Construction method and application of hMRGPRX3 humanized mouse model and atopic dermatitis model

Abstract

The invention discloses a construction method and application of an hMRGPRX3 humanized mouse model and an atopic dermatitis model, and relates to the technical field of disease model construction. The whole gene sequence of the human MRGPRX3 or a vector containing the gene sequence of the hMRGPRX3 is transferred into mouse genome DNA (Deoxyribonucleic Acid) by utilizing a transgenic technology, and a functional human hMRGPRX3 receptor can be simulated and expressed in a mouse model more truly by transferring upstream and downstream regulatory sequences of the hMRGPRX3 gene. The pruritus signal transduction mediated by the receptor in human dorsal root ganglion sensory neurons can be accurately simulated, and signal deviation caused by species difference of a traditional mouse model is overcome.

Description

Technical Field

[0001] This invention relates to the field of disease model construction technology, and more specifically, to a method for constructing an hMRGPRX3 humanized mouse model and an atopic dermatitis model, and their applications. Background Technology

[0002] hMRGPRX3 (human Mas-associated G protein-coupled receptor X3) is a subtype of the Mas-associated G protein-coupled receptor (MRGPRs) family. This family consists of specific orphan receptors primarily expressed in sensory neurons of humans and primates, belonging to the delta branch of rhodopsin-like class A GPCRs, and includes subtypes hMRGPRX1, 2, 3, and 4. The MRGPPR family members exhibit high structural diversity and differences across species. Based on sequence homology, they are mainly divided into nine subfamilies (MRGPR AH and X). Evolutionary studies of the MRGPR family show that the MRGPRA, B, C, and H subfamilies are primarily found in rodents and located on chromosome 7; the MRGPRX subfamily exists only in humans and primates and is located on chromosome 11; while the DG subfamily is evolutionarily conserved across species.

[0003] Different subtypes of hMRGPRX play important roles in physiological and pathological processes such as pain, itching, inflammation, and immune regulation, and have attracted widespread attention. hMRGPRX1 is the only MRGPR subtype in humans that is clearly expressed in primary sensory neurons and is mainly involved in pain transmission. Its activation triggers strong action potential discharges, thereby enhancing neuronal excitability. hMRGPRX2 and 4 are mainly expressed in mast cells and are key receptors for allergic and inflammatory responses. After binding to ligands such as BAM8-22 and γ2-MSH, they can activate mast cell degranulation and release inflammatory mediator pathways. hMRGPRX3 is mainly expressed in small-diameter sensory neurons, especially IB4-positive neurons in the dorsal root ganglion (DRG). It activates the phosphatidylinositol-calcium signaling pathway by binding to G proteins (such as Gq and G11), thereby regulating the function of pain neurons.

[0004] Itching is defined as an "unpleasant skin sensation that triggers the urge or reflex to scratch." Primary sensory neurons in the dorsal root ganglion (DRG) detect itch stimuli through their peripheral axons located on the skin and mucous membranes, and transmit signals to the spinal cord via their central axons, thus playing a crucial role in the generation of itch. hMRGPRA3 is considered an important receptor in this process. The relationship between MRGPRA3 and itch in mice has been reported multiple times. MRGPRA3 is specifically expressed in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) of the peripheral nervous system and is a specific receptor for the antimalarial drug chloroquine. The itch-inducing side effect of chloroquine is mainly achieved through MRGPRA3. Specific activation of MRGPRA3-positive neurons in the DRG region through photoactivation, chloroquine activation, and histamine activation can all induce scratching movements in mice. In various chronic pruritus models (xeroderma, contact dermatitis), the expression of pruritus receptors in MRGPRA3 neurons was significantly upregulated and excitability increased, further confirming the link between MRGPRA3 and pruritus.

[0005] However, the mouse MRGPRA3 gene and the human MRGPRX3 gene are not 1:1 homologous genes. There are differences in their gene sequences and their gene functions cannot be completely matched. The mouse MRGPRA3 cannot accurately simulate the itch signaling mediated by this receptor in human dorsal root ganglion sensory neurons.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for constructing a humanized hMRGPRX3 mouse model and an atopic dermatitis model to construct a humanized hMRGPRX3-transformed mouse model, thereby accurately simulating the pruritus signal transduction mediated by this receptor in human dorsal root ganglion sensory neurons (such as Gαq / Gαi pathway activation, TRPV1 sensitization and neuropeptide release), overcoming the signal bias caused by species differences in traditional mouse models.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides a method for constructing an hMRGPRX3 humanized mouse model, comprising the following steps: The human hMRGPRX3 gene or a vector containing the hMRGPRX3 gene sequence was transferred into mouse zygotes. The nucleotide sequence of the hMRGPRX3 gene includes the upstream sequence shown in SEQ ID NO: 2, the hMRGPRX3 gene coding sequence shown in SEQ ID NO: 3, and the downstream sequence shown in SEQ ID NO: 4.

[0009] Secondly, the present invention also provides a method for constructing an atopic dermatitis model, comprising: inducing an hMRGPRX3 humanized mouse model constructed by the above-mentioned method for constructing an hMRGPRX3 humanized mouse model by inducing the model with at least one substance selected from OVA, MC903, DNCB, DNFB and house dust mites.

[0010] Thirdly, the present invention also provides a method for constructing a psoriasis model, comprising: inducing an hMRGPRX3 humanized mouse model using imiquimod to construct the above-mentioned method for constructing a humanized hMRGPRX3 mouse model.

[0011] Fourthly, the present invention also provides a method for constructing a humanized hMRGPRX3 mouse model and the application of the constructed hMRGPRX3 humanized mouse model in screening agonists and / or antagonists targeting hMRGPRX3.

[0012] Fifthly, the present invention also provides a method for constructing an hMRGPRX3 humanized mouse model and the application of the constructed hMRGPRX3 humanized mouse model in screening antipruritic drugs or chronic dermatitis treatment agents.

[0013] The present invention has the following beneficial effects: This invention utilizes transgenic technology to transfer the human hMRGPRX3 gene or a vector containing the hMRGPRX3 gene sequence into mouse genomic DNA. The transferred vector includes the hMRGPRX3 coding gene, upstream sequence, and downstream sequence. By transferring the upstream and downstream regulatory sequences, the functional human hMRGPRX3 receptor can be expressed more realistically. This accurately simulates the itch signal transduction mediated by this receptor in human dorsal root ganglion sensory neurons (such as Gαq / Gαi pathway activation, TRPV1 sensitization, and neuropeptide release), overcoming the signal bias caused by species differences in traditional mouse models.

[0014] In addition, this invention also constructed an atopic dermatitis model. The hMRGPRX3 humanized mice in the atopic dermatitis model showed more severe skin pathological changes and behavioral symptoms, suggesting that the hMRGPRX3 gene may play an important role in the pathogenesis of atopic dermatitis.

[0015] The hMRGPRX3 humanized mouse model can accurately mimic the pharmacological properties of the human receptor. It allows for the screening of agonists and / or antagonists targeting hMRGPRX3, as well as antipruritic drugs or treatments for chronic dermatitis. This accelerates the preclinical evaluation of antipruritic drugs and atopic dermatitis treatments, significantly improving drug development efficiency.

[0016] The model provided by this invention can offer an in vivo research tool for elucidating the "neuro-immune-skin" regulatory axis of Mas-related GPCRs in diseases such as atopic dermatitis and psoriasis. By comparing the neuropeptide release profiles of wild-type and transgenic mice, a new pathway for itch signal transduction can be revealed. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a diagram illustrating the strategy for constructing the hMRGPRX3 humanized mouse model of this invention. Figure 2 A plasmid map of the constructed hMRGPRX3 gene; Figure 3 Diagram of PCR identification strategy for positive mice; Figure 4 Electrophoresis patterns of the 5' and 3' ends of hMRGPRX3-TG-target F0 mice (numbers represent mouse tail numbers, P is a positive control obtained from a correctly sequenced plasmid; WT is C57BL / 6JGpt, N is a blank control, and M is a DNA marker). Figure 5 The following is a statistical chart showing the expression of MRGPRX3 (A, MRGPRX3 expression in various human tissues from the GTEx Portal database; B, MRGPRX3 expression in various cell lines from The Human Protein Atlas (HPA) database; C, MRGPRX3 expression in skin tissues of WT mice and hMRGPRX3 mice; D, MRGPRX3 expression in various tissues of hMRGPRX3 mice). Figure 6 Figures showing the experimental results of AD models in hMRGPRX3 and WT mice (A, representative ear images and ADI score statistics of the two groups at different time points in the AD model; B, ear thickness measured with calipers at different time points in the two groups; C, scratching frequency of the two groups at different time points; D, HE staining results and epidermal thickness statistics of mouse ears collected on Day 14; E, HE staining results and epidermal thickness statistics of mouse back skin collected on Day 14. Scale bar = 200 μm, n = 5 per group). Figure 7 Electrophoresis image for PCR identification of F0 positive mice after propagation and establishment of the line (P: positive control; B6: negative control; N: blank control). Detailed Implementation

[0019] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0020] 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] Definition of noun The term "vector" is used herein in its most common sense and includes any intermediate medium for nucleic acids that enables the nucleic acids to be introduced, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. Vectors of this type preferably replicate and / or are expressed in cells. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. The term "plasmid," as used herein, generally refers to a construct of extrachromosomal genetic material, typically a circular double-stranded DNA that can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and remain stable within the host.

[0022] In one alternative embodiment, the vector is an expression vector, and an important feature of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0023] In a first aspect, the present invention provides a method for constructing an hMRGPRX3 humanized mouse model, comprising the following steps: The vector containing the hMRGPRX3 gene sequence was transferred into mouse zygotes. The nucleotide sequence of the hMRGPRX3 gene includes the upstream sequence shown in SEQ ID NO: 2, the hMRGPRX3 gene coding sequence shown in SEQ ID NO: 3, and the downstream sequence shown in SEQ ID NO: 4.

[0024] The hMRGPRX3 gene is located on human chromosome 11, is 17,534 bases long, contains 3 exons and 1 coding region (CDS), and ultimately encodes a protein of 322 amino acids (aa). Its gene ID is NCBI117195, HGNC ID is 17980, and OMIM ID is 607229 (Table 1).

[0025] Table 1 hMRGPRX3 gene information

[0026] In a preferred embodiment of the present invention, the nucleotide sequence of the vector is shown in SEQ ID NO: 1.

[0027] In a preferred embodiment of the present invention, the hMRGPRX3 gene or a vector containing the hMRGPRX3 gene sequence is transferred into mouse zygotes by microinjection.

[0028] In a preferred embodiment of the present invention, after the hMRGPRX3 gene or a vector containing the hMRGPRX3 gene sequence is transferred into mouse fertilized eggs, the surviving fertilized eggs are transplanted into pseudopregnant female mice, resulting in pregnancy and offspring, and positive hMRGPRX3 humanized mice are obtained through screening.

[0029] In a preferred embodiment of the present invention, the mouse zygote is a zygote with a C57BL / 6JGpt background.

[0030] Secondly, the present invention also provides a method for constructing an atopic dermatitis model, comprising: inducing an hMRGPRX3 humanized mouse model constructed by the above-mentioned method for constructing an hMRGPRX3 humanized mouse model by inducing the model with at least one substance selected from OVA, MC903, DNCB, DNFB and house dust mites.

[0031] MC903: Calcipotriol. DNCB: 2,4-Dinitrochlorobenzene. DNFB: 2,4-Dinitrofluorobenzene.

[0032] In a preferred embodiment of the present invention, the hMRGPRX3 humanized mouse model is induced using MC903, with the working concentration of MC903 being 20 μM-70 μM. For example, it is 20-60 μM, 30-70 μM, 40-70 μM, or 45-70 μM.

[0033] In some embodiments, the working concentration of MC903 is 40-50 μM, preferably 43-47 μM.

[0034] In a preferred embodiment of the present invention, a preset working concentration of MC903 is applied to the skin of mice daily for 14 consecutive days. For example, it is applied to the skin on the dorsal side of both ears, the ventral side, and the back of the mice.

[0035] An atopic dermatitis model was constructed using the above methods. The hMRGPRX3 humanized mice exhibited more severe skin pathological changes and behavioral symptoms in the atopic dermatitis model, suggesting that the hMRGPRX3 gene may play an important role in the pathogenesis of atopic dermatitis.

[0036] The hMRGPRX3 humanized mouse model can accurately mimic the pharmacological properties of the human receptor. It allows for the screening of agonists and / or antagonists targeting hMRGPRX3, as well as antipruritic drugs or treatments for chronic dermatitis. This accelerates the preclinical evaluation of antipruritic drugs and atopic dermatitis treatments, significantly improving drug development efficiency.

[0037] The model provided by this invention can offer an in vivo research tool for elucidating the "neuro-immune-skin" regulatory axis of Mas-related GPCRs in diseases such as atopic dermatitis. By comparing the neuropeptide release profiles of wild-type and transgenic mice, a new pathway for itch signal transduction can be revealed.

[0038] Thirdly, the present invention also provides a method for constructing a psoriasis model, comprising: inducing an hMRGPRX3 humanized mouse model using imiquimod to construct the above-mentioned method for constructing a humanized hMRGPRX3 mouse model.

[0039] Imiquimod, also known as IMQ, can rapidly induce lesions on the skin of mice that are very similar to those in humans when applied topically.

[0040] Fourthly, the present invention also provides a method for constructing a humanized hMRGPRX3 mouse model and the application of the constructed hMRGPRX3 humanized mouse model in screening agonists and / or antagonists targeting hMRGPRX3.

[0041] Fifthly, the present invention also provides a method for constructing an hMRGPRX3 humanized mouse model and the application of the constructed hMRGPRX3 humanized mouse model in screening antipruritic drugs or chronic dermatitis treatment agents.

[0042] In a preferred embodiment of the present invention, chronic dermatitis is selected from at least one of the following: atopic dermatitis, extrinsic dermatitis, seborrheic dermatitis, asteatotic dermatitis, stasis dermatitis, neurodermatitis, and discoid eczema.

[0043] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0044] Example 1 This embodiment constructs an hMRGPRX3 humanized mouse model, and the specific construction steps are as follows: (1) Design strategy of hMRGPRX3 humanized mouse model Sequence analysis of the human BAC clones with transcript Ensembl numbers RP11-113D6 or CH17-160M24 revealed two non-coding gene regions upstream of the hMRGPRX3 coding gene. An upstream sequence of approximately 14 kb was found upstream of the first non-coding gene region, and a downstream sequence of approximately 4 kb was found downstream of the hMRGPRX3 coding gene (design strategy referenced). Figure 1 (As shown). In this invention, approximately 14 kb of upstream sequence, the hMRGPRX3 coding gene, and approximately 4 kb of downstream sequence are recombined to construct a transgenic vector, which is then transformed into mice to establish an hMRGPRX3 humanized mouse model.

[0045] The humanized hMRGPRX3 mouse model constructed using this strategy retains the upstream 14Kb and downstream 4.0Kb sequences of the hMRGPRX3 gene. Figure 1 In the text, 1 and 2 refer to the non-coding region of hMRGPRX3, and 3 refers to the coding region of hMRGPRX3.

[0046] (2) Construction of transgenic vectors.

[0047] The corresponding transgenic vector was designed and constructed according to the transgenic protocol. The correctness of the vector sequence was then verified by enzyme digestion and sequencing. The transgenic vector diagram is shown below. Figure 2 As shown.

[0048] The nucleotide sequence of the vector is shown in SEQ ID NO: 1. Sequences 1-394 bp and 36695-37381 bp constitute the vector backbone sequence and the homologous arms with deleted flanking genes. This sequence is primarily used for the efficient cloning of exogenous fragments obtained through PCR or enzyme digestion, and also supports subsequent operations such as fragment replication, selection, and plasmid extraction in *E. coli*. Sequences 395 bp to 14806 bp are the upstream sequence of hMRGPRX3, as shown in SEQ ID NO: 2; 14807 bp to 32332 bp are the coding sequence of the hMRGPRX3 gene, as shown in SEQ ID NO: 3; and 32333 bp to 36694 bp are the downstream sequence of hMRGPRX3, as shown in SEQ ID NO: 4. Introducing these upstream and downstream regulatory sequences allows for more accurate expression of the functional human hMRGPRX3 receptor. It can accurately simulate the itch signal transduction mediated by this receptor in human dorsal root ganglion sensory neurons (such as Gαq / Gαi pathway activation, TRPV1 sensitization and neuropeptide release), overcoming the signal bias caused by species differences in traditional mouse models.

[0049] (3) Positive mice were obtained by injection.

[0050] The transgenic vector sample was microinjected into the fertilized eggs of C57BL / 6J mice. The surviving fertilized eggs were then transplanted into 0.5-day-old pseudopregnant C57BL / 6J mice, and F0 generation mice were obtained after the mice became pregnant and gave birth.

[0051] (4) Genotyping of F0 generation mice.

[0052] The following are some of the reagents involved in F0 generation mouse genotyping: Rat tail digestion solution: 25 ml of 1M Tris-HCl (pH 7.4), 100 ml of 0.5M EDTA (pH 8.0), 2.925 g of NaCl, 50 ml of 10% SDS, and deionized water to a final volume of 500 mL.

[0053] The toes of the obtained F0 generation mice were clipped and numbered at 5-7 days of age. Approximately 5 mm of the tail tip was harvested and digested with mouse tail digestion solution for 8-12 hours. The supernatant of the digestion solution was washed with anhydrous ethanol to obtain genomic DNA. The tail genomic DNA from the obtained F0 mice was then identified by PCR at both ends after target placement using two pairs of primers. The identification strategy is as follows: Figure 3As shown, 5' connector primers were designed targeting the CDS region of the human fragment from the upstream promoter and donor, with upstream primer F1 located in the promoter region and downstream primer R1 located in the CDS region. 3' connector primers were also designed targeting the CDS region of the human fragment from the donor and the 3' end of the PolyA sequence, with upstream primer F2 located in the CDS region and downstream primer R2 located in the PolyA region.

[0054] Primers MRGPRX3-TG-5tF1A / MRGPRX3-TG-5tR1A are located outside the 5' homologous arm and inside the human fragment of the donor. If this primer pair produces PCR products, it indicates that the target donor has been effectively inserted into the 5' end of the mouse genome. MRGPRX3-TG-3tF1A / MRGPRX3-TG-3tR1A are located inside the human fragment of the donor and outside the 3' homologous arm. If this primer pair produces PCR products, it indicates that the target donor has been effectively inserted into the 3' end of the mouse genome. The PCR amplification primer sequences, reaction system, and reaction conditions are listed in Tables 2, 3, and 4, respectively.

[0055] Table 2. F0 Identification Primer Sequences

[0056] Table 3 PCR Reaction System

[0057] Table 4 PCR Reaction Conditions

[0058] The products amplified at the 3' and 5' ends were identified by electrophoresis using a 1.5% (m / v) agarose gel at 140V 400mA for 25-30 min. F0 positive mice were identified based on the size and position of the obtained bands. Figure 4 The electrophoresis results show that a total of 7 positive F0 mice were obtained, with corresponding numbers 140, 141, 142, 143, 149, 150, and 152.

[0059] (5) Propagate and establish a lineage from the obtained F0 positive mice. The specific steps are as follows: The F0 positive mice with different serial numbers were bred separately with wild-type mice of the same background (one-way breeding; offspring of different F0 positive mice could not be bred together). Successful line establishment was indicated by a PCR identification protocol that confirmed a 1:1 ratio of positive mice to wild-type mice. Figure 7As shown, mice numbered 42, 43, and 45 are positive mice; mice numbered 44, 46, and 47 are WT mice, meeting the requirement of a 1:1 probability of positive mice, indicating that the lineage was successfully established.

[0060] Example 2 This embodiment evaluates the expression level of hMRGPRX3 in hMRGPRX3 positive mice.

[0061] Comparison of MRGPRX3 expression in various human tissues from the GTEx Portal database showed that MRGPRX3 was highly expressed in human skin and other tissues, and its expression was significantly higher in skin cancer-related cell lines than in other cell lines (Figure 5A). Figure 5 (B). qPCR detection was performed on skin tissues of wild-type (WT) and humanized hMRGPRX3 mice after propagation and establishment, with GAPDH as an internal control for standardization.

[0062] The qPCR detection method is as follows: Use SYBR Green qPCR premixed reagent. Prepare the reaction system in a 96-well qPCR plate (3 technical replicates per sample): 10 μL SYBR Green premix, 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer (10 μM), 2 μL cDNA template (diluted 10-fold before use), and add nuclease-free water to a total volume of 20 μL.

[0063] Using a real-time quantitative PCR instrument, run the following amplification program: Pre-denaturation: 95°C, 30 seconds. PCR cycles (40 cycles): 95°C for 5 seconds, 60°C for 30 seconds (fluorescence signal is acquired during this stage).

[0064] Melting curve analysis: 95°C for 15 seconds, 60°C for 1 minute, then slowly increased from 60°C to 95°C (heating rate 0.05°C / second), while continuously acquiring fluorescence signals.

[0065] The qPCR primer information is shown in the table below:

[0066] Figure 5 The results in Figure C showed that the expression level of human MRGPRX3 in the hMRGPRX3 group was significantly higher than that in WT mice (P = 0.00016).

[0067] The qPCR detection of various tissues of hMRGPRX3 humanized mice according to the above detection method also showed that hMRGPRX3 was expressed in skin tissue (D in Figure 5). Although the expression level was low, its functional activation under specific pathological conditions may have an amplification effect, regulating a variety of immune responses and neural signals.

[0068] Example 3 This embodiment constructs an hMRGPRX3 humanized mouse atopic dermatitis model (AD).

[0069] Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease characterized by itching and impaired skin barrier function, with significant involvement of immune inflammation. To investigate the potential role of the MRGPRX3 gene in the pathogenesis of human AD, this example uses MC903 to induce an atopic dermatitis model in hMRGPRX3 humanized mice constructed in Example 1, and performs functional validation.

[0070] In this embodiment, MC903 (MCE, HY-10001) was applied topically to skin-induced AD models in WT mice and hMRGPRX3 humanized mice, respectively. The working concentration of MC903 was 45 μM. Specifically, 12.5 μL of MC903 was applied once daily to the dorsal and ventral skin of both ears, for a total volume of 25 μL. Additionally, 25 μL of MC903 was applied to approximately one-quarter of the skin surface area on the back. This treatment was repeated for 14 consecutive days.

[0071] During the experiment, the Adverse Dermatitis Index (ADI) was evaluated. The ADI score included the following four indicators: (1) erythema / bleeding, (2) dryness / desquamation, (3) edema / swelling, and (4) erosion / scratching. The severity of each symptom was scored as follows: 0 = none, 1 = mild, 2 = moderate, and 3 = severe. The total score after adding up all the scores was the ADI.

[0072] During the experiment, scratching behavior was observed. One scratching behavior was defined as the time from when the mouse raised its hind leg to when it put its hind leg down.

[0073] The results are as follows Figure 6 As shown in Figure A, compared to the WT-AD model obtained by MC903-induced wild-type (WT) mice, the hMRGPRX3-AD model obtained by MC903-induced hMRGPRX3 humanized mice exhibits a more severe AD-like phenotype (higher AID score). Meanwhile, according to Figure B, Figure 6The study also showed that, compared with wild-type (WT) mice, hMRGPRX3 mice had significantly increased auricular thickness on days 7 and 14 of treatment, and also significantly increased the number of spontaneous scratchings, suggesting that their itch behavior was more pronounced.

[0074] Histological analysis further supported the aforementioned phenotypic differences. HE staining was performed on mouse ears and back skin samples taken on Day 14.

[0075] The HE detection method is as follows: (1) Baking, dewaxing and rehydration a. Drying the slices: Place the collected slices in an oven to dry the surface moisture (37℃, overnight); b. Baking the slices: Arrange the dried slices in a basket, and then melt the paraffin wax in an oven (65℃, bake for 4-10 hours). c. Dewaxing and rehydration: Treat with the following reagents in sequence: xylene, 10 min; xylene, 10 min; anhydrous ethanol, 5 min; anhydrous ethanol, 5 min; 95% ethanol, 5 min; 95% ethanol, 5 min; 75% ethanol, 5 min; wash with distilled water 3 times, 5 min each time; (2) Hematoxylin staining a. Immerse in hematoxylin for 5 minutes; b. Rinse with pure water until no color remains in the water; (3) Color separation a. Differentiate with hydrochloric acid alcohol, once in each direction; b. Rinse with pure water; c. Observe under a microscope to see if the color is appropriate (if not, return to step (2) to re-stain with hematoxylin); (4) Return to Blue a. Ammonia solution turns blue, 1 minute; b. Rinse with pure water; (5) Eosin staining a. Stain with eosin for 2 minutes; b. Rinse with pure water; c. Observe under a microscope to see if the color is suitable (if not, return to step (2) and re-stain with hematoxylin); (6) Dehydrated and transparent a. Treat with anhydrous ethanol for 5 min; b. Treat with xylene for 5 min.

[0076] (7) Sealing: Seal the film with neutral quick-drying adhesive. HE staining results showed that the epidermis of hMRGPRX3 mice was significantly thickened and the basal layer structure was disordered, indicating a more severe inflammatory response (D, E in Figure 6). These results suggest that hMRGPRX3 expression may enhance susceptibility to MC903-induced inflammation, thereby exacerbating skin inflammation and itching symptoms.

[0077] In conclusion, hMRGPRX3 humanized mice exhibited more severe skin pathological changes and behavioral symptoms in the AD model, suggesting that this gene may play an important role in the pathogenesis of AD.

[0078] In summary, this invention marks the first successful insertion of the hMRGPRX3 gene into mice, resulting in a humanized hMRGPRX3 mouse model. As a novel pruritus-related receptor, hMRGPRX3 in transgenic mice accurately mimics the pharmacological characteristics of the human receptor. By constructing an agonist / antagonist screening model, preclinical evaluation of anti-pruritus drugs and atopic dermatitis treatments can be accelerated, significantly improving drug development efficiency. The model provided by this invention can offer an in vivo research tool for elucidating the "neuro-immune-skin" regulatory axis of Mas-related GPCRs in diseases such as atopic dermatitis and psoriasis. By comparing the neuropeptide release profiles of wild-type and transgenic mice, novel pruritus signaling pathways can be revealed.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0080] SEQ ID NO: 1

[0081] SEQ ID NO:2

[0082] SEQ ID NO:3

[0083] SEQ ID NO:4

Claims

1. A method for constructing an hMRGPRX3 humanized mouse model, characterized in that, Includes the following steps: The human hMRGPRX3 gene or a vector containing the hMRGPRX3 gene sequence is transferred into mouse zygotes. The nucleotide sequence of the hMRGPRX3 gene includes the upstream sequence shown in SEQ ID NO: 2, the hMRGPRX3 gene coding sequence shown in SEQ ID NO: 3, and the downstream sequence shown in SEQ ID NO:

4.

2. The method for constructing the hMRGPRX3 humanized mouse model according to claim 1, characterized in that, The nucleotide sequence of the vector is shown in SEQ ID NO:

1.

3. The method for constructing the hMRGPRX3 humanized mouse model according to claim 1, characterized in that, The hMRGPRX3 gene or a vector containing the hMRGPRX3 gene sequence was transferred into mouse zygotes via microinjection.

4. The method for constructing the hMRGPRX3 humanized mouse model according to claim 1 or 3, characterized in that, After the hMRGPRX3 gene or a vector containing the hMRGPRX3 gene sequence was transferred into mouse zygotes, the surviving zygotes were transplanted into pseudopregnant female mice, resulting in pregnancy and offspring. Positive hMRGPRX3 humanized mice were obtained through screening.

5. The method for constructing the hMRGPRX3 humanized mouse model according to claim 4, characterized in that, The mouse zygotes were zygotes with a C57BL / 6JGpt background.

6. A method for constructing an atopic dermatitis model, characterized in that, It includes: an hMRGPRX3 humanized mouse model constructed by inducing the construction method of the hMRGPRX3 humanized mouse model according to any one of claims 1-5 with at least one substance selected from OVA, MC903, DNCB, DNFB and house dust mites.

7. The method for constructing an atopic dermatitis model according to claim 6, characterized in that, The hMRGPRX3 humanized mouse model was induced using MC903 at working concentrations of 20 μM-70 μM.

8. The method for constructing an atopic dermatitis model according to claim 7, characterized in that, Apply MC903 to the skin of mice daily for 14 consecutive days.

9. A method for constructing a psoriasis model, characterized in that, It includes: The hMRGPRX3 humanized mouse model was constructed using the method for constructing the hMRGPRX3 humanized mouse model according to any one of claims 1-5, induced by imiquimod.

10. The application of the hMRGPRX3 humanized mouse model constructed by the method of any one of claims 1-5 in screening agonists and / or antagonists targeting hMRGPRX3.

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