Method for constructing NPPK mouse model based on CRISPR / Cas9 technology and application thereof

Abstract

The invention discloses a method for constructing an NPPK mouse model based on a CRISPR / Cas9 technology and application of the NPPK mouse model, and belongs to the technical field of genetic engineering and disease model construction. The preparation method comprises the following steps: performing targeted knockout on a mouse Serpina12 gene through a CRISPR / Cas9 gene editing technology to obtain a Serpina12 gene-deficient mouse; an acetone-diethyl ether mixed solution (formed by mixing acetone and diethyl ether according to the volume ratio of 1: 1) and double distilled water are sequentially and externally applied to the skin surface of the Serpina12 gene defect mouse, and the NPPK mouse model is obtained. The method has the characteristics of simplicity and convenience in operation, high efficiency and stable phenotype, and the NPPK mouse model obtained by the method can be used for researching inflammatory keratotic dermatosis and screening drugs for treating NPPK or IL-17 related inflammatory dermatosis.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and disease model construction technology, specifically involving a method for constructing an NPPK mouse model based on CRISPR / Cas9 technology and its application. Background Technology

[0002] Nagashima-type palmoplantar keratosis (NPPK) is an autosomal recessive inherited skin disease characterized by significant hyperkeratosis, erythema, scaling, and inflammation of the palms and soles. The lesions extend beyond the palms and soles, potentially reaching the wrists, dorsum of the foot, and Achilles tendon area. The elbows and knees are frequently affected, and the condition is often accompanied by palmoplantar hyperhidrosis, severely impacting the patient's mental health. The lesions swell and turn white upon contact with water, exhibiting a spongy appearance. Some patients may also experience widespread erythematous keratosis on the face, limbs, and waist. Epidemiological studies have shown that NPPK significantly affects patients' quality of life. Currently, clinical treatments for NPPK are extremely limited, primarily focusing on symptom relief, such as topical keratolytic agents, corticosteroids, or physical therapy, but these treatments are often short-lived and do not provide a cure. Therefore, elucidating the molecular pathogenesis of NPPK and developing targeted therapeutic strategies based on this understanding has become a crucial direction in current dermatological research.

[0003] Recent studies have found that Serpina12 The genetically encoded Vaspin protein plays a crucial role in the pathogenesis of NPPK. Vaspin belongs to the serine protease inhibitor family and possesses anti-inflammatory and metabolic regulatory functions. Clinical evidence shows that... Serpina12 Loss-of-function mutations are significantly associated with the occurrence of NPPK, but the specific molecular mechanisms remain unclear, especially how Vaspin regulates skin barrier function and inflammatory signaling pathways (such as the IL-17 pathway), which still need further investigation.

[0004] Mechanistic studies of NPPK primarily rely on in vitro cell models, but these methods suffer from limitations in phenotypic mimicry: existing chemically induced or transgenic mouse models cannot fully replicate the typical pathological features of NPPK patients, such as palmoplantar keratosis and excessive activation of the IL-17 pathway, leading to insufficient relevance of research results to clinical practice. Due to the lack of suitable animal models, how Vaspin regulates downstream signaling pathways (such as serine protease networks or inflammatory cytokine cascades) remains unclear, limiting mechanistic research and severely hindering the development of targeted drugs. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for constructing an NPPK mouse model based on CRISPR / Cas9 technology and its application, so as to solve the technical problem of insufficient phenotypic simulation in the existing NPPK mouse model.

[0006] To achieve the above objectives, the present invention employs the following technical solution: The first aspect of this invention discloses a method for constructing an NPPK mouse model based on CRISPR / Cas9 technology. Serpina12 NPPK mouse model was obtained by sequentially applying a mixture of acetone-ether and double-distilled water to the skin surface of gene-deficient mice. The acetone-ethyl ether mixture is formed by mixing acetone and ethyl ether in a volume ratio of 1:1.

[0007] Preferably, in Serpina12 The skin of the genetically defective mice was first coated with an acetone-ether mixture for 15 seconds, and then coated with double-distilled water for 30 seconds.

[0008] Preferably, the acetone-ether mixture and double-distilled water are applied topically twice daily for 5 consecutive days.

[0009] Preferably, the NPPK mouse model is obtained by applying a mixture of acetone-ether and double-distilled water to induce an increased skin surface roughness and increased erythema and scaling in mice.

[0010] Preferably, the Serpina12 Gene-deficient mice are mice that have been targeted for knockout using CRISPR / Cas9 gene editing technology. Serpina12 The gene was obtained later; the gRNA plasmids used in CRISPR / Cas9 gene editing include gRNA-A1, gRNA-A2, gRNA-B1 and gRNA-B2, with nucleotide sequences shown as SEQ ID NO.1~SEQ ID NO.4 respectively.

[0011] Preferably, after modeling is completed, the model is evaluated by HE, RT-qPCR and immunofluorescence detection.

[0012] Further preferably, HE showed increased epidermal thickness and hyperkeratosis, and RT-qPCR and immunofluorescence detection results showed upregulated expression levels of inflammatory factors in mouse skin tissue, indicating that the NPPK mouse model was successfully constructed.

[0013] More preferably, the inflammatory factors are characterized by elevated mRNA and / or protein expression levels of Cxcl2, S100a8, S100a9, Lcn2, Il-1β, Mmp9, and Mmp13.

[0014] In a second aspect, the present invention discloses an NPPK mouse model obtained by the above method.

[0015] In a third aspect, the present invention discloses the application of the above-mentioned NPPK mouse model in the study of inflammatory keratotic skin diseases.

[0016] Preferably, the inflammatory keratotic dermatitis is NPPK.

[0017] A fourth aspect of the present invention discloses a method for screening candidate drugs for treating NPPK or IL-17-related inflammatory skin diseases, comprising the following steps: 1) The above-mentioned NPPK mouse model was used as the disease group, and a blank control group and a treatment group were set up at the same time; 2) The test drug was administered to the mice in the treatment group; 3) By comparing the macroscopic skin score, TEWL value, epidermal thickness, mRNA and protein expression levels of inflammatory factors, and CD4 expression levels of mice in each group, the following parameters were compared: + T cell infiltration was assessed to evaluate the efficacy of the test drug in improving NPPK or IL-17-related inflammatory skin diseases.

[0018] In a fifth aspect, this invention discloses a research system for studying the molecular mechanisms of skin inflammation, using the aforementioned NPPK mouse model for the following analysis: The ultrastructure of keratinocytes and abnormalities of the epidermal junction complex were observed using transmission electron microscopy. Disorders in the expression of epidermal layering markers were detected by multicolor immunofluorescence staining. The number of Th17 cells, neutrophils, dendritic cells and macrophages infiltrating the skin lesions was detected by flow cytometry or immunofluorescence. Changes in phosphorylation levels of key molecules in the IL-17 signaling pathway were detected by Western blotting or immunofluorescence.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The method for constructing an NPPK mouse model based on CRISPR / Cas9 technology provided by this invention, through the analysis of... Serpina12 Complete gene knockout ( Serpina12 - / -Sequential treatment of mice with acetone-ether mixture and ddH2O induced a phenotype highly consistent with that of NPPK patients, including: skin barrier dysfunction (increased skin surface roughness, increased erythema and scaling; HE staining showed significant epidermal thickening, hyperkeratosis and parakeratosis, dermal capillary dilation, and perivascular lymphocyte infiltration); and activated inflammatory response (significant upregulation of the IL-17 signaling pathway, accompanied by elevated levels of pro-inflammatory factors such as IL-1β and TNF-α). This method successfully reproduced the key pathological features of human NPPK in the mouse model. Serpina12 Gene deletion plays a crucial role in the development and progression of xeroderma by exacerbating skin inflammation and disrupting the skin barrier function, which provides a basis for further analysis. Serpina12 This method provides important experimental evidence for the molecular mechanisms of NPPK regulation in the skin barrier. Therefore, it fills a technological gap in NPPK research. By sequentially treating mice with an acetone-ether mixture and ddH2O, it induces a high degree of pathological similarity to human NPPK, enabling systematic in vivo studies of the role of vaporin in skin keratinization, barrier homeostasis, and inflammatory responses. It addresses the technical problem of insufficient phenotypic simulation in existing NPPK mouse models, and can be used for research on inflammatory keratotic skin diseases, the development of drugs for treating NPPK or IL-17-related inflammatory skin diseases, laying the foundation for further exploration of the mechanisms by which vaporin regulates the skin barrier and inflammatory pathways. It also promotes the development of targeted drugs (such as serine protease inhibitors or IL-17 antagonists), providing an irreplaceable tool for elucidating the pathological mechanisms of this disease and developing precision treatment strategies. This method has significant scientific value and promising clinical application prospects. Attached Figure Description

[0020] Figure 1 The CRISPR targeting method invented by this company Serpina12 Diagram of gene knockout plasmid construction; Figure 2 PCR identification invented by this party Serpina12 Genotyping results; (a) shows agarose gel electrophoresis, and (b) shows sequencing results. Figure 3 RT-qPCR, Western Blot and immunohistochemical validation of the present invention Serpina12 Gene knockout efficiency graph; where (a) is the RT-qPCR result, (b) is the Western Blot result, and (c) is the immunohistochemistry result; Figure 4 shows the results of the construction and phenotypic analysis of the AEW-stimulated NPPK mouse model of the present invention; where (a) is a picture of mouse skin after chemical induction, (b) is the HE staining result, (c) is the RT-qPCR result, (d) is the immunofluorescence staining result, (e) is the transcriptomics result, (f) is the KEGG pathway enrichment analysis result, and (g) is the GSEA analysis result. Detailed Implementation

[0021] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0022] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0023] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0024] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0025] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0026] This invention provides a method for constructing an NPPK mouse model based on CRISPR / Cas9 technology, which uses CRISPR / Cas9 gene editing technology to target and knock out mice. Serpina12 Genes, obtained Serpina12 Gene-deficient mice; in Serpina12NPPK mouse model was obtained by sequentially applying a mixture of acetone-ether and double-distilled water (ddH2O) to the skin surface of gene-deficient mice. This induced skin roughness, increased erythema and scaling, skin barrier defects and abnormal differentiation, and activation of the IL-17 signaling pathway. The acetone-ethyl ether mixed solution is formed by mixing acetone and ethyl ether in a volume ratio of 1:1.

[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0028] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0029] I. Construction based on CRISPR / Cas9 technology Serpina12 Gene knockout mouse model 1. Constructing gRNA plasmids like Figure 1 As shown, CRISPR / Cas-mediated genetic engineering technology was used to target mice. Serpina12 Four gRNA plasmids were designed from the exon 3 region of the gene (NCBI: 68054; NM_026535.2): gRNA-A1, gRNA-A2, gRNA-B1 and gRNA-B2, with nucleotide sequences shown in Table 1 as SEQ ID NO.1~SEQ ID NO.4.

[0030] Table 1 Nucleotide sequence list

[0031] 2. CRISPR / Cas9 gene editing The four gRNA plasmids constructed in step 1 were transcribed into RNA via microinjection, and then injected into the pronucleus of C57BL / 6J mouse zygotes along with Cas9 mRNA to knock out the target region (1227 bp deletion) through CRISPR / Cas9-mediated gene editing. After injection, the zygotes were transplanted into the oviducts of C57BL / 6J pseudopregnant mice and bred. F0 generation positive mice were obtained by PCR amplification and agarose gel electrophoresis.

[0032] 3. Obtain F1 generation mice The F0 generation positive mice obtained in step 2 were bred and mated with wild-type C57BL / 6J mice to obtain offspring mice.

[0033] DNA was extracted from the tails of offspring mice for PCR amplification. The PCR primers (F1, R1, and F2) are shown in Table 1 (SEQ ID NO. 5-SEQ ID NO. 7), the PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3. After PCR amplification, a 1.0% agarose gel was prepared. The PCR product was mixed with the loading buffer and then spotted into the wells along with the DNA molecular weight standard. Electrophoresis was performed at a constant voltage of 100-120V until the indicator migrated to two-thirds of the gel. After electrophoresis, the gel was imaged under UV light. The product size was determined by comparing the sample bands with the DNA standard. After verifying the correct size of the PCR product by agarose gel electrophoresis, a purification kit was used to remove residual primers and dNTPs. The purified PCR product was used directly as a sequencing template, and Sanger sequencing was performed using the same primers. The reaction system included BigDyeTerminator v3.1 cyclic sequencing reagents. After the sequencing PCR program, the extension products were purified by ethanol / EDTA precipitation to remove unincorporated dye terminators. The purified sequencing products were then subjected to capillary electrophoresis on an ABI 3500 series genetic analyzer. The obtained raw sequencing data were compared with wild-type... Serpina12 Gene sequences are compared, and features such as sequence breaks, overlaps, or frame shifts in the sequencing map are analyzed to confirm whether the expected deletion mutation exists at the target site, thus verifying the successful targeting of the F1 generation. Serpina12 -KO heterozygous mice ( Serpina12 + / - ).

[0034] Table 2 PCR reaction system

[0035] Table 3 PCR reaction conditions

[0036] 4. Obtain F2 generation mice For F1 generation Serpina12 -KO heterozygous mice were fed with F1 generation mice. Serpina12 -KO heterozygous mice and F1 generation Serpina12 -KO heterozygous mice were used for breeding and mating, and the F1 generation... Serpina12 -KO heterozygous mice and F1 generation Serpina12-KO heterozygous mice were bred and mated with wild-type C57BL / 6J mice to produce F2 generation mice. The tails of the F2 generation mice were collected, and DNA was extracted using the method described in step 3 for PCR amplification, agarose gel electrophoresis, and sequencing. F2 generation mice were then selected. Serpina12 -KO homozygous mice ( Serpina - / - ) and F2 generation ​ -KO heterozygous mice ( ​ + / - ).

[0037] 5. Obtain F3 generation mice F2 generation breeding ​ -KO homozygous mice and F2 generation ​ -KO heterozygous mice, allowing the F2 generation ​ -KO homozygous / heterozygous mice and F2 generation ​ -KO homozygous / heterozygous mice were bred and mated, specifically as follows: ​ - / - ​ - / - , ​ - / - ​ + / - , ​ + / - ​ + / - F3 generation mice were generated. DNA was extracted from the tails of F3 mice using the method described in step 3 for PCR amplification. Primers F1 / R1 were used to specifically detect knockout-positive individuals (345 bp band), and primers F2 / R1 were used to specifically detect wild-type individuals (466 bp and 1572 bp bands). The PCR products were subjected to agarose gel electrophoresis, and the gel was imaged under UV light after electrophoresis. By comparing the sample bands with DNA standards, a single, sharp, bright band at the expected size was observed, confirming successful amplification of the specific Serpina12 gene fragment. Simultaneously, the negative control wells were checked for bands to ensure the results were not contaminated. The identification results are as follows: ​ As shown in (a), ​ -KO homozygous F3 mice ( ​ - / - The results showed a 345 bp band, while the wild-type (WT) showed bands of 466 bp and 1572 bp, indicating that... ​The gene has been knocked out. After verifying the correct size of the PCR products by agarose gel electrophoresis, residual primers and dNTPs were removed using a purification kit. The purified PCR products were used directly as sequencing templates, and Sanger sequencing reactions were performed using the same primers. The reaction system included BigDye Terminator v3.1 cyclic sequencing reagents. After the sequencing PCR program was completed, the extension products were purified by ethanol / EDTA precipitation to remove unincorporated dye terminators. The purified sequencing products were then subjected to capillary electrophoresis on an ABI 3500 series genetic analyzer. The obtained raw sequencing data were compared with wild-type... ​ Gene sequences are compared, and by analyzing features such as sequence breaks, overlaps, or frame shifts in the sequencing map, it is confirmed whether the target site contains the expected deletion mutation (1227 bp deletion), thereby achieving [the desired mutation]. ​ -KO homozygous F3 mice ( ​ - / - Accurate identification of genotype ( ​ (b) 6. RT-qPCR and Western Blot Validation ​ Gene knockout efficiency RT-qPCR (verification of mRNA levels) method steps: 1) Sample preparation: Total RNA was extracted from the skin tissue of wild-type (WT) and knockout (KO) mice, and the concentration was determined. The total RNA was reverse transcribed into cDNA using a reverse transcription kit. 2) qPCR reaction: Primers: Design ​ Specific primers and internal reference gene primers (e.g., Gapdh). System: Amplification was performed using the SYBR Green method on a qPCR instrument. 3) Data analysis: [The text abruptly ends here, so the translation also ends here.] The method calculates relative expression levels. Knockout efficiency = 1 - (relative expression level in the KO group / relative expression level in the WT group). Success criterion: KO group ​ mRNA expression levels should be significantly reduced (usually >90%).

[0038] Western Blot (protein level verification) procedure: First, total protein is extracted from the skin of wild-type and gene knockout models, and its concentration is determined; then, equal volumes of protein samples are separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane; next, after blocking with a blocking buffer, a protein targeting Vaspin (…) is used. ​ The sample is incubated with a specific primary antibody for gene expression and then identified with a corresponding enzyme-labeled secondary antibody. Finally, it is developed by a chemiluminescence system and semi-quantitatively analyzed by analyzing the gray value of the target band. If the Vaspin protein band signal is significantly weakened or completely disappeared in the knockout sample, while the internal control (GAPDH) band shows no significant change, it indicates that the knockout efficiency at the protein level is good.

[0039] RT-qPCR and Western Blot results showed that ​ - / - mouse skin ​ Complete absence of mRNA and Vaspin protein expression, further confirmed by immunohistochemical staining, confirmed the absence of Vaspin protein expression in skin tissue. ​ ).

[0040] It should be noted that during the construction of the NPPK mouse model, ​ Gene knockout mice can be obtained from sources including, but not limited to, those constructed using the methods described above, or from commercially available sources. ​ Gene knockout mice, such as C57BL / 6JCya- from Cyagen (Suzhou) Biotechnology Co., Ltd. ​ em1 / Cya mice, product number: S-KO-16850.

[0041] II. Constructing an NPPK mouse model 1. Pretreatment Shave the 8-week-old baby with an electric shaver. ​ / Hair removal cream was applied to the back hair (2 cm × 3 cm area) of wild-type C57BL / 6J mice (half male and half female) and left for 1 minute to completely remove residual hair. (Purchased from Cyagen (Suzhou) Biotechnology Co., Ltd.)

[0042] 2. Chemical induction Four days after shaving, mice in each group underwent chemical induction treatment to establish a long island-type palmoplantar keratosis model. The specific steps were as follows: Acetone and ether were mixed in a 1:1 volume ratio. A cotton pad was thoroughly soaked in the mixture and gently applied to the shaved area on the mouse's back for 15 seconds. Immediately afterwards, a cotton pad soaked in double-distilled water (ddH2O) was gently pressed onto the same area for 30 seconds. Finally, residual liquid was blotted dry with sterile gauze. This treatment was performed twice daily (12-hour intervals) for 5 consecutive days, ultimately obtaining WT-AEW model mice and NPPK model mice (i.e.,...). ​ - / - -AEW mice).

[0043] 3. Model Evaluation 1) Macroscopic scoring: Observe the skin lesions of mice in each group daily and score them according to the following criteria: Erythema: 0 points = no erythema, normal skin color; 1 point: light pink, faintly visible; 2 points: red, clear borders; 3 points: dark red.

[0044] Surface roughness: 0 points = smooth skin; 1 point = slight texture, no bumps; 2 points = obviously rough, with visible fine particles; 3 points = significantly thickened skin, resembling pebbles or moss.

[0045] Scales: 0 points = no scales; 1 point = fine scales; 2 points = medium scales; 3 points = large amount of thick scales.

[0046] Cracks: 0 points = no cracks; 1 point = dry cracks but no breakage; 2 points = obvious linear breakage with no bleeding; 3 points = deep cracks with punctate or linear bleeding.

[0047] The skin manifestations of mice in each group are shown in Figure 4(a)). Compared with the WT-AEW group, ​ - / - The AEW group mice showed more severe skin damage, specifically increased skin surface roughness and increased erythema and scaling.

[0048] 2) Laboratory tests: After macroscopic scoring of mice in each group, HE, RT-qPCR and immunofluorescence detection were performed.

[0049] The HE method involves the following steps: Paraffin sections are thoroughly dewaxed and hydrated using xylene and graded ethanol to remove the embedding medium and allow water to enter the tissue. After pretreatment, the sections undergo hematoxylin staining (to stain the nuclei), differentiation (to remove non-specific background staining), bluing (to restore the bright blue color of the nuclei), and brief eosin counterstaining (to stain the cytoplasm and other components). After staining, the sections are rigorously dehydrated and cleared using graded ethanol and xylene. Finally, the sections are mounted with neutral resin, and the classic staining results can be observed under a microscope: the nuclei appear as a clear blue, while the cytoplasm, collagen fibers, and other components show varying degrees of pink or red.

[0050] The steps for the RT-qPCR method are the same as before.

[0051] The steps of the immunofluorescence detection method are as follows: (1) Preparation of paraffin sections: Take the skin lesions of each group of mice and immediately put them into 4% paraformaldehyde fixative. After fixation, the tissues are dehydrated by ethanol of low to high concentrations, and then placed in xylene for clearing. The tissues are then immersed in paraffin at 56℃. The paraffin-immersed tissues are placed in the embedding mold, melted paraffin is poured in, and the paraffin is placed in cold water to solidify into blocks. The paraffin blocks are cut into 4 μm thin slices using a microtome. After the slices are flattened in a warm water bath, they are picked up with a glass slide and attached. They are then dried in a 37℃ incubator. (2) Multicolor immunofluorescence staining: The paraffin sections are placed in xylene I and xylene II in sequence, and then dewaxed by ethanol of gradient concentrations. After the alcohol is dried, the sections are washed with water and rinsed with distilled water. The sections are placed in antigen retrieval solution (citric acid buffer with a pH of 6.0), boiled in a microwave oven for antigen retrieval for 20 minutes, cooled, and washed 3 times with PBS. The sections are then placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark for 15 minutes. The sections are then washed 3 times with PBS. Draw circles around the tissue with a histochemical pen, add blocking solution, and block at room temperature for 30 minutes. Discard the blocking solution, add diluted primary antibody, incubate overnight at 4°C, and place in a humidified chamber protected from light. The next day, wash the sections three times with PBS, add HRP secondary antibody of the corresponding species, incubate at room temperature protected from light for 50 minutes, and wash three more times with PBS. Add TYR fluorescent dye and react for 15 minutes, then wash three times with PBS. Repeat the antigen retrieval step, using different TYR fluorescent dyes for the next round of labeling. Finally, mount the sections with anti-fluorescence quenching mounting medium (containing DAPI), observe and photograph under a fluorescence microscope or other equipment.

[0052] 4. Results Compared to WT-AEW mice, ​ - / - -AEW mice exhibited more severe skin lesions, characterized by increased skin surface roughness and increased erythematous scaling (Figure 4(a)). HE staining analysis of the lesion tissue showed that... ​ - / - -AEW mice showed significant epidermal thickening, hyperkeratosis and parakeratosis, dermal capillary dilation (black arrows), and perivascular lymphocytic infiltration (Figure 4(b)). Further RT-qPCR analysis revealed that... ​ - / - Inflammatory factors (such as) in the skin tissue of AEW mice ​ The mRNA expression level was significantly upregulated (Figure 4(c)). Furthermore, immunofluorescence staining results showed that... ​ - / - The expression levels of inflammatory factors (Il-17a, Il-6, Il-1β, Mmp13) in AEW mice were significantly higher than those in WT-AEW mice, accompanied by increased Cd4+ cell infiltration (*). P <0.05, *** P <0.001, **** P<0.0001 (in Figure 4(d)). ​ - / - Transcriptomic analysis of AEW mouse skin showed significant upregulation of IL-17 signaling pathway-related inflammatory factors (Cxcl2, Mmp9, Mmp13, Il-1β, etc.) (Figure 4(e)). KEGG pathway enrichment analysis indicated that... ​ - / - -AEW mouse skin showed significant upregulation of the IL-17 signaling pathway and cytokine-cytokine receptor interaction pathway (Figure 4(f)). GSEA analysis further validated the activation of the IL-17 signaling pathway (Figure 4(g)). These results indicate that... ​ Gene deletion exacerbates skin inflammation, which provides a basis for further analysis. ​ This provides important experimental evidence for the molecular mechanisms involved in the regulation of skin inflammation.

[0053] III. Drug screening using the constructed NPPK mouse model 1. Experimental Grouping Blank control group: WT-AEW mice (n=6); Model control group: NPPK mice (n=6); Treatment group: NPPK mice (n=6) were given an IL-17A inhibitor.

[0054] 2. Evaluation Methods 1) Skin erythema, surface roughness, scaling, and fissure score (same as above).

[0055] 2) Skin barrier function: After 14 days of treatment, transepidermal water loss (unit: g·m) was measured in mice using a TEWL measuring instrument. - ²·h - ¹).

[0056] 3) Phenotypic analysis: First, the thickness of mouse epidermis was observed by HE staining; second, key inflammatory factors (such as...) were quantitatively analyzed using RT-qPCR technology. ​ The transcriptional levels of IL-17A and IL-6 were measured; finally, immunofluorescence staining was used to locate and assess cytokines such as IL-17A and IL-6, as well as CD4+. + The expression and infiltration of T cells in tissues.

[0057] 3. Results Compared with the blank control group, the TEWL value in the skin lesions of mice in the model control group was increased, and RT-qPCR quantification ( ​ ​ Increased expression of mRNAs such as Il-17a and Il-6; immunofluorescence staining results showed increased expression of inflammatory factors (Il-17a, Il-6, etc.) and CD4+. +T cell infiltration was significantly increased in both groups. Compared with the model control group, the TEWL value of the NPPK mice in the treatment group was lower than that in the model group. ​ Downregulation of mRNA expression and reduction in epidermal thickness indicate that the NPPK mice constructed using this method can be used for screening NPPK therapeutic drugs.

[0058] IV. Mechanistic studies using the constructed NPPK mouse model 1. Observe the abnormalities of keratinocytes and epidermal junctions in NPPK mice using transmission electron microscopy. Investigate epidermal differentiation abnormalities by labeling the basal layer (KRT14, KRT5), spinous layer (KRT10, KRT1), granular layer (IVL, TGM1), and stratum corneum (FLG) of the NPPK mouse epidermis using multicolor immunofluorescence staining; label Th17 cells (CD3+) using multicolor immunofluorescence and flow cytometry. + CD4 + IL-17A + ), neutrophils (CD45) + CD11b + CD16 + ), dendritic cells (CD11c + MHCII + ), macrophages (CD11b + CD68 + ), B cells (CD19) + CD20 + The number of infiltrating cells was counted to investigate the type of inflammation. Key downstream effector molecules of the IL-17 signaling pathway (IL-6, CXCL8, GM-CSF, etc.) were labeled to explore the role of the IL-17 signaling pathway in xeroderma modeling. ​ - / - Skin activation status in mice.

[0059] 2. Transcriptomic and proteomic sequencing were performed on the skin of NPPK and WT-AEW mice. Gene ontology enrichment analysis and KEGG pathway enrichment analysis were performed on differentially expressed genes to establish gene-protein association networks and to identify key molecules and pathways in the IL-17 pathway.

[0060] 3. Detection of IL-17 signaling pathway activation in NPPK mouse skin: Key molecules of the IL-17 signaling pathway, including initiation molecules (IL-17A, IL-17F), IL-17 receptor-related markers (IL-17R, Act1), and signal transduction molecules (Stat3, p-Stat3, p38, p-p38, c-Jun, pc-Jun, c-Fos, pc-Fos, etc.), were labeled using multicolor tissue immunofluorescence to investigate their expression levels, phosphorylation levels, and nuclear translocation. Western blotting was used to detect the protein expression and phosphorylation levels of IL-17 signaling molecules (STAT3, p-STAT3, p38, p-p38, c-Jun, pc-Jun, c-Fos, pc-Fos, etc.) in the epidermis of NPPK mice.

[0061] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for constructing a NPPK mouse model based on CRISPR / Cas9 technology, characterized in that, In Serpina12 The skin surface of the genetically deficient mice was sequentially applied with a mixture of acetone and ether and double distilled water to obtain the NPPK mouse model. The acetone-ether mixed solution is formed by mixing acetone and ether in a volume ratio of 1:

1.

2. The method for constructing a NPPK mouse model based on CRISPR / Cas9 technology according to claim 1, characterized in that, In Serpina12 The skin surface of the genetically deficient mouse was first applied with a mixture of acetone and ether for 15 seconds, and then applied with distilled water for 30 seconds. 3.The method of claim 1, wherein the NPPK mouse model is constructed based on CRISPR / Cas9 technology. The acetone-ether mixed solution and double distilled water are applied externally twice a day for 5 consecutive days. 4.The method for constructing a NPPK mouse model based on CRISPR / Cas9 technology according to any one of claims 1 to 3, wherein, The acetone-ether mixed solution and double distilled water are applied externally to induce the phenotype of increased roughness of the skin surface, increased erythema and scaling in mice, obtaining the NPPK mouse model.

5. The method for constructing a NPPK mouse model based on CRISPR / Cas9 technology according to any one of claims 1-3, wherein, After modeling, the model is evaluated by HE, RT-qPCR and immunofluorescence detection.

6. The method for constructing a NPPK mouse model based on CRISPR / Cas9 technology according to any one of claims 1-3, wherein, The Serpina12 Gene-deficient mice are targeted knockout mice by CRISPR / Cas9 gene editing technology Serpina12 Genes are obtained after; gRNA plasmids used in CRISPR / Cas9 gene editing include gRNA-A1, gRNA-A2, gRNA-B1 and gRNA-B2, and the nucleotide sequences are shown in SEQ ID NO. 1~SEQ ID NO. 4, respectively.

7. The NPPK mouse model obtained by the method of any one of claims 1-6.

8. The use of the NPPK mouse model obtained by the method of any one of claims 1-6 in the study of inflammatory keratotic skin diseases.

9. A method for screening a candidate drug for treating NPPK or IL-17 related inflammatory skin diseases, characterized by, The method comprises the following steps: 1) Take the NPPK mouse model of any one of claims 1-6 as the disease group, and set up a blank control group and a treatment group at the same time; 2) Give the test drug to the mice in the treatment group; 3) by comparing the skin macroscopic scores, TEWL values, epidermal thickness, inflammatory factor mRNA and protein expression levels, and CD4 + T cell infiltration in each group of mice, to evaluate the improvement effect of the test drug on NPPK or IL-17 related inflammatory skin diseases.

10. A research system for investigating molecular mechanisms of skin inflammation, characterized by, The NPPK mouse model of any one of claims 1-6 is used for the following analysis: Observe the ultrastructure of keratinocytes and the abnormality of epidermal junction complex by transmission electron microscopy; Detect the expression disorder of epidermal stratification markers by multicolor immunofluorescence staining; Detect the infiltration number of Th17 cells, neutrophils, dendritic cells and macrophages in the skin lesions by flow cytometry or immunofluorescence; Detect the change of the phosphorylation level of key molecules of the IL-17 signaling pathway by Western Blot or immunofluorescence.

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