JP7385842B2 - Observation methods, measurement methods, analysis methods, quantitative methods, and kits - Google Patents

Description

The present invention relates to a method for observing, measuring, analyzing, and quantifying Meis1 protein, and a kit for performing the same.

Many people have problems with hair loss, such as hair loss due to aging or genetic factors, or alopecia areata due to excessive stress. According to the 2017 edition of the Medical Treatment Guidelines for Male and Female Pattern Hair Loss (Japan Dermatological Association Guidelines), the incidence of male pattern hair loss, androgenetic alopecia (AGA), is approximately 30% on average for all ages. .
In addition, according to the 2014 government statistics patient survey (injury/disease classification change), the total number of patients who visited hospitals due to alopecia alone exceeded 100,000, and it is believed that there are many more potential alopecia patients. It will be done.

Hair follicles, which are the organs that form hair, have the unique property of repeatedly regenerating and degenerating, and in recent years, hair regeneration from mature skin tissue has been intensively studied. Hair follicles have a growth phase (Anagen) in which the hair grows, a catagen phase (Catagen) in which the hair prepares to fall out, and a telogen phase (Telogen) in which the hair is ready to fall out. The cycle is called the hair cycle. During the growth phase, dermal papilla cells in the bulb of the hair follicle are activated, causing hair matrix cells to proliferate and differentiate, and elongate as a hair shaft, resulting in hair growth. After that, when the catagen phase begins, hair growth declines, and the hair irreversibly enters the telogen phase, where hair growth stops and hair loss eventually occurs. The hair cycle is a delicate biological mechanism that has its own cycle for each hair follicle and is controlled by various genes and their products, or the environment around the hair follicle.

Non-Patent Document 1 describes that a protein called Meis1, which is a transcription factor, is expressed in the bulge region within the hair follicle (region where hair follicle stem cells exist) and dermal papilla cells during the resting phase of mouse skin. ing. In the same literature, it was confirmed that conditional deletion of Meis1 protein on epithelial cells including stem cells during the resting phase caused epidermal hyperplasia and a significant decrease in epithelial stem cells in the bulge region. .

K. Okumura et al. PLoS One. 2014 Jul 11;9(7)

Regarding the role of Meis1 protein in the skin, it has been reported that it is necessary for the maintenance of epithelial stem cells in the hair follicle bulge region, but it is necessary for the maintenance of epithelial stem cells in the hair follicle bulge region. There are no reports yet on its role in the regression phase.
An object of the present invention is to perform detailed observation of Meis1 protein at each stage of the hair cycle.

The present inventors focused on the relationship between Meis1 protein in dermal papilla cells and the hair growth mechanism. By observing the Meis1 protein in the hair follicle, the present inventors found that the location of the Meis1 protein in the dermal papilla cells of the hair bulb changes depending on the hair cycle.
The present invention relates to observation methods, measurement methods, analysis methods, quantitative methods, and kits for carrying out these methods regarding Meis1 protein (sometimes abbreviated as Meis1 in this specification).

That is, the present invention provides, for example, the following [1] to [17].
[1]
Method for observing Meis1 protein in the hair bulb.
[2]
The observation method according to [1], wherein Meis1 protein in dermal papilla cells contained in the hair bulb is observed.
[3]
The observation method according to [2], which comprises observing the location of Meis1 protein in the dermal papilla cells.
[4]
The observation method according to any one of [1] to [3], which involves observing Meis1 protein stained by fluorescent immunostaining.
[5]
The observation method according to [4], further comprising staining the nucleus in the observation method.
[6]
The observation method according to [5], wherein tissue staining is further performed in the observation method.
[7]
A measurement method of observing Meis1 protein in dermal papilla cells and measuring the number of dermal papilla cells in which Meis1 protein is localized in the nucleus.
[8]
The measurement method described in [7], in which Meis1 protein stained by fluorescent immunostaining is observed.
[9]
The measurement method according to [7] or [8], wherein tissue staining is further performed before the observation.
[10]
A method for analyzing the hair cycle of hair follicles by observing Meis1 protein in dermal papilla cells and observing the number of cells in which Meis1 protein is present in the nucleus of dermal papilla cells.
[11]
A method for analyzing the hair cycle of a hair follicle, which comprises observing the location of Meis1 protein in the dermal papilla cells and calculating the ratio of the number of dermal papilla cells in which Meis1 protein is present in the nucleus to the total number of dermal papilla cells.
[12]
A quantitative method for quantifying the amount of Meis1 protein present in the nucleus by immunofluorescently staining Meis1 protein in dermal papilla cells and observing the obtained fluorescent signal.
[13]
A quantitative method for quantifying the amount of Meis1 protein present in the nucleus of dermal papilla cells by the following steps.
(a) Step of isolating dermal papilla cells (b) Step of separating a nuclear fraction from the dermal papilla cells (c) Step of quantifying Meis1 protein contained in the nuclear fraction and other fractions
[14]
A method of analyzing the hair cycle of hair follicles using the quantitative method of [12] or [13].
[15]
A kit for carrying out the observation method according to any one of [3] to [6], comprising at least one of the following (a) and (b) and (c).
(a) an agent for isolating dermal papilla cells from hair;
(b) a drug for performing tissue staining;
(c) Labeling agent for specifically labeling Meis1 protein:
[16]
A kit for carrying out the quantitative method described in [13], comprising the following (a') to (c').
(a') an agent for isolating dermal papilla cells from hair;
(b') an instrument for fractionating the nuclear fraction and other fractions of the dermal papilla cells;
(c') Labeling agent for specifically labeling Meis1 protein:
[17]
The kit according to [15] or [16], wherein the labeling agent is an anti-Meis1 antibody.

According to the observation method of the present invention, by observing the hair bulb, it is possible to observe the location where Meis1 protein is present, and by quantifying it, it is possible to analyze the hair cycle of the hair bulb.

FIG. 1(a) is a schematic diagram showing changes in the location of Meis1 in each hair cycle of mouse dermal papilla cells. FIG. 1(b) is a diagram showing morphological changes in mouse body hair during each hair cycle. FIG. 2 is a diagram showing changes in the intracellular location of Meis1 in each hair cycle of mouse dorsal skin. (a) HE-stained images using mouse skin sections at each hair cycle. (b) Fluorescent immunostaining images for Meis1 in each hair cycle in the bulge region of the hair follicle in the mouse dorsal skin. (c) Fluorescent immunostaining images for Meis1 in each hair cycle in the hair bulb. FIG. 3(a) is a photograph showing whisker hair follicles of a wild type mouse (C57BL/6) at each hair cycle. FIG. 3(b) is a fluorescent immunostaining image for Meis1 of dermal papilla cells of whisker hair follicles in each hair cycle. FIG. 3(c) is a graph showing the relationship between changes in the percentage of cells in which Meis1 is present in the nucleus and the hair cycle. Figure 4 shows bright field images showing changes in hair shaft growth over time in the whiskers of a mouse that does not lack Meis1 (left image) and the whiskers of a mouse that lacks Meis1 (right image), and It is a graph showing changes over time in the length of whisker hair shafts of mice. FIG. 5(a) is a stained image showing the results of EdU staining of the whisker hair follicles of a mouse that does not lack Meis1 and a mouse that lacks Meis1. FIG. 5(b) is a graph quantifying the results of FIG. 5(a). FIG. 5(c) is a graph showing the thickness of the region where hair matrix cells exist on the Auber's line of the body hair of each mouse. Figure 6(a) shows the results of EdU staining of whisker hair follicles from Meis1-deficient mice cultured with the culture supernatant of wild-type mouse dermal papilla cells or the culture supernatant of Meis1-deficient dermal papilla cells. This is a stained image. FIG. 6(b) is a graph quantifying the results of FIG. 6(a). FIG. 6(c) is a graph showing the thickness of the region where hair matrix cells exist on the Auber's line of the body hair of each mouse. FIG. 7 is a schematic diagram showing the behavior and mechanism of action of Meis1 protein in dermal papilla cells during the growth phase, catagen phase, and resting phase based on the obtained findings.

<Observation method>
The "observation method" of the present invention is carried out by observing Meis1 protein in dermal papilla cells contained in the hair bulb, preferably by observing Meis1 protein in dermal papilla cells contained in the hair bulb. carried out by

In the "observation method" of the present invention, it is preferable that the Meis1 protein be observed by specifically labeling the Meis1 protein by fluorescent immunostaining.
The "target substance" of the fluorescent immunostaining used in the present invention is the Meis1 protein, and the specimen to be stained includes the hair bulb of hair or body hair, dermal papilla cells contained in the hair bulb, and the hair bulb. Preferably, the cells are dermal papilla cells isolated from skin tissue or the hair bulb.

In the "observation method" of the present invention, it is preferable to observe the location of Meis1 protein in dermal papilla cells included in the hair bulb. As for the location where Meis1 protein is present, the location where Meis1 protein is present in one dermal papilla cell may be observed, or the location where Meis1 protein is present in multiple dermal papilla cells may be observed. , the location where Meis1 protein is present in all dermal papilla cells included in the hair bulb may be observed.

Furthermore, when observing a single dermal papilla cell, for example, 80% to 100%, more preferably 90% to 100% of the Meis1 protein is present in the nucleus, and Meis1 protein is localized in the nucleus. It is preferable to determine that the cells are existing dermal papilla cells. Specifically, for example, when Meis1 protein is fluorescently immunostained and the nucleus is stained with DAPI, if 90% or more of the signal derived from the fluorescent immunostaining overlaps with the nucleus, the Meis1 protein is detected in the nucleus in the dermal papilla cell. It can be determined that it is localized within the

As mentioned above, the hair cycle includes a growth phase (Anagen) in which hair grows, a catagen phase (Catagen) in which hair prepares to fall out, and a resting phase (Telogen) which is a period until hair falls out. The growth period (Anagen) is roughly divided into the Very Early Growth Period (Very Early Anagen), the Early Growth Period (Early Anagen), the Mid Growth Period (Mid Anagen), and the Late Growth Period (Late Anagen). According to studies by the present inventors, Meis1 protein moves from outside the nucleus into the nucleus during the early growth period, early growth period, and middle growth period. Then, after the middle growth phase ends, the Meis1 protein moves from the nucleus to the outside of the nucleus during the late growth phase, catagen phase, and resting phase.

FIG. 1 shows a schematic diagram of changes in the location of Meis1 in each hair cycle and morphological changes in mouse body hair in each hair cycle. As mentioned above, dermal papilla cells in which 80% to 100%, more preferably 90% to 100%, of Meis1 protein is located in the nucleus can be said to be dermal papilla cells in which Meis1 protein is localized in the nucleus; When such dermal papilla cells account for 90% or more of all dermal papilla cells in a hair follicle, the hair follicle can be said to be in the middle growth phase. Furthermore, Meis1 protein present in the nucleus moves from the inside of the nucleus to the outside of the nucleus from the middle growth stage to the late growth stage. In the catagen phase, the proportion of dermal papilla cells in which Meis1 protein is localized in the nucleus is usually less than 10%, and in the telogen phase, it is 0 to 1%. Then, as the hair cycle enters the growth phase again, the Meis1 protein gradually re-translocates into the nucleus.

The "observation method" of the present invention may be performed by observing Meis1 protein stained by fluorescent immunostaining. Fluorescent labeling of Meis1 protein in this fluorescent immunostaining may be performed directly or indirectly. As a specific method, the Meis1 protein may be specifically labeled by reacting the anti-Meis1 antibody with a fluorescent dye directly chemically modified with the Meis1 protein on the sample; By reacting the Meis1 antibody with a specimen such as a hair root, and then using a fluorescently modified anti-IgG antibody, the fluorescently modified anti-IgG antibody is reacted with the anti-Meis1 antibody bound to the Meis1 protein on the specimen. Meis1 protein may be specifically labeled.

In the process of observing specimens that have undergone fluorescent immunostaining, excitation light corresponding to the fluorescent dye used for fluorescent immunostaining is irradiated onto the specimen sample under a microscope at a desired magnification, and the immunofluorescence generated by the fluorescent dye is detected. Observe and photograph each stained image. Irradiation with these excitation lights can be performed, for example, by using a laser light source included in a fluorescence microscope and, if necessary, an optical filter for excitation light that selectively transmits a predetermined wavelength. Moreover, when photographing a fluorescent immunostained image, it can be carried out using, for example, a digital camera included in a fluorescent microscope. When taking fluorescence immunostained images, if necessary, use a fluorescence optical filter that selectively transmits predetermined wavelengths, so that only the desired fluorescence is included, and unintended fluorescence and excitation that becomes noise are eliminated. It is possible to take immunostained images excluding light and other light.

Fluorescent dyes are irradiated with electromagnetic waves (X-rays, ultraviolet rays, or visible light) of a predetermined wavelength, and when the energy is absorbed, electrons are excited, and when they return from the excited state to the ground state, they release excess energy. Refers to substances that emit electromagnetic waves. Furthermore, "fluorescence" has a broad meaning, and includes phosphorescence, which has a long luminescence life and continues to emit light even after irradiation with electromagnetic waves for excitation is stopped, and fluorescence in a narrow sense, which has a short luminescence life.

The fluorescent dye is not particularly limited, and examples thereof include rhodamine dyes, squarylium dyes, cyanine dyes, aromatic ring dyes, oxazine dyes, carbopyronine dyes, and pyromecene dyes. Alternatively, Alexa Fluor (registered trademark, manufactured by Invitrogen) pigments, BODIPY (registered trademark, manufactured by Invitrogen) pigments, Cy (registered trademark, manufactured by GE Healthcare) pigments, DY (registered trademark, manufactured by DYOMICS) HiLyte (registered trademark, manufactured by Anaspec) pigments, DyLight (registered trademark, manufactured by Thermo Scientific) pigments, ATTO (registered trademark, manufactured by ATTO-TEC) pigments, MFP (registered trademark, manufactured by ATTO-TEC) pigments, (manufactured by Mobitec), etc. can be used. The generic name of such dye molecules is based on the main structure (skeleton) in the compound or the registered trademark, and those skilled in the art will need extensive trial and error to determine the range of fluorescent dyes that belong to each category. This is something that can be properly grasped without any problems.

Further, it is preferable to specifically fluorescently label the Meis1 protein and stain the nucleus at the same time, and it is more preferable to stain the nucleus fluorescently. By simultaneously staining the nucleus with fluorescence, it is possible to determine whether the Meis1 protein is present in the nucleus, outside the nucleus, or both inside and outside the nucleus. Methods for fluorescently staining the nucleus are not particularly limited, but include DAPI (4',6-diamidino-2-phenylindole), propidium iodide, Hoechst stain, SYTO (registered trademark), TO-PRO (registered trademark)-3, and Nuclear stains such as SYTOX (registered trademark) are widely known, and DAPI is preferably used from the viewpoint of binding particularly strongly to DNA.

In the "observation method" of the present invention, it is preferable to further perform tissue staining in addition to Meis1 protein staining. In this specification, tissue staining refers to staining that enables observation of the skin around hair roots, the morphology of hair roots, cells contained in hair roots, etc. in a bright field. Tissue staining can be performed according to conventional methods. For example, staining using eosin is standard, which stains cytoplasm, stroma, various fibers, red blood cells, and keratinocytes in red to deep red. . In addition, staining using hematoxylin, which stains cell nuclei, calcareous parts, cartilage tissue, bacteria, and mucus in blue-indigo to pale blue, is also standardly used. (also known as eosin staining (HE staining)). When tissue staining is performed, it may be performed after the immunostaining process by using different sections successively cut from the same immunostained specimen, or it may be performed before the immunostaining process. good.

Furthermore, in the "observation method" of the present invention, the Meis1 protein can also be observed by specifically labeling the Meis1 protein by immunostaining using a dye. Specifically, for example, the Meis1 protein may be specifically labeled by immunostaining using an antibody that specifically labels the Meis1 protein combined with an arbitrary dye; After immunostaining using a combination of an antibody that specifically labels Meis1 and an enzyme that develops color with an arbitrary chromogenic substrate, the Meis1 protein can be specifically labeled by reacting the sample with the chromogenic substrate. good. When immunostaining using a dye is performed in this way, observation can be performed using a bright field microscope as appropriate.

<Measurement method>
In the "measuring method" of the present invention, the Meis1 protein in the dermal papilla cells is observed, and the number of dermal papilla cells in which the Meis1 protein is localized in the nucleus is measured.
Preferably, the number of dermal papilla cells in which Meis1 protein is localized in the nucleus is measured by fluorescent immunostaining. Although the measuring method is not particularly limited, it may be measured automatically using an arbitrary program, or may be measured visually. At this time, the criteria for determining whether Meis1 protein is localized in the nucleus of dermal papilla cells can be arbitrarily set, but preferably 80% to 100%, more preferably 90% to 100%. It is preferable to judge a dermal papilla cell in which Meis1 protein is localized in the nucleus to be a dermal papilla cell in which Meis1 protein is localized in the nucleus. Specifically, for example, when Meis1 protein is fluorescently immunostained and the nucleus is stained with DAPI, when 90% of the signal derived from the fluorescent immunostaining overlaps with the nucleus, it is assumed that the Meis1 protein is localized within the nucleus in that cell. It can be determined that there is a In observing Meis1 protein in this measurement method, tissue staining is preferably performed before the observation.

<Analysis method>
In the "analysis method" of the present invention, the amount of Meis1 protein present in the nucleus of dermal papilla cells is observed by the method of observing Meis1 protein in dermal papilla cells. This is a method to analyze the hair cycle.

As described above, in dermal papilla cells, Meis1 protein moves from outside the nucleus to inside the nucleus during the early growth period, early growth period, and middle growth period. Then, after the middle growth phase ends, the Meis1 protein moves from the nucleus to the outside of the nucleus again during the late growth phase, catagen phase, and resting phase.
In one embodiment of the "analysis method," the location of Meis1 protein in dermal papilla cells is observed by a method of observing Meis1 protein in dermal papilla cells, and the dermal papilla cells in which Meis1 protein is present in the nucleus are determined. A method for analyzing the hair cycle of a hair follicle includes a step of calculating the ratio between the total number of dermal papilla cells and the total number of dermal papilla cells. For example, if the number of dermal papilla cells in which Meis1 protein is localized in the nucleus/total number of dermal papilla cells is 90%, it can be analyzed that the hair follicle is in the middle growth phase.

<Quantitative method>
The "quantification method" of the present invention quantifies the amount of Meis1 protein present in the nucleus by observing the fluorescent signal obtained by performing fluorescent immunostaining on Meis1 protein in dermal papilla cells. It is a quantitative method.
In one embodiment of the "quantification method," the amount of Meis1 protein present in the nucleus is quantified using a fluorescent signal obtained by performing fluorescent immunostaining on Meis1 protein in dermal papilla cells.
For example, in a photographed fluorescent immunostained image, a fluorescent labeling signal corresponding to a fluorescent dye that labels the Meis1 protein is measured based on image processing, and a fluorescent labeling signal corresponding to the target biological substance outside the nucleus is identified.

An example of software that can be used for image processing is "ImageJ" (open source: http://imagej.nih.gov/ij/). By using such image processing software, you can extract bright spots with a predetermined wavelength (color) from a fluorescent immunostained image and calculate the sum of their brightness, or calculate the number of bright spots with a predetermined brightness or higher. Processing such as measurement can be performed semi-automatically and quickly.

Further, in another aspect of the "quantification method" of the present invention, (a) a step of isolating dermal papilla cells, (b) a step of separating a nuclear fraction from the dermal papilla cells, (c) a step of the nuclear fraction The amount of Meis1 protein present in the nucleus of dermal papilla cells is quantified by the step of quantifying Meis1 protein contained in fractions and other fractions.

The method for subjecting the step (a) to isolating dermal papilla cells is not particularly limited, and can be carried out by any conventional method. For example, after washing a piece of skin with body hair with phosphate buffered saline (PBS) or the like, if necessary, use a proteolytic enzyme or the like to remove the epidermis layer and dermis layer to leave only the subcutaneous fat layer, and then use tweezers etc. Dermal papilla cells can be isolated by cutting out the hair bulb from the isolated hair follicle and exposing the dermal papilla from the lower part of the hair bulb.

The method used in the step (b) of separating the nuclear fraction from dermal papilla cells is not particularly limited; for example, a commercially available kit such as the 8-min Cytoplasmic & Nuclear Protein Extraction Kit (Cosmo Bio) may be used. It can be carried out.

The method used in the step (c) of quantifying the Meis1 protein contained in the nuclear fraction and other fractions is not particularly limited, and may be a method that is normally used for quantifying a specific protein, such as electrical This can be carried out using any method such as staining using electrophoresis. Furthermore, as an optional step, it is also preferable to quantify the amount of total protein collected from the sample before separating the nuclear fraction and other fractions in (b) above.

The hair cycle of the hair follicle described above can also be analyzed using the "quantification method". Specifically, by quantifying the amount of Meis1 protein in the nuclear fraction and other fractions of the dermal papilla cells separated in step (b), it can be determined that the hair cycle in the dermal papilla cells is in the growth phase. It can be determined whether the animal is in a catagen or telogen phase. For example, in mice, if the Meis1 protein present in the nuclear fraction is 50% or more and less than 80% of the total amount of Meis1 protein, the cell is in the early growth phase, and if it is 80% or more and less than 100%, the cell is in the middle phase. It can be said that it is possible to judge whether a child is in a growth period or not. Note that the numerical value varies greatly depending on animal species, individuals, individuals, etc., so it is preferable to set it arbitrarily.

Note that the proportion of Meis1 protein contained in the nuclear fraction and other fractions in the early growth phase and the regression phase may be similar. In this case, which hair cycle the hair root belongs to can be easily determined based on the morphology of the hair root (see FIG. 1).

According to studies conducted by the present inventors, it has been found that in dermal papilla cells, whether Meis1 is present in the cell nucleus or in the cytoplasm is important in the hair cycle. For example, as described in the Examples below, by using whisker hair follicles in the early growth stage in which Meis1 was conditionally knocked out, the proliferation of hair matrix cells, which are important for hair shaft formation, was significantly reduced due to the deletion of Meis1. Furthermore, the growth inhibition is partially rescued by the addition of dermal papilla cell culture supernatant containing Meis1, but there is no change in the culture supernatant derived from dermal papilla cells in which Meis1 has been conditionally knocked out. It was shown that Therefore, it can be inferred that the function of Meis1 in dermal papilla cells is regulated depending on whether it is present in the cell nucleus or the cytoplasm.

<Kit>
One embodiment of the "kit" of the present invention is a kit for performing the above-mentioned observation method, which includes (a) a drug for isolating dermal papilla cells from hair, and (b) a drug for tissue staining. and (c) a labeling agent for specifically labeling Meis1 protein.
One embodiment of the "kit" of the present invention is a kit for performing the above-mentioned quantitative method, which comprises: (a') an agent for isolating dermal papilla cells from hair; (b') the dermal papilla cells; Equipment for fractionating the nuclear fraction and other fractions; (c') Contains a labeling agent for specifically labeling Meis1 protein.
The labeling agent for specifically labeling Meis1 protein is not particularly limited, but it is preferable to use an anti-Meis1 antibody. For example, when the above kit is used for observation or quantification by fluorescent immunostaining, it is preferable to select an anti-Meis1 antibody that is directly or indirectly modified with a fluorescent dye.

The anti-Meis1 antibody is not particularly limited, but is preferably a monoclonal antibody from the viewpoint of binding strength with Meis1 protein. Furthermore, the antibody may be an antibody fragment or a derivative instead of a natural full-length antibody as long as it has the ability to specifically recognize and bind to the Meis1 protein. That is, the term "antibody" as used herein includes not only full-length antibodies, but also antibody fragments such as Fab, F(ab)'2, Fv, and scFv, chimeric antibodies (humanized antibodies, etc.), and multifunctional antibodies. Derivatives such as

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
[Preparation example 1]
<Preparation of Meis1 conditional knockout mouse (Meis1cKO mouse)>
We investigated the effect on the hair cycle by deleting the Meis1 gene from the whisker hair follicles of mice during the growth phase.
All animal experiments below were conducted in accordance with protocols approved by the Tokyo University of Science Animal Experiment Committee.
A mouse with a fixed allele of the Meis1 gene (Meis1 fl/fl) was created (Ariki et al., 2014; Hirayama et al., 2014), and the mouse was transformed into a Rosa26-CreERT2 transgenic mouse (Kawai et al., 2018). ), a Rosa26-CreERT2;Meis1 flox mouse was created in which a Cre recombinase induction sequence was introduced into the Rosa26 gene locus on the chromosome and a loxP sequence was introduced into the Exon8 region, which is the functional region of Meis1.
In these mice, the Meis1 gene is conditionally deleted in the mouse body hair by administration of 4-hydroxytamoxifen (4-OHT).

[Example 1]
First, in order to confirm the detailed expression of Meis1 in hair follicles, fluorescent immunostaining was performed using an anti-Meis1 antibody. The dorsal skin of wild-type mice (C57BL/6) aged 24 to 51 days was collected, and the skin at each hair cycle was subjected to HE staining (upper panel of Figure 2(a)) and fluorescent immunization with anti-Meis1 antibody. Staining (lower diagram of FIG. 2(a)) was performed.
HE staining was performed using the following method.
Skin from the back of the back was harvested and fixed in 4% paraformaldehyde overnight at 4°C, followed by gradual replacement with 15% then 30% sucrose. Thereafter, the skin piece was embedded in Tissue Tech (registered trademark) OCT compound (Sakura Fine Tech Japan Co., Ltd.) and frozen to prepare a specimen block. A section of 8 to 12 μm was prepared from the specimen block, and then stained with hematoxylin and eosin according to a conventional method.
Fluorescent immunostaining was performed using the following method.

The skin sections prepared above were subjected to cell membrane permeabilization for 20 minutes with 0.5% Triton/PBS, and blocked for 1 hour with blocking buffer (PBS containing 3% BSA and 0.1% Tween 20). The sections were reacted overnight using rabbit anti-Meis1 antibody (Abcam) as a primary antibody at a ratio of 1:100, and after washing, a secondary antibody (Alexa Fluor 647-conjugated anti-rabbit IgG antibody (Cell Signaling Technology)) was incubated. Fluorescent immunostaining of Meis1 was performed by using a ratio of 1:1000 and reacting for 1 hour. Furthermore, each section stained with Meis1 was mounted with DAPI-containing ProLong (registered trademark) Gold (Thermo Fisher Scientific), which is an anti-fading mounting medium. Images were acquired using a HS all-in-one fluorescence microscope BIOREVO BZ9000 (Keyence Corporation) or a confocal laser scanning microscope FV1000-D (Olympus Corporation).

The HE staining results shown in Figure 2(a) show that the skin tissue (dermis and fat layer) is thicker in the middle growth stage compared to the early growth stage, and the shape of the hair bulb is also larger. I can confirm that it is. In the catagen phase, the hair bulb becomes smaller in shape, and in the telogen phase, the hair bulb shrinks, and the dermis and fat layer also become thinner, as observed in the early growth phase.
FIG. 2(b) is a region including the outer root sheath and bulge (Bu) in each hair cycle, and FIG. 2(c) is a fluorescent immunostained image of Meis1 in the hair bulb (region containing dermal papilla cells). In any hair cycle, Meis1 is expressed in dermal papilla cells, the bulge region, and the outer root sheath, and it has been confirmed that Meis1 in the bulge region and outer root sheath is present outside the nucleus within the cell. (Figure 2(b)). On the other hand, in dermal papilla cells, it was confirmed that Meis1 moves between the cytoplasm and nucleus during the hair cycle, and its location within the cell changes. (Figure 2(c)). Arrows indicate locations where Meis1 is present outside the nucleus, and arrowheads indicate locations where Meis1 is accumulated within the nucleus.
That is, in the early growth phase of dermal papilla cells, most of Meis1 is present in the cytoplasm, but in the middle growth phase, it was confirmed that Meis1 is present in the nucleus in many cells. It was also confirmed that when the hair cycle enters the catagen phase, a portion of Meis1 in the nucleus migrates out of the nucleus, and when the hair cycle enters the telogen phase, most Meis1 is present in the cytoplasm.
In other words, in the hair follicle, only in dermal papilla cells, Meis1 changes its location within the cell by moving in and out of the cell nucleus during a series of hair cycles, and Meis1 accumulates in the cell nucleus during the growth phase. This was confirmed.

[Example 2]
In order to investigate whether changes in the location of Meis1 in dermal papilla cells occur regardless of the location or type of hair, we conducted a test using hair follicles from mouse whiskers. Whisker hair follicles can be surgically isolated, and their hair cycle can be observed in the same way as the hair follicles on the back of mice. Furthermore, isolated hair follicles can be cultured on a dish, making it possible to observe and analyze the hair cycle over time, from hair growth to regression.
Hair follicles were obtained by cutting out the cheek tissue (Whisker pad) of a 4- to 8-week-old wild-type mouse (C57BL/6), then cutting out the whisker hair follicles from the tissue pieces, and examining the hair cycle of each hair follicle using a microscope. Classified according to. Hair follicles in the early growth stage, early growth stage, middle growth stage, late growth stage, and regression stage are shown in FIG. 3(a), respectively.
Using the same method as in Example 1, dermal papilla cells in each hair cycle were subjected to fluorescent immunostaining using an anti-Meis1 antibody. The results are shown in FIG. 3(b). Arrows indicate Meis1 existing outside the nucleus, and arrowheads indicate Meis1 accumulated within the nucleus. Similar to hair follicles in the dorsal skin, Meis1 in dermal papilla cells accumulates in the cell nucleus as growth progresses from the early growth stage, Meis1 in the cell nucleus decreases in the late growth stage, and most of it exists outside the nucleus during the catagen stage. It was shown that Even in mouse whisker hair follicles, the location of Meis1 changes between the cytoplasm and nucleus in dermal papilla cells with the hair cycle. It was shown that this phenomenon can occur in both the cheek tissue (beard hair follicles) and the cheek tissue (beard hair follicles).
In addition, by using ImageJ software, we extracted the DAPI-positive region (nucleus) of dermal papilla cells and the cells where the nucleus and Meis1-positive region overlap, respectively, using ImageJ software for the fluorescent immunostained images with anti-Meis1 antibody obtained using the same method. and counted using a multipoint tool. From the counting results, the proportion of cells in which the nucleus and Meis1-positive region overlap with respect to the total number of dermal papilla cells (the number of DAPI-positive cells) was calculated to determine the proportion of dermal papilla cells in which Meis1 was present in the nucleus. A graph showing changes in the proportion of cells in which Meis1 is present in the nucleus during each hair cycle is shown in FIG. 3(C). The figure on the left shows data on hair follicles collected from the back of a mouse, and the figure on the right shows data on hair follicles collected from the whiskers of a mouse. It was shown that in all hair follicles, the proportion of cells in which Meis1 is present in the nucleus is high in the growth phase, especially in the middle growth phase.

[Example 3]
Hair growth was observed using the Rosa26-CreERT2; Meis1 flox mouse produced in Production Example 1. Hair follicles in the early growth phase of a 30-day-old mouse were isolated from the whisker region and cultured in serum-free basal medium for 2 days. 1 μM of 4-OHT and ethanol (EtOH) were added as a control to the hair follicles in which hair growth was confirmed, cultured for 6 days, and hair growth was measured under a stereomicroscope. The results are shown in Figure 4.
In control mice (wild-type mice and EtOH added), constant hair shaft growth was observed until day 8 of culture, whereas in Meis1 conditional knockout mice, hair shaft growth was observed on day 7 of culture, which is the fifth day after addition of 4-OHT. Hair growth was significantly delayed from day one onwards.

[Example 4]
EdU (5-ethynyl-2'-deoxyuridine, 5-ethynyl-2'-deoxyuridine) staining was performed in the same manner as in Example 3 using whisker hair follicles of Rosa26-CreERT2; Meis1 flox mice. This stain stains cells in the cell proliferation phase. Specifically, after adding 1 μM of 4-OHT or ethanol, whisker hair follicles of mice on the 5th day of culture were incubated with 10 μM of EdU for 1 hour at 37°C under 5% CO ₂ , and then Click-iT EdU was added. -The incorporated EdU was fluorescently labeled using the Alexa-Fluor488 kit (Thermo Fisher Scientific). The obtained fluorescence image is shown in Figure 5(a), the graph showing the number of EdU positive hair matrix cells is shown in Figure 5(b), and the thickness of the area where hair matrix cells exist is shown in Figure 5(c). . Cells that were EdU positive and DAPI positive cells in the hair follicle were extracted using ImageJ software (http://imagej.nih.gov/ij/) and counted using a multipoint tool. In addition, the thickness of the region where hair matrix cells exist in the hair bulb was quantified by digital image analysis with respect to Auber's line (a line passing through the maximum diameter in the direction perpendicular to the hair axis).
Hair follicles in which Meis1 was deleted by 4-OHT were shown to have fewer EdU-positive cells, especially in the upper part of the hair bulb. On the other hand, in the hair follicles of the control EtOH group, many EdU-positive cells were observed in the hair bulb. Furthermore, in whisker hair follicles in which Meis1 was deleted by 4-OHT, the growth inhibition in the hair bulb was remarkable in the upper part of the hair bulb. In particular, there was a significant decrease in EdU-positive cells above Auber's line (FIG. 5(a)). FIG. 5(b) is a graph prepared by quantifying the number of EdU-positive cells in FIG. 5a. It was also confirmed that the thickness of the region where hair matrix cells exist at this location was significantly thinner in whisker hair follicles in which Meis1 was deleted (FIG. 5(c)). To summarize the above, it can be said that deletion of Meis1 suppresses the proliferation of the region where hair matrix cells exist.

[Example 5]
Since Meis1 is localized in the nucleus in dermal papilla cells in the mid-anagen phase, it is possible that it plays some function as a transcription factor.
Therefore, the present inventors considered that it is highly likely that Meis1 controls the production of some secreted factors in dermal papilla cells and promotes the proliferation of hair matrix cells. To examine this possibility, we added culture medium of whisker dermal papilla cells from wild-type mice or mice lacking Meis1 to whisker hair follicles lacking Meis1 and cultured them. Only when a culture solution of dermal papilla cells (normal dermal papilla culture solution) was added, the number of EdU-positive cells in Meis1-deficient whisker hair follicles was recovered (FIG. 6(a)). FIG. 6(b) is a graph showing the number of EdU-positive cells above or below Auber's line. Particularly above the Auber's line, it was shown that the number of EdU-positive cells was significantly recovered by the addition of the normal dermal papilla culture solution.
The hair matrix in Auber's line also increased in thickness by adding the dermal papilla cell culture medium, but the matrix thickness did not change in the Meis1-deficient dermal papilla cell culture medium (Fig. 6c). These data showed that this was caused by an interaction between dermal papilla cells and hair matrix. This suggests the possibility that some secreted factors derived from dermal papilla cells that are regulated by the transcriptional activity of Meis1 in the nucleus indirectly promote the proliferation of hair matrix cells above Auber's line. do.

(Consideration)
From the above findings, it can be inferred that the function of Meis1 in dermal papilla cells is regulated depending on whether it is present in the cell nucleus or outside the nucleus. Specifically, at the start of the growth phase, Meis1 translocates into the nucleus in dermal papilla cells, and as a result, Meis1 in the nucleus as a transcription factor in dermal papilla cells is involved in the transcription of some secreted proteins, and the secreted proteins It is suggested that the proliferative activity of hair matrix cells existing in the vicinity and hair growth are promoted. FIG. 7 shows a schematic diagram showing the behavior and mechanism of action of Meis1 in dermal papilla cells during the growth phase, catagen phase, and telogen phase.

Claims (8)

A measurement method of observing Meis1 protein in dermal papilla cells and measuring the number of dermal papilla cells in which Meis1 protein is localized in the nucleus. The measuring method according to claim 1 , wherein the Meis1 protein stained by fluorescent immunostaining is observed. The measuring method according to claim 1 or 2 , further comprising performing tissue staining before said observation. A method for analyzing the hair cycle of hair follicles by observing Meis1 protein in dermal papilla cells and observing the number of cells in which Meis1 protein is present in the nucleus of dermal papilla cells. The hair follicle according to claim 4 , wherein the location of Meis1 protein in the dermal papilla cells is observed, and the ratio of the number of dermal papilla cells in which Meis1 protein is present in the nucleus to the total number of dermal papilla cells is calculated. How to analyze periods. A quantitative method for quantifying the amount of Meis1 protein present in the nucleus by immunofluorescently staining Meis1 protein in dermal papilla cells and observing the obtained fluorescent signal. A quantitative method for quantifying the amount of Meis1 protein present in the nucleus of dermal papilla cells by the following steps.
(a) Step of isolating dermal papilla cells (b) Step of separating a nuclear fraction from the dermal papilla cells (c) Step of quantifying Meis1 protein contained in the nuclear fraction and other fractions A method for analyzing the hair cycle of a hair follicle using the quantitative method according to claim 6 or 7 .