in step S20b, the concentration of the neutral enzyme is 0.1%, and the neutral enzyme treatment time is 45min; the concentration of trypsin is 0.25%, and the trypsin treatment time is 10min; the filtration was carried out using a 70 μm filter.

Further, the step S30 specifically includes the following steps:

s30a, subjecting the second generation hair papilla cells to trypsin/EDTA digestion treatment, and then neutralizing by adopting DMEM to obtain hair papilla cell suspension;

s30b, centrifuging the hair papilla cell suspension, and discarding the supernatant; then adopting DMEM to resuspend, and performing cell count;

s30c, inoculating in 96-well ultra-low adsorption plate, inoculating 3000 hair papilla cells in each well, and placing at 37deg.C and 5% CO ₂ Is cultured in an incubator until the cells aggregate into papilla cell balls having a diameter of 200. Mu.m.

Further, step S30a specifically includes: subjecting the second generation hair papilla cells to digestion treatment by using 0.25% (w/v) trypsin/EDTA, and then neutralizing by using DMEM (medium-electron microscope) containing 10% fetal bovine serum (v/v) to obtain hair papilla cell suspension;

preferably, the centrifugal speed in S30b is 1000rpm;

preferably, the DMEM used for the resuspension in S30b contains 10% fetal bovine serum (v/v).

Further, the step S40 specifically includes the following steps:

s40a, placing 10 3D cultured papilla cell spheres in an upper chamber of a transwell culture dish;

s40b, putting 4000 outer root sheath cells into serum-free DMEM one day in advance, inoculating the outer root sheath cells into a lower chamber of a transwell culture dish pretreated by 10 mug/mL human fibronectin, and adding human epidermal keratinocyte culture medium (KSFM);

s40c, changing a human epidermal keratinocyte culture medium (KSFM) after 1 day, and then adding 1-100 mu M dihydrotestosterone to obtain a cell model of androgenetic alopecia;

preferably, 10. Mu.M dihydrotestosterone is added in step S40 c.

The invention has the beneficial effects that: the diameter of the hair papilla cells cultured by the invention is similar to that of hair papilla balls in vivo, the real arrangement form of the hair papilla cells in vivo is reduced, and the communication between cells and extracellular matrixes is promoted. The cell model of androgenetic alopecia built by the invention is stable, the number of the sheath cells of the outer root is reduced after successful modeling, the change trend of the androgenetic alopecia related markers is consistent with that of the inner body, and the cell model has higher reference value for testing androgenetic alopecia drugs or treatment means and is suitable for popularization and application. Compared with an androgenetic alopecia animal model, the method is convenient and quick, and has the advantages of lower cost, no species difference, shortened modeling time and reduced modeling risk.

Drawings

FIG. 1 shows immunofluorescence experiments of Androgen Receptor (AR) expression by generation 2, 4, 6, and 8 hair papilla cells.

FIG. 2 shows the experiments of protein immunoblotting of Androgen Receptor (AR) expressed by the generation 2, 4, 6 and 8 hair papilla cells.

Fig. 3 is a graph showing the relative numbers of external root sheath cells (ORS) in 2D and 3D androgenic alopecia cell models.

FIG. 4 shows protein immunoblotting experiments of 0, 1uM, 100uM, 10 uM Dihydrotestosterone (DHT) -treated hair papilla cells expressing Ki67, clear caspase3 in 2D and 3D androgenic alopecia cell models.

FIG. 5 shows the gene expression levels of 2D and 3D hair papilla cells with respect to extracellular matrix receptor interactions.

Fig. 6 is a change in androgenic alopecia related biomarkers after addition of dihydrotestosterone to hair papilla cells in a 3D model of androgenic alopecia.

FIG. 7 shows the construction flow and subsequent sequencing analysis of 2D and 3D androgenic alopecia cell models according to the present invention.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments.

The various starting materials of the present invention are commercially available, or may be prepared according to methods conventional in the art, unless specifically indicated.

Example 1

Step one, obtaining a hair bulb, inoculating, culturing and passaging.

1. The human hair follicle is taken from the bald (frontal) area of a male patient undergoing a hair transplantation procedure;

2. placing hair follicles into DPBS for three times, shearing hair bulb parts of the hair follicles under a split type microscope by using microsurgery scissors, flushing the hair bulb parts with the DPBS for three times, adding 3ml of collagenase of 2mg/ml for digestion for 30min after the DPBS is absorbed, and blowing once every 10min to obtain hair bulb suspension;

3. the hair papilla pellet suspension was centrifuged at 1000rpm for 5min, the supernatant was discarded, resuspended in 1% (v/v) penicillin-streptomycin and DMEM medium containing 20% (v/v) fetal bovine serum, the hair papilla pellet was inoculated into a cell culture flask, and 1% (v/v) penicillin-streptomycin and DMEM medium containing 20% (v/v) fetal bovine serum were added and left to stand at 37℃and 5% CO ₂ Culturing in an incubator;

4. once the cells grew beyond 80% of the bottom of the flask, the adherent papilla cells were digested with 0.25% (w/v) trypsin/EDTA and transferred to a new dish at a 1:2 ratio for the second generation.

Step two, separating out the outer root sheath cells from the hair follicle of the androgenetic alopecia patient, inoculating and culturing.

1. Human hair follicles are taken from non-bald (occipital) areas of male patients undergoing hair transplantation surgery;

2. placing the hair follicle into DPBS, flushing for three times, and shearing the carina region of the hair follicle under a split type microscope by using microsurgery scissors; the dermis was treated with 0.1% neutral enzyme for 45min, then the dermis sheath was separated and removed under a stereoscope, and the dermis component was treated with 0.05% trypsin for 10min.

3. After the digestion was terminated, the sample was filtered through a 70 μm filter. The filtered outer root sheath cells were placed in a culture dish pre-coated with 10. Mu.g/mL human fibronectin and co-cultured in human epidermal keratinocyte medium (KSFM).

Thirdly, performing 3D culture on the papilla cells to form cell spheres, and inoculating the cell spheres into the upper chambers of 6 transwell culture dishes.

1. The hair papilla cells of the second generation were digested with 0.25% (w/v) trypsin/EDTA for 3min, and neutralized with 3ml DMEM containing 10% fetal bovine serum (v/v), to obtain hair papilla cell suspension;

2. the hair papilla cell suspension was centrifuged at 1000rpm for 5min, the supernatant was discarded, resuspended in 10% fetal bovine serum (v/v) DMEM, and the cells counted. Inoculating cells in a 96-well ultra-low adsorption plate; each well was seeded with 3000 cells and placed at 37℃and 5% CO ₂ Culturing in an incubator;

after 3.1 days, the cells aggregated into spheres (papilla cell spheres) with a diameter of about 200 μm;

4. 10 3D cultured hair papilla cell pellets were placed in the upper chamber of 6 transwell dishes.

Step four, inoculating the outer root sheath cells into the lower chamber of a transwell culture dish to obtain a 3D cell model (control-3D).

The outer root sheath cells were placed in serum-free DMEM one day in advance, and then inoculated into the lower chamber of a transwell culture dish pretreated with 10 μg/mL human fibronectin, 4000 cells per well, and human epidermal keratinocyte medium (KSFM) was added to obtain the cell model (control-3D) of the example.

Step five, control-3D groups 3 transwell dishes were filled with 10. Mu.M dihydrotestosterone to give 2 groups of models (control-3D, DHT-3D). Wherein DHT-3D is an androgenic alopecia cell model of the present invention.

Example 2

This example is identical to the procedure of example 1 above, except that the dihydrotestosterone concentration is 100 μm.

Example 3

This example is identical to the procedure of example 1 above, except that the dihydrotestosterone concentration is 1 μm.

Comparative example 1

The comparative example was the same as example 1 above, except that the hair papilla cells digested in step three were not subjected to 3D culture, and were directly inoculated into the upper chamber of 6 transwell dishes with a cell count of 3X 10 per well ⁴ 。

Comparative example 2

This comparative example was identical to example 1 above, except that the papilla cells obtained in step one were cultured to the fourth generation.

Comparative example 3

This comparative example was identical to example 1 above, except that the papilla cells obtained in step one were cultured until the sixth generation.

Comparative example 4

This comparative example was identical to example 1 above, except that the papilla cells obtained in step one were cultured until the eighth generation.

Immunofluorescent staining and immunoblotting experiments were performed on the hair papilla cells of example 1 and comparative examples 2 to 4 expressing Androgen Receptor (AR), respectively. As shown in FIGS. 1 and 2, the results show that the second generation hair papilla cells in example 1 showed the highest androgen receptor expression compared with comparative examples 2 to 4, and the androgen receptor expression decreased with the increase of the algebra of the cells, indicating that the second generation hair papilla cells cultured in vitro were consistent with the expression of the androgen receptor in the hair papilla cells in patients with androgenic alopecia, and therefore the second generation hair papilla cells were selected.

Protein immunoblotting experiments with Ki67, clear caspase3 was expressed on the hair papilla cells of example 1 and examples 2 and 3, respectively. As shown in fig. 3, the dihydrotestosterone can inhibit proliferation of hair papilla cells and promote apoptosis, and the concentration of dihydrotestosterone in example 1 has the most remarkable effect of inhibiting hair papilla cells compared with examples 2 and 3, so that 10 μm of dihydrotestosterone is selected as the optimal concentration.

The change in the number of outer root sheath cells in control-3D and DHT-3D in example 1 and control-2D and DHT-2D in comparative example 1 after 3 days of culture was calculated, respectively, and the results are shown in FIG. 4: control-3D is more abundant than control-2D in cell number; the cell numbers of the DHT-2D and DHT-3D groups were smaller than those of the control-2D and control-3D groups, respectively.

After 3 days of incubation of the samples of example 1 and comparative example 1, control-2D, DHT-2D, control-3D, DHT-3D four groups of papilla cells were removed, RNA extracted, pooled and sequenced. As shown in FIG. 5, the expression of 21 related genes of extracellular matrix receptor interaction of the control-3D group is increased relative to the expression of the control-2D group, and the expression of 2 related genes is reduced, so that the 3D model proves that the communication between cells and extracellular matrix is increased, and the in-vivo situation is reflected more truly.

Finally, the two groups of androgen-related markers of control-3D and DHT-3D in example 1 were analyzed, and as shown in FIG. 6, IGF-1 expression was decreased, DKK1, IL-6, TGF-. Beta.1, PTGDS, CXXC5 expression was increased, consistent with changes in vivo, indicating that the cell model mimics the in vivo situation to some extent.

A flow chart of the construction and verification method of example 1 and comparative example 1 is shown in fig. 7.

The above examples are only for illustrating the detailed method of the present invention, and the present invention is not limited to the above detailed method, i.e., it does not mean that the present invention must be implemented depending on the above detailed method. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. The method for constructing the androgenetic alopecia cell model is characterized by comprising the following steps of:

s10a, taking hair follicles of a bald area of the forehead of a patient suffering from androgenetic alopecia and flushing;

s10b, taking the hair bulb part of the washed hair follicle, adding collagenase with the concentration of 2mg/ml for digestion, and obtaining a hair papilla;

s10c, centrifuging the hair papilla, and discarding supernatant; resuspension was performed using 1% (v/v) penicillin-streptomycin and DMEM medium containing 20% (v/v) fetal bovine serum, followed by inoculation into cell culture flasks, and 1% (v/v) penicillin-streptomycin and DMEM medium containing 20% (v/v) fetal bovine serum were added at 37 ℃ and 5% co ₂ Culturing in an incubator;

s10d, when the cells grow to be more than 80% of the bottle bottom area, digesting the attached papilla cells by trypsin/EDTA, transferring the cells into a new culture dish in a ratio of 1:2, and transferring the cells to a second generation;

s20a, taking hair follicles of a non-bald area of a pillow part of an androgenetic alopecia patient subjected to a hair transplantation operation, and rinsing;

s20b, taking the carina region of the hair follicle, adopting neutral enzyme with the concentration of 0.1% to treat for 45min, then separating and removing dermis sheath, adopting trypsin with the concentration of 0.25% to treat dermis component for 10min and filtering;

s20c, placing the filtered outer root sheath cells into a culture dish pre-coated with 10 mug/mL human fibronectin, and adding a human epidermis keratinocyte culture medium (KSFM) into the culture dish;

s30a, subjecting second generation hair papilla cells to digestion treatment by using 0.25% (w/v) trypsin/EDTA, and then neutralizing by using DMEM (medium-high-concentration polyethylene) containing 10% fetal bovine serum (v/v) to obtain hair papilla cell suspension;

s30c, inoculating in 96-well ultra-low adsorption plate, inoculating 3000 hair papilla cells in each well, and placing at 37deg.C and 5% CO ₂ Culturing in an incubator until the cells aggregate into papilla cell balls with a diameter of 200 μm;

s40b, putting 4000 outer root sheath cells into serum-free DMEM one day in advance, inoculating the outer root sheath cells into a lower chamber of a transwell culture dish pretreated by 10 mu g/mL human fibronectin, and adding a human epidermal keratinocyte culture medium (KSFM);

and S40c, replacing a human epidermal keratinocyte culture medium (KSFM) after 1 day, and then adding 10 mu M dihydrotestosterone to obtain a cell model of androgenetic alopecia.

2. The method of constructing a model of androgenic alopecia according to claim 1, wherein in step S10c, the centrifugation condition is centrifugation at 1000rpm for 5min.

3. The method of constructing an androgenic alopecia cell model according to claim 1, wherein in the step S20b, a 70 μm filter is used for the filtration.

4. The method of constructing an androgenic alopecia cell model according to claim 1, wherein the centrifugal rotation speed in S30b is 1000rpm.

5. The method of constructing an androgenic alopecia cell model according to claim 1, wherein the DMEM for the resuspension in S30b contains 10% fetal bovine serum (v/v).