Methods for constructing, scoring, and modeling a mouse model of androgenetic alopecia
By combining shaving with depilatory cream and using detailed back skin scoring indicators, the limitations of existing androgenetic alopecia mouse models in drug delivery and evaluation were overcome, achieving more accurate drug screening results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING XINGHUI REGENERATION TECH CO LTD
- Filing Date
- 2023-12-01
- Publication Date
- 2026-07-31
AI Technical Summary
Existing mouse models of androgenetic alopecia have limitations in drug delivery methods and lack of objective and accurate evaluation methods, resulting in poor drug screening outcomes.
Hair removal was performed using a combination of shaving and depilatory cream, and DHT solution was injected intraperitoneally. The results were evaluated using detailed back skin scoring indicators and image recognition software to optimize drug delivery and evaluation methods.
This achieves a gentler hair removal method, reduces stimulation to hair follicles, extends the observation window, and improves the accuracy and reliability of drug screening.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the medical field, and more particularly to a method for constructing, scoring, and modeling a mouse model of androgenetic alopecia. Background Technology
[0002] Androgenic alopecia (AGA) is the most common progressive hair loss disease, characterized by specific hair loss in both men and women. Generally, AGA is characterized by a shortened anagen phase, a prolonged catagen phase, and miniaturization of hair follicles. Although its mechanisms are not fully understood, androgens, particularly dihydrotestosterone (DHT), which bind to receptors (ARs) in the dermal dermal papilla cells (HFDPs) of the bald area, initiating a series of reactions leading to AGA, have been identified as the main factor in its development. The main treatments for AGA include hair transplantation surgery, oral medications, and LDL laser irradiation, but to date, no treatment can completely cure the disease. Animal models play an important role in exploring the potential mechanisms of disease development and evaluating new therapies. Therefore, the establishment of androgenic alopecia models is beneficial for further exploring the pathological mechanisms of androgenic alopecia and for drug screening.
[0003] Animal models used to study hair growth include the spontaneous baldness model of rhesus monkeys with detached tails, the androgen-dependent mouse model, the human bald scalp transplant nude mouse model, and the hamster lateral ventral organ model.
[0004] Currently, transgenic mouse models and androgen-dependent mouse models are the main animal models used to study hair growth because using stump-tailed macaques is impractical, and alternative models are insufficient. In existing studies, K5-hAR transgenic mice were obtained by inserting human AR (hAR) into mice. Human AR (hAR) can be expressed in the basal epidermis and outer root sheath (ORS) of transgenic mice, but AR is not expressed in the dermal papilla cells of hair follicles, while AR is expressed in human dermal papilla cells (HFDP). This indicates that the K5-hAR transgenic mouse has significant limitations as a model of the AGA phenotype.
[0005] Androgen-induced mouse models have been used to screen new drugs. However, these models are not comparable to the clinical AGA phenotype, and studies have shown that their drug delivery methods are flawed, requiring further improvement in evaluation methods. Topical application of DHT is less effective at inhibiting hair growth, possibly because the drug solution is wiped off the mouse's back skin, limiting its effectiveness. Subcutaneous injection of DHT is more commonly used, but its limited absorption capacity in the subcutaneous layer leads to the accumulation and deposition of unabsorbed solvent under the skin. Furthermore, it is difficult to administer and can cause secondary damage to the mouse's skin, making it less than optimal for DHT delivery.
[0006] Although these models have been used to test the efficacy of various drug candidates, it remains unclear whether they mimic the mechanisms and characteristics of human AGA, and no wild-type mouse model of AGA has been described to date. DHT targets AR in wild-type mouse HFDP. Therefore, some studies suggest that this wild-type mouse model may be similar to human AGA.
[0007] Intraperitoneal injection of DHT into wild-type mice is another method, but the preparation of AGA mouse models still faces many challenges. For example, the method of hair removal significantly impacts hair growth in mice. Hair removal methods include rosin wax plucking, shaving, and depilatory creams, but each method has limitations. Rosin wax plucking is difficult to perform and can easily cause secondary damage such as burns and strains to the skin and hair follicles. Furthermore, the mechanical pulling of this method significantly stimulates the hair follicles, accelerating the hair follicle regrowth cycle and shortening the observation window for AGA mouse models. Shaving may lead to irregular and slow hair growth, making it difficult to observe. Depilatory creams may cause asynchronous hair regrowth (i.e., asynchronous hair growth), easily leading to unstable results; all of these methods are not conducive to drug screening. Secondly, the effectiveness of the evaluation methods for AGA mouse models is also a crucial factor in determining the suitability of an AGA mouse model. Summary of the Invention
[0008] Technical issues
[0009] In view of this, the technical problem to be solved by the present invention is how to provide a method for constructing, scoring and modeling a mouse model of androgenetic alopecia.
[0010] The hair removal method used in this invention for the AGA mouse model is a combination of shaving and depilatory cream. Since the depilatory cream uses its chemical substances to dissolve the hair structure to achieve the hair removal effect, unlike the intense mechanical stimulation of rosin and paraffin wax plucking, it causes less stimulation to the hair follicles in the short term and has less impact on the hair follicle growth cycle. This invention has continuously explored and obtained an optimal DHT dosage (1-2 mg / kg / day) and established a more detailed and quantifiable back skin scoring index. At least three people simultaneously score the skin and the average is taken to minimize subjective influence. Alternatively, image recognition software can be used for more objective area statistics to improve the feasibility of the back skin scoring.
[0011] Solution
[0012] To solve the above technical problems, the present invention provides the following technical solution:
[0013] In a first aspect, the present invention provides a method for constructing a mouse model of androgenetic alopecia, comprising the following steps:
[0014] 1) Take adaptively cultured mice and inject them intraperitoneally with DHT solution; the dosage is 0.5-5 mg / kg / day;
[0015] 2) Three days after administering the medication as in step 1), perform hair removal treatment. The hair removal method is to shave the back of the mouse, apply hair removal cream, leave it on for 1-5 minutes, and then wipe off the hair removal cream.
[0016] 3) Continue to inject DHT solution into the peritoneum daily as in step 1), and continue culturing to obtain a mouse model of androgenetic alopecia.
[0017] Furthermore, in step 1), the DHT solution is administered once a day.
[0018] Further, in step 1), the dosage is 1 to 3 mg / kg / day, and optionally 1 to 2 mg / kg / day.
[0019] Furthermore, in step 2), after hair removal, mice with darker back skin color or obvious local black spots are removed to make the back skin color of the mice basically uniform.
[0020] Further, in step 2), the hair removal cream contains potassium thioglycolate and calcium hydroxide, optionally including cetearyl alcohol, liquid paraffin, potassium thioglycolate, calcium hydroxide and cetearyl alcohol polyether-20, optionally Veet hair removal cream.
[0021] Furthermore, in step 3), the observation window for the androgenic alopecia mouse model is 12–18 days.
[0022] Furthermore, it also includes a mouse dorsal skin scoring system to assess the relative effectiveness of the drug to be screened compared to the model group, dividing the dorsal skin score into at least four levels:
[0023] Level 1: Scoring is based on the coverage area of the gray area in the hair removal skin. The gray area refers to the part of the skin that appears gray relative to the original pink skin. The corresponding score ranges from 2.0 to 3.0. The coverage area of the gray area in the skin is further subdivided into 4 to 8 levels from 0% to 100%. The score difference between adjacent subdivision levels is 0.125 to 0.25 points.
[0024] Second level: Scoring is based on the coverage area of the black area in the hair removal skin. The black area refers to the part of the skin that is black relative to the original pink skin. The corresponding score ranges from 3.0 to 4.0. The coverage area of the black area in the skin is further subdivided into 4 to 8 levels from 0% to 100%. The score difference between adjacent subdivision levels is 0.125 to 0.25 points.
[0025] The third level is scored based on the coverage area of the hair-penetrating area in the treated skin. The hair-penetrating area refers to the area where hair sprouts from the skin. The corresponding score ranges from 4.0 to 5.0. The coverage area of the hair-penetrating area in the skin is further subdivided into 4 to 8 levels from 0% to 100%. The score difference between adjacent subdivision levels is 0.125 to 0.25 points.
[0026] Level 4: Scoring is based on the area of hair coverage in the treated skin. The hair coverage area refers to the area where hair clearly grows out of the skin and has a certain length. The corresponding score ranges from 5.0 to 6.0. The area of hair protruding from the skin is further subdivided into 4 to 8 levels from 0% to 100%, with a score difference of 0.125 to 0.25 points between adjacent subdivision levels.
[0027] Optionally, the scoring process uses at least three parallel trials and at least three evaluation results, and calculates the average value.
[0028] Secondly, a scoring method for the dorsal skin of a mouse model of androgenetic alopecia is provided for evaluating or screening drugs for the prevention and / or treatment of androgenetic alopecia. The dorsal skin score is divided into at least four levels, namely:
[0029] Level 1: Scoring is based on the coverage area of the gray area in the hair removal skin. The gray area refers to the part of the skin that appears gray relative to the original pink skin. The corresponding score ranges from 2.0 to 3.0. The coverage area of the gray area in the skin is further subdivided into 4 to 8 levels from 0% to 100%. The score difference between adjacent subdivision levels is 0.125 to 0.25 points.
[0030] Second level: Scoring is based on the coverage area of the black area in the hair removal skin. The black area refers to the part of the skin that is black relative to the original pink skin. The corresponding score ranges from 3.0 to 4.0. The coverage area of the black area in the skin is further subdivided into 4 to 8 levels from 0% to 100%. The score difference between adjacent subdivision levels is 0.125 to 0.25 points.
[0031] The third level is scored based on the coverage area of the hair-penetrating area in the treated skin. The hair-penetrating area refers to the area where hair sprouts from the skin. The corresponding score ranges from 4.0 to 5.0. The coverage area of the hair-penetrating area in the skin is further subdivided into 4 to 8 levels from 0% to 100%. The score difference between adjacent subdivision levels is 0.125 to 0.25 points.
[0032] Level 4: Scoring is based on the area of hair coverage in the treated skin. The hair coverage area refers to the area where hair clearly grows out of the skin and has a certain length. The corresponding score ranges from 5.0 to 6.0. The area of hair protruding from the skin is further subdivided into 4 to 8 levels from 0% to 100%, with a score difference of 0.125 to 0.25 points between adjacent subdivision levels.
[0033] Optionally, the scoring process uses at least three parallel trials and at least three evaluation results, and calculates the average value.
[0034] In the first or second aspect mentioned above, the coverage area of gray area, black area, hair protrusion area or hair coverage area in the back skin of the first, second, third or fourth level is gradually refined into 5 levels from 0% to 100%, with 20%, 40%, 60%, 80% and 100% as nodes, and the score difference between adjacent refined levels is 0.2 points.
[0035] In the first or second aspect mentioned above, in the second level, the black area represents the area where the hair follicles have not yet sprouted but have already turned noticeably black;
[0036] And / or, in the third level, the hair emergence area refers to the area where the hair has just sprouted, with an emergence length of about 0.01 to 2 mm;
[0037] And / or, in the fourth level, the hair-covered area refers to the area where the hair grows out to a length of approximately ≥2.01 mm.
[0038] Thirdly, a mouse model for screening drugs to prevent and / or treat androgenetic alopecia is provided, the model being constructed by the method described in the first aspect.
[0039] Beneficial effects
[0040] The hair removal method of "shaving + depilatory cream" in this invention is gentler than "rosin and paraffin wax plucking," causing less irritation to hair follicles and thus not accelerating the normal hair growth cycle. The AGA mouse model (drug-treated group) constructed in this invention shows more significant and visually discernible differences compared to the control group, with a longer difference window period, approaching 18 days. This is more beneficial for subsequent drug screening applications. The dorsal skin scoring index of this invention is more detailed and quantifiable, facilitating objective statistical analysis of differences and enabling better evaluation of drug efficacy and drug screening.
[0041] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, and to make the above and other objects, technical features and advantages of the present invention easier to understand, one or more preferred embodiments are listed below and described in detail with reference to the accompanying drawings. Attached Figure Description
[0042] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.
[0043] Figure 1 This is a flowchart illustrating the construction of the AGA mouse model in Example 1 of the present invention.
[0044] Figure 2 The images are of mice taken on Day 3, Day 5, Day 8, Day 11, Day 13, and Day 15 after hair removal in Example 1 of the present invention.
[0045] Figure 3 The scoring index for the back skin of mice after hair removal in Example 1 of the present invention.
[0046] Figure 4 The following are the statistical results of the back skin scores of mice on Day 3, Day 5, Day 8, Day 11, Day 13, Day 15, Day 17, Day 19, and Day 23 after hair removal in Example 1 of the present invention.
[0047] Figure 5 HE staining results of longitudinal sections of the back skin of mice on Day 8, Day 13, and Day 23 after hair removal in Example 1 of the present invention.
[0048] Figure 6 Images of mice taken on Day 3, Day 5, Day 8, and Day 11 after hair removal in Example 2 of this invention.
[0049] Figure 7 Example 3 of the present invention: A photograph of a mouse taken on Day 1 after hair removal. Detailed Implementation
[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated elements or components, and does not exclude other elements or other components.
[0051] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.
[0052] The hair removal method used in this invention for the AGA mouse model is a combination of shaving and depilatory cream. Since the depilatory cream uses chemical substances to dissolve the hair structure to achieve the hair removal effect, unlike the intense mechanical stimulation of rosin and paraffin wax plucking, it causes less stimulation to the hair follicles in a short period and has less impact on the hair follicle growth cycle. The specific steps are as follows: After anesthetizing the mice, the longer hair on the mouse's back is shaved off using an electric shaver. Then, the depilatory cream is evenly applied to a fixed area and left for about 1-2 minutes. The depilatory cream and dissolved hair are then wiped away in one direction with a soft tissue, exposing the mouse's back skin.
[0053] This invention also establishes a more detailed and quantifiable back skin scoring index, reducing the problems caused by evaluating the model group and control group solely based on back skin color—the back skin color difference is not obvious, it is difficult to distinguish the color difference with the naked eye, and it is also difficult to count the specific difference value, resulting in the inability to quantify the evaluation.
[0054] In the following examples, all mice were provided by Beijing Huafukang Biotechnology Co., Ltd., and all animal experiments were conducted in the mouse room of the Animal Experiment Center of Zhongyan Zichuang (Beijing) Biotechnology Co., Ltd. Skin tissue section staining was performed by Bios-Changyan Pathology. The day of hair removal treatment was defined as "Day 0", and so on. A brief experimental procedure is provided below. Figure 1 .
[0055] In the following examples, all raw materials were commercially available. For example, DHT was purchased from Solarbio, ID0310; rosin and paraffin were conventional commercially available products; hair removal cream was purchased from Veet Pure Hair Removal Cream; and corn oil (pharmaceutical grade) was purchased from Aladdin, C116025.
[0056] In the following examples, mice were required to undergo acclimatization before being grouped for the experiment: 21 healthy male C57BL / 6 mice weighing between 18-22g at 7 weeks of age (45 days of age) were selected, ear-tagged and weighed, and then began acclimatization for about 3 days.
[0057] Example 1
[0058] 1. Grouping and starting administrationTwenty-one healthy male C57BL / 6 mice that had undergone adaptive culture for 3 days were selected. After weighing (the weight difference between mice did not exceed 4g), all mice were randomly divided into three groups: control group, treatment group 1 (DHT, 1mg / kg / d), and treatment group 2 (DHT, 2mg / kg / d). Treatment groups 1 and 2 were intraperitoneally injected with DHT solution (solvent: pharmaceutical-grade corn oil) at a dose of 50ul, 1mg / kg / d and 2mg / kg / d, respectively, once daily until the end of the experiment. The control group was injected with corn oil at the same dose and frequency.
[0059] 2. Day 0: Three days after administration, shaving and depilatory treatment were performed: All mice were anesthetized with 1.5%-3% isoflurane. First, longer hair on the backs of the mice was shaved using an electric shaver. Then, depilatory cream (purchased from Veet Pure Depilatory Cream) was evenly applied to the area and left for 1-2 minutes. Afterward, the depilatory cream and dissolved hair were wiped away in one direction with a soft tissue to expose the mouse's back skin. Mice with darker back skin or obvious local black spots were removed to ensure all mice had entered the telogen phase, at which point the back skin color was generally uniform, being pink. Administration was continued according to the same method described in step 1.
[0060] 3. Take photos and rate On days 3, 5, 8, 11, 13, 15, 17, 19, and 23 after hair removal treatment, all mice were anesthetized with 1.5%-3% isoflurane. Photos were taken of each mouse at fixed height, angle, light source, and camera parameters. The results for days 3 through 15 are shown below. Figure 2 Then, scoring is conducted: at least three people score the skin based on back skin scoring criteria (such as...). Figure 3 As shown, "**" represents a P-value < 0.01, "***" represents a P-value < 0.001, and "****" represents a P-value < 0.0001 (n ≥ 5). The mouse images were scored by back skin evaluation, and the scoring results are as follows: Figure 4 As shown.
[0061] The dorsal skin scoring index of the present invention, such as Figure 3 Specifically: the back skin score is divided into four levels, namely:
[0062] Level 1: Scoring is based on the coverage area of the gray area in the treated skin. The gray area refers to the part of the skin that appears gray compared to the original pink skin (representing the start of hair growth in the hair follicle). The corresponding score is 2.0 to 3.0 points. The coverage area of the gray area in the skin is further subdivided into 0.2 points at the nodes of 20%, 40%, 60%, 80%, and 100%. For example, when the gray area covers 0 to 20% of the skin, the score is 2.0 points, and when the gray area covers 60 to 80% of the skin, the score is 2.6 points.
[0063] The second level is scored based on the coverage area of the black area on the treated skin. The black area refers to the part of the skin that appears black relative to the original pink skin (meaning that the hair in the hair follicle has not yet sprouted but has already turned obviously black). The corresponding score is 3.0 to 4.0. The coverage area of the black area on the skin is further subdivided into 0.2 points at the nodes of 20%, 40%, 60%, 80%, and 100%. For example, when the black area covers 0 to 20% of the skin, the score is 3.0, and when the black area covers 60 to 80% of the skin, the score is 3.6.
[0064] The third level is scored based on the coverage area of the hair-penetrating area in the treated skin. The hair-penetrating area refers to the area where hair sprouts from the skin (i.e., the hair has just sprouted, with a sprouting length of about 0.01 to 2 mm). The corresponding score is 4.0 to 5.0 points. The coverage area of the hair-penetrating area in the skin is further subdivided into 0.2 points at the nodes of 20%, 40%, 60%, 80%, and 100%. For example, when the coverage area of the hair-penetrating area in the skin is 0 to 20%, the score is 4.0 points, and when the coverage area of the hair-penetrating area in the skin is 60 to 80%, the score is 4.6 points.
[0065] Level 4: Scoring is based on the area of hair coverage in the treated skin. The hair coverage area refers to the area where hair clearly grows out of the skin and has a certain length (i.e., the length of the hair growth is approximately ≥2.01mm). The corresponding score is 5.0 to 6.0. The coverage area of the hair coverage area in the skin is further subdivided into 0.2 points at the nodes of 20%, 40%, 60%, 80%, and 100%. For example, when the hair coverage area accounts for 0-20% of the skin coverage area, the score is 5.0, and when the hair coverage area accounts for 60-80%, the score is 5.6.
[0066] In the above evaluation criteria, there is a transition between adjacent levels, which is difficult to strictly distinguish, so there is some overlap when assigning scores.
[0067] Combination Figure 3 The score corresponding to the mouse's back skin was used as a scoring reference. The mouse images were scored based on their back skin, and the results are as follows: Figure 4 As shown.
[0068] Combination Figure 2 Starting from Day 8, the color of the back skin of mice in the treatment group and the control group began to show obvious differences, which were visible to the naked eye. By Day 15, the hair on the back skin of mice in the control group had basically grown out, while the hair growth cycle of mice in the treatment group was significantly delayed, and the hair coverage was less than half. The delaying effect of treatment group 2 (DHT, 2mg / kg / d) was better than that of treatment group 1 (DHT, 1mg / kg / d).
[0069] Depend on Figure 4 As can be seen, starting from Day 8, the difference in back skin scores between the treatment group and the control group was highly significant, and the observation window for the difference was close to 18 days. Furthermore, the delayed effect of treatment group 2 (DHT, 2 mg / kg / d) was better than that of treatment group 1 (DHT, 1 mg / kg / d). This is related to... Figure 2 The results were consistent.
[0070] 5. Skin tissue section staining On Day 8, Day 13, and Day 23: One mouse was randomly selected from each group, euthanized by cervical dislocation, and a 1cm x 1cm piece of skin from the hairless area was taken and placed in a labeled EP tube containing formalin fixative. The tissue was then embedded in paraffin, sectioned longitudinally, and stained with hematoxylin and eosin (HE) using a hematoxylin and eosin (HE) staining kit, purchased from Solarbio, G1120. The results are as follows: Figure 5 As shown. By Figure 5 It can be seen that on Day 8, Day 13, and Day 23, the hair follicles of the mice in the drug treatment group were in an earlier growth cycle than those of the control group, indicating that the hair follicle growth cycle of the mice in the drug treatment group was significantly delayed.
[0071] In summary, although the hair removal method of shaving and depilatory cream resulted in patches on the hair growth area of the mouse back skin, when the growth of the mouse back skin was evaluated using the above scoring criteria at the same time and with the same dosage, the scoring results were similar and there was no instability. That is, the hair removal method of shaving and depilatory cream combined with the above scoring criteria can obtain the AGA mouse model.
[0072] Example 2
[0073] 1. Grouping and starting administration After weighing, all mice were randomly divided into two groups: a control group and a treatment group 1 (DHT, 1 mg / dbody weight). The treatment group received intraperitoneal injections of DHT solution (Solarbio, ID0310, corn oil as solvent) at the prescribed dose, with a volume of 50 μL, once daily until the end of the experiment; the control group received corn oil injections at the same dose and frequency.
[0074] 2. Day 0: Three days after drug administration, rosin and paraffin wax plucking was performed: A 1:1 solution of rosin / paraffin wax, heated and melted, was prepared in advance. All mice were anesthetized with 1.5%-3% isoflurane. While still warm, the preheated rosin and paraffin wax solution was evenly applied to the backs of the mice. After 1-2 minutes, once the rosin and paraffin wax had hardened, it was immediately peeled off along with the hair, exposing the mouse's back skin. Mice with darker back skin or obvious localized black spots were removed, ensuring that all mice had entered the telogen phase of hair growth. At this point, the back skin of the mice was generally uniformly pink.
[0075] 3. Photography: On Day 3, Day 5, Day 8, and Day 11, all mice were anesthetized with 1.5%-3% isoflurane. Each mouse was photographed individually at a fixed height, angle, light source, and camera parameters. The results are as follows: Figure 6 As shown in the figure, although the rosin and paraffin hair removal method resulted in good synchronization of the long-cycle hair growth on the back of mice, the skin color difference between the treatment group and the control group was not significant and was almost indistinguishable to the naked eye, making it difficult to statistically analyze the difference.
[0076] In addition, through Figure 2 control group and Figure 6 The comparison with the control group showed that the mice treated with rosin paraffin had their hair fully grown by Day 11, while the mice in the depilatory cream group had not grown their hair by Day 15. This indicates that rosin paraffin treatment has a greater stimulating effect on hair follicles and will accelerate the normal hair growth cycle of hair follicles, resulting in a shorter observation window for subsequent differences, which is not conducive to subsequent drug screening and application.
[0077] Example 3
[0078] 1. Day 0: After administering the medication for 3 days according to step 1 of Example 1, the mice were treated with shaving and depilatory cream: After anesthetizing the mice with 1.5%-3% isoflurane, the longer hair on the back of the mice was shaved off with an electric shaver, and then depilatory cream was evenly applied to the area and left for 2 minutes, 5 minutes and 10 minutes respectively. Afterwards, the depilatory cream and dissolved hair were wiped off in one direction with a soft tissue to expose the back skin of the mice.
[0079] 2. Day 1: After anesthetizing all mice with 1.5%-3% isoflurane, photographs were taken of each mouse for observation. The results are as follows: Figure 7 As shown in the results, when using hair removal cream, applying it evenly and leaving it on for about 2 minutes yields the best results, leaving the back skin clean and without any inflammation or other abnormalities the next day; leaving it on for about 5 minutes is more likely to cause mild inflammation the next day; leaving it on for 10 minutes (the instructions for the hair removal cream recommend 5-10 minutes) will cause some skin irritation, and skin damage was observed the next day.
[0080] In summary, the hair removal method of "shaving + hair removal cream" in this invention is gentler than "rosin and paraffin hair removal" and causes less stimulation to the hair follicles, thus not accelerating the normal hair growth cycle of the hair follicles.
[0081] The AGA mouse model constructed in this invention shows a more significant and visually discernible difference between the treatment group and the control group, with a longer difference window period, approaching 18 days. This is more beneficial for subsequent drug screening applications.
[0082] The back skin scoring index of this invention is more detailed and quantitative, making it easier to objectively count differences.
[0083] The foregoing description of specific exemplary embodiments of the present invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. Any simple modifications, equivalent changes, and alterations made to the foregoing exemplary embodiments should fall within the scope of protection of the present invention.
Claims
1. A method for constructing an androgenic alopecia mouse model, characterized by, Includes the following steps: 1) Take adaptively cultured mice and inject them intraperitoneally with DHT solution; the dosage is 1~3 mg / kg / day; the frequency of DHT solution administration is once a day; 2) Three days after administering the medication as in step 1), perform hair removal treatment. The hair removal method is to shave the back of the mouse, apply hair removal cream, leave it on for 1-2 minutes, and then wipe off the hair removal cream. The hair removal cream contains potassium thioglycolate, calcium hydroxide, cetearyl alcohol, liquid paraffin, and cetearyl alcohol polyether-20. After hair removal, mice with darker back skin color or obvious local black spots were removed to make the back skin color of the mice basically uniform. 3) Continue to inject DHT solution intraperitoneally daily as in step 1), and continue culturing to obtain a mouse model of androgenetic alopecia; It also includes a mouse dorsal skin scoring system to assess the relative effectiveness of the drug to be screened compared to the model group. The dorsal skin score is divided into at least four levels: Level 1: Scoring is based on the coverage area of the gray area in the hair removal skin. The gray area refers to the part of the skin that appears gray relative to the original pink skin. The corresponding score ranges from 2.0 to 3.
0. The coverage area of the gray area in the skin is further subdivided into 4 to 8 levels from 0% to 100%. The score difference between adjacent subdivision levels is 0.125 to 0.25 points. Second level: Scoring is based on the coverage area of the black area in the hair removal skin. The black area refers to the part of the skin that appears black relative to the original pink skin. The corresponding score ranges from 3.0 to 4.
0. The coverage area of the black area in the skin is further subdivided into 4 to 8 levels from 0% to 100%, with a score difference of 0.125 to 0.25 points between adjacent subdivision levels. The black area represents the area where the hair follicles have not yet sprouted but have already turned obviously black. The third level is scored based on the coverage area of the hair-penetrating area in the treated skin. The hair-penetrating area refers to the area where hair sprouts from the skin. The corresponding score ranges from 4.0 to 5.
0. The coverage area of the hair-penetrating area in the skin is further subdivided into 4 to 8 levels from 0% to 100%. The score difference between adjacent subdivision levels is 0.125 to 0.25 points. The hair-penetrating area refers to the area where the hair has just sprouted, with a sprouting length of 0.01 to 2 mm. Level 4: Scoring is based on the area of hair coverage in the treated skin. The hair coverage area refers to the area where hair clearly grows out of the skin and has a certain length. The corresponding score ranges from 5.0 to 6.
0. The hair coverage area in the skin is further subdivided into 4 to 8 levels from 0% to 100%, with a score difference of 0.125 to 0.25 points between adjacent subdivision levels. The hair coverage area is defined as an area where the length of hair that grows out is ≥2.01mm. The scoring process uses at least three parallel trials and at least three evaluation results, and calculates the average value.
2. The construction method according to claim 1, characterized in that, In step 1), the dosage is 1~2 mg / kg / day.
3. The construction method according to claim 1, characterized in that, In step 2), the hair removal cream is Veet hair removal cream.
4. The construction method according to claim 1, characterized in that, In step 3), the observation window for the androgenic alopecia mouse model is 12-18 days.
5. The construction method according to any one of claims 1 to 4, characterized in that, In the first, second, third or fourth levels, the coverage area of gray area, black area, hair protrusion area or hair-covered area in the back skin is gradually refined into 5 levels from 0% to 100%, with 20%, 40%, 60%, 80% and 100% as nodes, and the score difference between adjacent refined levels is 0.2 points.
6. A scoring method for the dorsal skin of a mouse model of androgenetic alopecia, used to evaluate or screen drugs for the prevention and / or treatment of androgenetic alopecia, characterized in that, The back skin is graded into at least four levels, namely: Level 1: Scoring is based on the coverage area of the gray area in the hair removal skin. The gray area refers to the part of the skin that appears gray relative to the original pink skin. The corresponding score ranges from 2.0 to 3.
0. The coverage area of the gray area in the skin is further subdivided into 4 to 8 levels from 0% to 100%. The score difference between adjacent subdivision levels is 0.125 to 0.25 points. Second level: Scoring is based on the coverage area of the black area in the hair removal skin. The black area refers to the part of the skin that appears black relative to the original pink skin. The corresponding score ranges from 3.0 to 4.
0. The coverage area of the black area in the skin is further subdivided into 4 to 8 levels from 0% to 100%, with a score difference of 0.125 to 0.25 points between adjacent subdivision levels. The black area represents the area where the hair follicles have not yet sprouted but have already turned obviously black. The third level is scored based on the coverage area of the hair-penetrating area in the treated skin. The hair-penetrating area refers to the area where hair sprouts from the skin. The corresponding score ranges from 4.0 to 5.
0. The coverage area of the hair-penetrating area in the skin is further subdivided into 4 to 8 levels from 0% to 100%. The score difference between adjacent subdivision levels is 0.125 to 0.25 points. The hair-penetrating area refers to the area where the hair has just sprouted, with a sprouting length of 0.01 to 2 mm. Level 4: Scoring is based on the area of hair coverage in the treated skin. The hair coverage area refers to the area where hair clearly grows out of the skin and has a certain length. The corresponding score ranges from 5.0 to 6.
0. The hair coverage area in the skin is further subdivided into 4 to 8 levels from 0% to 100%, with a score difference of 0.125 to 0.25 points between adjacent subdivision levels. The hair coverage area is defined as an area where the length of hair that grows out is ≥2.01mm. The scoring process uses at least three parallel trials and at least three evaluation results, and calculates the average value.
7. The scoring method according to claim 6, characterized in that, In the first, second, third or fourth levels, the coverage area of gray area, black area, hair protrusion area or hair-covered area in the back skin is gradually refined into 5 levels from 0% to 100%, with 20%, 40%, 60%, 80% and 100% as nodes, and the score difference between adjacent refined levels is 0.2 points.
8. A mouse model for screening drugs to prevent and / or treat androgenetic alopecia, characterized in that, The model is constructed by the method described in any one of claims 1 to 5.