Construction method of palmoplantar pustulopathy animal model, animal model and application

Abstract

The invention discloses a construction method of a palmoplantar pustulopathy animal model, the animal model and application of the animal model, and the construction method comprises the following steps: (1) constructing Krt14-CreERT2; tamoxifen is continuously injected into the Mettl3flox / flox mouse for 2 to 10 days; and (2) after 2-4 weeks, additionally injecting tamoxifen into the mouse in the step (1) for 3-10 days continuously, so as to obtain the palmoplantar pustulopathy animal model. By combining gene editing and chemical induction technologies, the mouse model capable of highly simulating pathological characteristics of human palmoplantar pustulopathy is successfully constructed. Lesion specificity: the palmoplantar depustulation parts of 100% of experimental group mice are limited to palmoplantar areas and are highly consistent with human diseases. Pathological goodness of fit: pustules are formed stably, and pathological characteristics are similar to those of human diseases. The application value is that skin inflammation is relieved after JAK inhibitor intervention, and it is proved that the model can be used for drug screening, mechanism research and new drug development.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a method for constructing an animal model of palmoplantar pustulosis, the animal model and its application. Background Art

[0002] Palmoplantar pustulosis (PPP) is a chronic, recurrent skin disease characterized by the formation of sterile pustules on the palms and soles, accompanied by erythema, scaling, and pain. The pathogenesis of PPP is not fully understood, but is currently believed to be closely related to immune system abnormalities, genetic factors, and environmental triggers such as infection and smoking. However, the lack of an ideal animal model significantly limits research on the pathogenesis of PPP and the development of new drugs.

[0003] Currently, research on palmoplant pustulosis mainly relies on clinical patient samples, but due to individual differences and disease severity among patients, the samples are highly heterogeneous, and confounding factors other than the pathogenesis of the disease cannot be ruled out. Although some existing animal models (such as mice and rats) can partially simulate skin inflammatory responses, they are significantly different from human palmoplant pustulosis in terms of pustule formation, pathological characteristics, and immune mechanisms. Therefore, developing an animal model that can highly simulate the pathological characteristics of human palmoplant pustulosis is of great significance for in-depth research on the pathogenesis of the disease and screening effective therapeutic drugs.

[0004] In 2009, a study used severe combined immunodeficiency (SCID) mice to intraperitoneally inject mononuclear cells prepared from tonsils removed from PPP patients with exacerbated rash into SCID mice, resulting in hair loss on the cheeks and forehead of the mice, and the development of a rash 4-8 weeks later. Epidermal thickening and blister formation were observed in the rash area. CD4 + T cells infiltrate in small amounts, but CD8 + T cells or B cells. (See Auris Nasus Larynx, 2009), however, this model is quite difficult to implement and can only reflect the role of tonsil cells in the pathogenesis of PPP, but cannot reflect the overall immune response of PPP.

[0005] Currently, there is no animal model that simulates palmoplantar pustulosis. The lesions of similar animal models of skin inflammation are not limited to the palm and plantar regions and are difficult to reflect the complexity of clinical PPP. Summary of the Invention

[0006] In order to overcome the shortcomings of the above-mentioned existing technical solutions, the following problems need to be solved:

[0007] 1. Provide an animal model in which the lesion is strictly confined to the palmar and plantar regions.

[0008] 2. Achieve stable sterile pustule formation and neutrophil infiltration, simulating the core pathological characteristics of PPP.

[0009] 3. Provide a reliable tool for drug screening and mechanism studies.

[0010] To address the above technical problems, the present invention provides a method for constructing an animal model of palmoplantar pustulosis, an animal model, and its application. By combining gene editing and chemical induction technologies, the present invention successfully constructed a mouse model that can highly simulate the pathological characteristics of human palmoplantar pustulosis. Lesion specificity: 100% of the experimental group mice developed localized palmoplantar pustules. The lesions were confined to the palmoplantar region, which is highly consistent with the human disease. Pathological consistency: Pustule formation was stable, and the pathological characteristics were similar to those of the human disease. Application value: Skin inflammation was alleviated after intervention with a JAK inhibitor, confirming that the model can be used for drug screening, mechanism research, and new drug development.

[0011] A first aspect of the present invention provides a method for constructing an animal model of palmoplantar pustulosis, comprising the following steps:

[0012] (1) Krt14-CreER T2 ;Mettl3 flox / flox Mice were injected with tamoxifen for 3 to 10 consecutive days;

[0013] (2) After 2 to 4 weeks, the mice in (1) were given additional injections of tamoxifen for 3 to 10 consecutive days to obtain a palmoplantar pustulosis animal model.

[0014] In a preferred embodiment, Krt14-CreER T2 ;Mettl3 flox / flox Krt14-CreER T2 Mice and Mettl3 flox / flox Mice are bred together, for example by two generations of hybridization.

[0015] In a preferred embodiment, Krt14-CreER T2 Mice can be purchased from Southern Model Organisms C57BL / 6Smoc-Krt14 em2(CreERT2-Wpre-polyA)Smoc (Catalog No. NM-KI-190024) strain.

[0016] In a preferred embodiment, Mettl3 flox / flox The mouse may be a Mettl3-CKO4-N conditional knockout mouse.

[0017] In a preferred embodiment, the construction process of the Mettl3-CKO4-N conditional gene knockout mouse is as follows: the Mettl3 gene (Ensembl number: ENSMUSG00000022160) is flox-modified by using the principle of homologous recombination and ES cell targeting.

[0018] In one specific embodiment, the Mettl3-CKO4-N conditional knockout mouse was constructed as follows: An ES cell targeting vector containing a 2.9 kb 5' homology arm, a 1.1 kb flox region, a PGK-Neo-polyA construct, a 3.0 kb 3' homology arm, and an MC1-TK-polyA negative selection marker was constructed by infusion. This vector was linearized and electroporated into JM8A3 ES cells. Long-range PCR was used to identify positive clones demonstrating correct homologous recombination. Positive ES cell clones were expanded and injected into blastocysts of C57BL / 6J mice to generate chimeric mice. High-proportion chimeric mice were mated with Flp mice to produce positive F1 generation Neo-depleted heterozygous mice.

[0019] In a preferred embodiment, Krt14-CreER T2 ;Mettl3 flox / flox The mice were C57BL / 6Smoc-Krt14 of the Southern model organisms em2(CreERT2-Wpre-polyA)Smoc The strain (catalog number NM-KI-190024) was bred with the Mettl3-CKO4-N conditional gene knockout mouse strain constructed by Southern Model Organisms.

[0020] In a preferred embodiment, Krt14-CreER T2 ;Mettl3 flox / flox Krt14-CreER T2 Mice and Mettl3 flox / flox The target mice were obtained by hybridization of two generations.

[0021] For example, in the first generation of hybridization, Krt14-CreER T2 Mice and Mettl3 flox / flox Mice were mated and the offspring genotype was Krt14-CreER T2 / + ;Mettl3 flox / + Mice; Second generation hybrid: Krt14-CreER T2 / + ;Mettl3 flox / + Mice and Mettl3 flox / flox The mice are mated, and target mice are selected from the offspring.

[0022] In a preferred embodiment, in step (1), the dose of tamoxifen injected is 50-100 mg / kg, for example, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg or 100 mg / kg.

[0023] In a preferred embodiment, in step (2), the dose of tamoxifen injected is 50-100 mg / kg, for example, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg or 100 mg / kg.

[0024] In a preferred embodiment, tamoxifen is injected intraperitoneally.

[0025] In a preferred embodiment, the mouse is a three-week-old mouse.

[0026] In a preferred embodiment, in step (1), tamoxifen is injected into the mouse for 3 consecutive days, 4 days, 5 days, 6 days, 7 days, 8 days or 9 days.

[0027] In a preferred embodiment, in step (2), the mice in (1) are given additional injections of tamoxifen for 3 consecutive days, 4 days, 5 days, 6 days, 7 days, 8 days or 9 days.

[0028] In a preferred embodiment, in step (2), the mice in (1) are continuously given additional injections of tamoxifen 15 days later, 17 days later, 21 days later, 25 days later, or 28 days later.

[0029] In a preferred embodiment, in step (2), 3 weeks later, the mice in step (1) are given additional injections of tamoxifen for 5 consecutive days.

[0030] In a preferred embodiment, in step (1), the dose of tamoxifen injected is 75 mg / kg, and in step (2), the dose of tamoxifen injected is 75 mg / kg.

[0031] The second aspect of the present invention provides an animal model of palmoplantar pustulosis, which is constructed by the construction method described in the first aspect of the present invention.

[0032] The third aspect of the present invention provides an application of the construction method described in the first aspect of the present invention in constructing an animal model of plantar pustulosis.

[0033] In a fourth aspect, the present invention provides an animal model of plantar pustulosis constructed by the construction method described in the first aspect of the present invention, or an animal model described in the second aspect of the present invention, for use in the field of medical technology.

[0034] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0035] Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0036] The positive progress effect of the present invention is:

[0037] By combining gene editing and chemical induction techniques, this study successfully constructed a mouse model that closely mimics the pathological features of human palmoplantar pustulosis. Lesion specificity: 100% of the experimental group mice developed localized palmoplantar pustules, with lesions confined to the palm and sole regions, highly consistent with the human disease. Pathological consistency: Pustule formation was stable, and the pathological features were similar to those of the human disease. Application value: Treatment with a JAK inhibitor reduced skin inflammation, confirming the model's potential for drug screening, mechanism studies, and new drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Diagram of the modeling strategy for the palmoplantar pustulosis mouse model.

[0039] Figure 2 Photos and H&E staining images of skin lesions in mice with palmoplantar pustulosis.

[0040] Figure 3 These are photos and H&E-stained images of the dorsal skin of mice with palmoplantar pustulosis.

[0041] Figure 4 Immunofluorescence staining of proliferation and differentiation indicators in the palmoplantar pustulosis mouse model.

[0042] Figure 5 Immunofluorescence staining of proliferation and differentiation indicators in the palmoplantar pustulosis mouse model.

[0043] Figure 6 Immunofluorescence staining of neutrophils in the mouse model of palmoplantar pustulosis.

[0044] Figure 7 Gene set enrichment analysis of differentially expressed genes in a mouse model of palmoplantar pustulosis.

[0045] Figure 8 This is the gene and enrichment analysis of the differentially expressed genes (GSE185856) that are upregulated in the palmoplantar pustulosis mouse model vs normal skin on the palms and soles of mice and palmoplantar pustulosis lesions vs normal control skin.

[0046] Figure 9 Gram staining of mouse skin tissue.

[0047] Figure 10 Diagram of the upadacitinib dosing strategy in the palmoplantar pustulosis mouse model.

[0048] Figure 11 Upadacitinib was used to treat mice with palmoplantar pustulosis.

[0049] Figure 12 Figure 2 is the expression level of neutrophil chemotaxis-related factors after silencing METTL3 expression in primary keratinocytes.

[0050] Figure 13 Figure 2 is the expression levels of neutrophil chemotaxis-related factors under inflammatory conditions after silencing METTL3 expression in primary keratinocytes.

[0051] Figure 14 5% imiquimod cream was applied externally to the palms and soles of C57BL / 6 mice.

[0052] Figure 15 Diagram of the single-dose tamoxifen induction strategy.

[0053] Figure 16 These are photos of the palms and soles of mice after a single injection of tamoxifen. DETAILED DESCRIPTION

[0054] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0055] Step 1: Construction of conditional knockout mice

[0056] At 3 weeks of age, Krt14-CreER T2 ;Mettl3 flox / flox Mettl3 was specifically knocked out in keratinocytes by intraperitoneal injection of tamoxifen in mice (cKO mice).

[0057] Key improvements: Maintaining continuous METTL3 deficiency through two tamoxifen injections (3 weeks apart) prevents spontaneous remission of inflammation.

[0058] Step 2: Pathological feature verification

[0059] Clinical manifestations: cKO mice had large amounts of yellow pus crusts on their palms and soles, while there were no obvious lesions on their backs.

[0060] Histopathology: H&E staining showed epidermal thickening and stratum corneum pustules in the palm and plantar regions.

[0061] Immunohistochemistry: A large number of neutrophils infiltrated the spontaneous inflammatory lesions on the palms and soles of cKO mice, and the proliferation ability of keratinocytes in the lesions was enhanced, while the differentiation ability was reduced.

[0062] Transcriptome analysis: The differentially expressed genes between the palm and plantar regions of cKO mice and WT mice were enriched in the IL-17 / TNF / JAK-STAT signaling pathway, which highly overlapped with the gene spectrum of PPP patients.

[0063] Step 3: Verification of sterile inflammation

[0064] Gram staining was negative, ruling out bacterial infection; inflammation was significantly alleviated after JAK inhibitor intervention, confirming that it was driven by an immune mechanism.

[0065] Step 4: In vitro mechanistic studies

[0066] Silencing the expression of METTL3 in primary human keratinocytes upregulated the expression of neutrophil chemokines such as CXCL1, CXCL8, and IL36G, suggesting that METTL3 deficiency promotes the formation of palmoplantar pustulosis-like lesions by enhancing the release of neutrophil chemokines.

[0067] The following is described with reference to specific examples. Unless otherwise specified, the mice used in the following examples are of the C57 strain.

[0068] Krt14-CreER used in the following examples T2 ;Mettl3 flox / flox The mice (cKO mice) were C57BL / 6Smoc-Krt14 from the Southern Model Organisms em2(CreERT2-Wpre-polyA)Smoc The strain (catalog number NM-KI-190024) was bred with the Mettl3-CKO4-N conditional gene knockout mouse strain constructed by Southern Model Organisms.

[0069] The Mettl3-CKO4-N conditional knockout mouse was constructed as follows: The Mettl3 gene (Ensembl ID: ENSMUSG00000022160) was floxed using homologous recombination and ES cell targeting. The procedure was briefly described as follows: an ES cell targeting vector containing a 2.9 kb 5' homology arm, a 1.1 kb floxed region, a PGK-Neo-polyA region, a 3.0 kb 3' homology arm, and an MC1-TK-polyA negative selection marker was constructed by infusion. After linearization, the vector was electroporated into JM8A3 ES cells. Long-range PCR was used to identify positive clones demonstrating correct homologous recombination. Positive ES cell clones were expanded and injected into blastocysts of C57BL / 6J mice to generate chimeric mice. High-proportion chimeric mice were mated with Flp mice to generate positive F1 generation Neo-dead heterozygous mice.

[0070] Example 1: Animal model construction and phenotypic verification

[0071] Animal Grouping:

[0072] Experimental group: three-week-old Krt14-CreER T2 ;Mettl3 flox / flox Mice (cKO mice) (n=10) were intraperitoneally injected with tamoxifen (75 mg / kg, for 5 consecutive days).

[0073] Control group: Mettl3 of the same litter, same sex, and same age flox / flox Mice (WT mice) (n=10) were injected with the same amount of tamoxifen in the same way.

[0074] Secondary induction: 3 weeks later, the experimental group received additional tamoxifen (SigmaAldrich, catalog number #T5638) injection (75 μg / g, for 5 consecutive days) ( Figure 1 ).

[0075] Results: Six weeks after tamoxifen injection, yellow pus crusts appeared on the palms and soles of the experimental group. H&E staining showed that a large number of lymphocytes infiltrated the lesions of cKO mice ( Figure 2 ), while there was no obvious skin lesion on the back ( Figure 3 ).

[0076] By immunofluorescence technique, the lesion area was found to be m 6 A modification and its methyltransferase METTL3 protein expression were significantly lower than those in the normal skin of the control group ( Figure 4 ).

[0077] At the same time, Ki67 +The number of keratinocytes increased significantly compared with the control group, indicating that the proliferation ability of keratinocytes in the lesions was enhanced; and the expression levels of filaggrin and keratin 10 (K10) proteins in the lesions were significantly lower than those in the control group, indicating that the differentiation ability of keratinocytes in the lesions was reduced. Figure 5 ).

[0078] In addition, a large amount of Ly6G can be observed in the lesions under the microscope. + CD11b + cells, indicating that neutrophils accumulate in large numbers in the lesions ( Figure 6 ).

[0079] Example 2: Transcriptomics and mechanism verification

[0080] Krt14-CreER T2 ;Mettl3 flox / flox The palm and plantar lesion areas of (cKO) mice and Mettl3 flox / flox High-throughput transcriptome analysis was performed on the normal skin of the palm and sole of WT rats, and gene set enrichment analysis (GSEA) was performed on the differentially expressed genes (|log2FC|>1, P adj<0.05). The results showed that the differentially expressed genes were significantly enriched in Cytokine-cytokine receptor interaction, Jak-Stat signaling pathway, TNF signaling pathway, IL-17 signaling pathway ( Figure 7 ).

[0081] In addition, GSEA results showed that the differentially regulated gene sets in the palm and plantar skin of PPP patients compared with normal skin (data from GEO database GSE164154) were significantly overlapped with the model lesion skin (P < 0.001), suggesting that the transcriptional profiles of spontaneous inflammation and PPP patients' skin lesions are similar ( Figure 8 ).

[0082] Example 3: Sterility Verification of Spontaneous Palmoplantar Pustulosis-like Skin Inflammation

[0083] To further verify that the spontaneous skin inflammation was sterile inflammation and to exclude bacterial infection, Gram staining was performed on the skin lesion tissue sections. The Gram staining results showed that no Gram-positive or Gram-negative bacteria were found in the skin lesions. This suggests that the large number of neutrophils gathered in the skin lesions was not caused by bacterial infection ( Figure 9 ).

[0084] Example 4: Feasibility Verification of Models for Drug Screening and New Drug Development

[0085] Small molecule targeted drugs, JAK inhibitors, have been reported to be effective in treating palmoplantar pustulosis in clinical practice. Next, cKO mice were given upadacitinib orally to treat palmoplantar pustulosis-type skin inflammation. The specific medication strategy is described in [1]. Figure 10 After 14 days of administration of upadacitinib (MCE, Catalog No. HY-19569) (3.69 mg / kg) to mice, the erythema, scales, and pus crusts on the palm and soles of the mice were significantly reduced compared with the untreated group, indicating that upadacitinib can significantly reduce the pustular skin inflammation on the palms and soles of the mice ( Figure 11 ), suggesting that this animal model can be used for subsequent basic research related to drug screening and new drug development for palmoplantar pustulosis.

[0086] Example 5: In vitro experimental mechanism verification

[0087] After silencing the expression of METTL3 in primary human keratinocytes, the mRNA expression of neutrophil chemokines such as CXCL1, CXCL8 and IL36G was detected by qRT-PCR technology, and it was found that their expression was significantly upregulated compared with the control group after silencing METTL3 in cells ( Figure 12 ); After silencing the expression of METTL3 in primary keratinocytes, the mRNA expression levels of CXCL1, CXCL8 or IL36G were significantly increased compared with the control group (siControl group) under the stimulation of 100ng / mL IL-17A, 100ng / mL TNF-α or 100ng / mL IL-36γ ( Figure 13 These results suggest that decreased METTL3 expression may promote the formation of palmoplantar pustulosis-like lesions by enhancing the release of neutrophil chemotactic factors.

[0088] Comparative Example 1: Imiquimod-induced model

[0089] Methods: 5% imiquimod cream was applied to the palms and soles of C57BL / 6 mice for 7 days.

[0090] Results: Only slight redness of the skin was seen, and no pustules were formed ( Figure 14 ).

[0091] Comparative Example 2: Single injection of tamoxifen induced model

[0092] Experimental group: 3-5 weeks old Krt14-CreER T2 ;Mettl3 flox / flox Mice (cKO mice) (n=10) were intraperitoneally injected with tamoxifen (75 mg / kg, for 5 consecutive days). Figure 15 .

[0093] Control group: Mettl3 of the same litter, same sex, and same age flox / flox Mice (WT mice) (n=10) were injected with the same amount of tamoxifen in the same way.

[0094] Results: Four weeks after tamoxifen injection, skin inflammation was observed in the palms and soles of the experimental group, and the inflammation subsided after another two weeks. Figure 16 ).

Claims

1. A method for constructing an animal model of palmoplantar pustulosis, characterized in that: The following steps are involved: (1) Krt14-CreER T2 ;Mettl3 flox / flox Mice were injected with tamoxifen for 3 to 10 consecutive days; (2) After 2 to 4 weeks, the mice in (1) were given additional injections of tamoxifen for 3 to 10 consecutive days to obtain a palmoplantar pustulosis animal model.

2. The construction method according to claim 1, wherein In step (1), the dosage of tamoxifen injected is 50 to 100 mg / kg, and / or, in step (2), the dosage of tamoxifen injected is 50 to 100 mg / kg.

3. The construction method according to claim 1 or 2, wherein: Tamoxifen is injected intraperitoneally.

4. The construction method according to any one of claims 1 to 3, wherein: The mice were three-week-old mice.

5. The construction method according to any one of claims 1 to 4, characterized in that: In step (1), the mice are injected with tamoxifen for 5 consecutive days; and / or, in step (2), 3 weeks later, the mice in (1) are given an additional injection of tamoxifen for 5 consecutive days.

6. The construction method according to any one of claims 1 to 5, wherein: In step (1), the dosage of tamoxifen injected is 60 to 90 mg / kg, and / or, in step (2), the dosage of tamoxifen injected is 60 to 90 mg / kg.

7. The construction method according to claim 6, wherein: In step (1), the dosage of tamoxifen injected is 75 mg / kg, and / or, in step (2), the dosage of tamoxifen injected is 75 mg / kg.

8. A palmoplantar pustulosis animal model constructed by the construction method according to any one of claims 1 to 7.

9. Use of the construction method according to any one of claims 1 to 7 in constructing an animal model of plantar pustulosis.

10. Use of the plantar pustulosis animal model constructed by the construction method according to any one of claims 1 to 7 or the animal model according to claim 8 in the field of medical technology.

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