Construction method of animal model of systemic lupus erythematosus

Abstract

The invention provides a construction method of a systemic lupus erythematosus animal model. The systemic lupus erythematosus animal model shows high autoantibody, low complement, high inflammatory factor and serious kidney injury. The systemic lupus erythematosus animal model provided by the invention is clear in pathogenesis, can cause innate immune and postnatal immune system abnormalities, is short in experimental period, and provides a new animal model for drug candidate screening, drug treatment effect evaluation and pathogenesis research of systemic lupus erythematosus.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and health, and relates to a method for constructing an animal disease model, in particular to a method for constructing an animal model of systemic lupus erythematosus and its application. Background Art

[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by abnormal production of autoantibody IgG, cytokine storm and neutropenia. Although SLE has been clinically recognized as an independent disease for over a hundred years, its pathogenesis remains incompletely understood. A large number of studies have shown that genetics, endocrine, infection, immune abnormalities and environmental factors are related to the onset of SLE. Existing medical means cannot completely cure SLE, and most patients can only control the condition by long-term application of hormones and immunosuppressants. Therefore, studying the pathogenesis of SLE is of great significance for understanding and treating SLE.

[0003] Animal models of human diseases are experimental objects and materials with manifestations similar to human diseases established in biomedical scientific research. Animal models help to more conveniently and effectively understand the occurrence and development laws of human diseases and study prevention and treatment measures. Female SPF-grade MRL / lpr mice are the most commonly used SLE models in the world at present, established by Murphy et al. in 1978. These mice are produced by complex mating of small numbers of LG / J, AKR / J, C3H, HeDi and C57BL / 6J strains. Due to the defect of the Fas gene in such mice, the mortality rate of T cells is reduced, and autoreactive lymphocytes cannot be cleared through the apoptotic pathway, resulting in swollen lymph nodes, enlarged spleen, and autoimmune disease symptoms, characterized by a large amount of anti-dsDNA, ANA antibodies, and severe glomerulonephritis. The onset is early and there is no gender difference. The MRL / lpr mouse mimics the functional abnormality caused by T cell apoptosis defect, characterized by swollen lymph and spleen. These two organs contain a large number of lymphocytes. Therefore, this model mainly mimics the abnormality of the adaptive immune system. And the innate immune system is normal, for example: IFN-alpha does not increase. Although MRL / lpr mice show increased anti-dsDNA antibodies, proteinuria, and hypocomplementemia, they do not fully conform to the characteristics of human diseases.

[0004] The NZB / NZW F1 mouse is the first-generation mouse resulting from the mating of NZB mice and NZW mice. It spontaneously exhibits a lupus phenotype, and neither of its parental strains shows lesions. It was discovered by Helyer in 1963. Symptoms of the disease in these model mice start to appear at 4 - 5 months, obvious symptoms of glomerulonephritis emerge at 5 - 6 months, and it progresses to severe lupus by 10 - 12 months, eventually leading to death due to renal failure. The disease symptoms in these mice are similar to those in humans, and sex hormones have a significant impact on these mice. Female mice develop the disease earlier and more severely than male mice. Its characteristics include high titers of anti-dsDNA and hypergammaglobulinemia. However, the reason for the lupus phenotype in NZB / NZW F1 mice is unknown, the disease onset cycle is long (6 months), it is susceptible to environmental factors, and the experimental process is not easy to control, which brings difficulties to the study of the disease pathogenesis.

[0005] Therefore, there is an urgent need to study a lupus model with a clear etiology, which can simultaneously cause abnormalities in both the innate and adaptive immune systems and has a short experimental cycle. Summary of the Invention

[0006] In view of this, in order to make up for the deficiencies of the prior art, the present invention is specifically proposed.

[0007] In a first aspect of the present invention, a method for constructing a non-human animal model of systemic lupus erythematosus is provided, and the construction method includes causing the deletion of Fth1 expression or activity in the non-human animal.

[0008] In some embodiments, the neutrophils of the non-human animal have a deletion of Fth1 expression or activity.

[0009] In some embodiments, the construction method further includes using a systemic lupus erythematosus-inducing drug to induce a non-human animal with a deletion of Fth1 expression or activity.

[0010] In the present invention, the Fth1 is ferritin heavy chain polypeptide 1.

[0011] In the present invention, the deletion of Fth1 expression refers to the reduction or loss of the Fth1 expression level, and the deletion of Fth1 activity refers to the reduction or loss of the Fth1 activity function. The effect of deleting Fth1 expression or activity can be achieved by knocking out and / or knocking down Fth1, or by administering an Fth1 inhibitor. No matter which method is used, as long as the effect of deleting Fth1 expression or activity is achieved, it falls within the protection scope of the present invention. In one embodiment of the present invention, the deletion of Fth1 expression or activity is achieved by knocking out and / or knocking down Fth1 in the non-human animal.

[0012] In some embodiments, the knockout and / or knockdown can be achieved using one or more of the following techniques, including but not limited to: Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS, phiC31, CRISPR-Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology, and interfering RNA.

[0013] In some embodiments, the techniques used for the knockout and / or knockdown are selected from CRISPR-Cas9 and Cre-LoxP.

[0014] In the present invention, there are 9 transcriptional variants of the Fth1 gene. Knocking out and / or knocking down the Fth1 gene can reduce or abolish the expression level or functional activity of any one or several of the transcriptional variants, preferably reducing or abolishing the expression level or functional activity of all transcriptional variants.

[0015] In some embodiments, the method for the knockout and / or knockdown includes the following steps:

[0016] Cross a non-human animal FTH1 containing the loxp sequence fl / fl with a Cre non-human animal with conditional knockout in neutrophils.

[0017] In some embodiments, using the CRISPR-Cas9 technology, loxp sequences are inserted upstream of exon 1 and downstream of exon 2 of the Fth1-203 transcript to obtain a non-human animal FTH1 fl / fl 。

[0018] In some embodiments, the Cre non-human animals with conditional knockout in neutrophils include Mrp8Cre non-human animals and LysMcre non-human animals.

[0019] In some embodiments, the Cre non-human animals with conditional knockout in neutrophils are selected from Mrp8Cre non-human animals.

[0020] In some embodiments, the method for the knockout and / or knockdown includes the following steps:

[0021] (1) Co-introduce a targeting vector containing homologous arms and loxp sequences, sgRNA, and Cas9-mRNA into the fertilized eggs of parental non-human animal 1;

[0022] (2) Transplant the fertilized eggs into a surrogate recipient to obtain F0 generation non-human animals through surrogacy;

[0023] (3) Backcross the F0 generation non-human animals with parental non-human animal 1 to obtain stably inherited F1 generation non-human animals FTH1 fl / fl;

[0024] (4) Cross the F1 generation non-human animal FTH1 fl / fl with a Cre non-human animal with conditional knockout of neutrophils to obtain an FTH1 fl / fl Mrp8cre + non-human animal.

[0025] In a specific embodiment, the sgRNA sequence is as shown in SEQ ID NO. 1-2.

[0026] In some embodiments, the method of co-introducing the targeting vector, sgRNA, and Cas9-mRNA into the fertilized eggs of the parental non-human animal includes, but is not limited to, electroporation, calcium phosphate method, liposome method, DEAE-dextran method, microinjection, and virus infection.

[0027] In a specific embodiment, the method is selected from microinjection.

[0028] In some embodiments, the vector includes, but is not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus, poxvirus, baculovirus, papillomavirus, polyomavirus, phage, and plasmid.

[0029] In some embodiments, the systemic lupus erythematosus inducing drug includes, but is not limited to, imiquimod, Pristane, peptide, lipopolysaccharide, Campylobacter jejuni, Freund's complete adjuvant, and lymphocyte active chromatin, alone or in combination.

[0030] In some embodiments, the systemic lupus erythematosus inducing drug is selected from imiquimod and Pristane.

[0031] In some embodiments, the dosage of imiquimod is 10-50 mg / ear.

[0032] In a specific embodiment, the dosage of imiquimod is 25 mg / ear.

[0033] In some embodiments, the frequency of using imiquimod is 1-5 times per week.

[0034] In a specific embodiment, the frequency of using imiquimod is 3 times per week.

[0035] In some embodiments, the use time of imiquimod lasts for 2-10 weeks and has or shows the characteristics of systemic lupus erythematosus disease or symptoms within 2-10 weeks.

[0036] In a specific embodiment, the use time of imiquimod lasts for 6 weeks and has or shows the characteristics of systemic lupus erythematosus disease or symptoms at 6 weeks.

[0037] In some embodiments, the method of using Pristane is a single injection of 0.2 - 1 ml.

[0038] In a specific embodiment, the method of using Pristane is a single injection of 0.5 ml.

[0039] In some embodiments, within 1 - 6 months after using Pristane, it has or shows the characteristics of systemic lupus erythematosus disease or symptoms.

[0040] In the present invention, the characteristics of the systemic lupus erythematosus disease or symptoms include but are not limited to congenital immune system abnormalities, acquired immune system abnormalities, elevated specific autoantibodies, reduced complement, elevated inflammatory factor levels, kidney damage, skin damage, joint damage, and central nervous system damage.

[0041] In the present invention, the non - human animal model refers to a non - human animal that has or shows the characteristics of a disease or symptoms.

[0042] In some embodiments, the non - human animal is a mammal.

[0043] In some embodiments, the mammals include but are not limited to rodents, carnivores, chiropterans, erinaceomorphs, insectivores.

[0044] In some embodiments, the mammal is selected from rodents.

[0045] In some embodiments, the rodents include but are not limited to Calomyscidae, Cricetidae, Muridae, Nesomyidae, Platacanthomyidae, Talpidae, Echimyidae, Petromuridae.

[0046] In some embodiments, the rodent is selected from Muridae.

[0047] In the present invention, the Muridae family includes but is not limited to black rats, brown rats, gerbils, New World rats, Old World rats, SD rats, Polynesian rats, tree rats, wood rats, stick rats, rice rats, kangaroo rats, climbing rats, shrew mice, Sri Lankan mice, Sikkim mice, Javan mice, Indian mice, field mice, brown mice, Kuhl's mice, Cypriot mice, South Indian mice, Thai mice, Yugoslavian mice, house mice, Burmese field mice, Hungarian mice, Mediterranean mice, earth - colored mice, Ivory Coast mice, toad mice, Angolan mice, highland mice, Botswana mice, dressed mice, Somali mice, Ghanaian mice, South African mice, pleasing mice, shrew - shaped mice, valley mice, Central African mice, bristly mice, Zambian mice, Ugandan mice, delicate mice, sea - god mice, Freudenthal's mice, Fea's mice, flat - haired mice, cave mice, Shaw's mice.

[0048] In some embodiments, the murine is selected from Mus musculus.

[0049] In some embodiments, the Mus musculus includes, but is not limited to, C57BL / 6 sub-strain mice.

[0050] In some embodiments, the C57BL / 6 sub-strain mice are selected from C57BL / 6JGpt sub-strain mice.

[0051] The second aspect of the present invention provides any one of the following methods:

[0052] (1) A method for screening drug candidates for the treatment of systemic lupus erythematosus, the method comprising:

[0053] a) Administering a reagent to be screened to a non-human animal with systemic lupus erythematosus prepared by the construction method described in the first aspect of the present invention;

[0054] b) Detecting the therapeutic effect of the reagent to be screened on systemic lupus erythematosus;

[0055] (2) A method for evaluating the therapeutic effect of a drug for the treatment of systemic lupus erythematosus, the method comprising:

[0056] a) Administering a drug to a non-human animal with systemic lupus erythematosus prepared by the construction method described in the first aspect of the present invention;

[0057] b) Detecting the therapeutic effect of the systemic lupus erythematosus on the drug;

[0058] (3) A method for studying the pathogenesis of systemic lupus erythematosus, which is to use a non-human animal with systemic lupus erythematosus prepared by the construction method described in the first aspect of the present invention to study the pathogenesis of systemic lupus erythematosus.

[0059] In the present invention, the term "treatment" refers to administering a compound or composition to control the progression of a disease. The control of the progression of a disease should be understood as achieving a beneficial or desired clinical outcome, including but not limited to alleviating symptoms, reducing the duration of the disease, stabilizing the pathological state (especially avoiding additional deterioration), delaying the progression of the disease, improving the pathological state, and remission (partial and complete). Compared with the expected survival without treatment, the control of the progression of a disease also involves an extension of the survival period.

[0060] In the present invention, drug candidates can be obtained from a vast variety of sources, including but not limited to synthetic, naturally occurring, or recombinantly produced molecules, including small molecules, peptides, antibodies, or other polypeptides. For example, a variety of organic compounds and biomolecules can be synthesized randomly and directionally, or natural compound libraries in the form of bacterial, fungal, plant, or animal extracts, or natural or synthetic libraries and compounds modified by conventional chemical, physical, or biochemical means, or known pharmacological agents can be chemically modified directionally or randomly, such as acylation, alkylation, esterification, amidation, etc. to generate structural analogs.

[0061] The third aspect of the present invention provides any one of the following applications:

[0062] (1) The application of the systemic lupus erythematosus non-human animal model prepared by the construction method described in the first aspect of the present invention in screening drug candidates for the treatment of systemic lupus erythematosus;

[0063] (2) The application of the systemic lupus erythematosus non-human animal model prepared by the construction method described in the first aspect of the present invention in evaluating the therapeutic effect of drugs for the treatment of systemic lupus erythematosus;

[0064] (3) The application of the systemic lupus erythematosus non-human animal model prepared by the construction method described in the first aspect of the present invention in studying the pathogenesis of systemic lupus erythematosus.

[0065] The fourth aspect of the present invention provides the application of Fth1 in constructing a systemic lupus erythematosus non-human animal model.

[0066] In some embodiments, a systemic lupus erythematosus non-human animal model is constructed by deleting the expression or activity of Fth1 in a non-human animal.

[0067] In a specific embodiment, a systemic lupus erythematosus non-human animal model is constructed by deleting the expression or activity of Fth1 in neutrophils of a non-human animal.

[0068] In some embodiments, the non-human animal is a mammal.

[0069] In some embodiments, the mammals include but are not limited to rodents, carnivores, chiropterans, erinaceomorphs, and insectivores.

[0070] In some embodiments, the mammal is selected from rodents.

[0071] In some embodiments, the rodents include but are not limited to Calomyscidae, Cricetidae, Muridae, Nesomyidae, Platacanthomyidae, Talpidae, Echimyidae, and Petromuridae.

[0072] In some embodiments, the rodent is selected from the Muridae.

[0073] In the present invention, the Muridae includes, but is not limited to, black rats, brown rats, gerbils, New World rats, Old World rats, Sprague-Dawley rats, Polynesian rats, tree rats, wood rats, stick rats, rice rats, kangaroo rats, climbing mice, shrew mice, Sri Lankan mice, Sikkim mice, Javan mice, Indian mice, field mice, brown mice, Kuhl's mice, Cypriot mice, South Indian mice, Thai mice, Yugoslavian mice, house mice, Burmese field mice, Hungarian mice, Mediterranean mice, earth-colored mice, Ivory Coast mice, toad mice, Angolan mice, hill mice, Botswana mice, masked mice, Somali mice, Ghanaian mice, South African mice, pleasing mice, shrew-like mice, valley mice, Central African mice, bristly mice, Zambian mice, Ugandan mice, delicate mice, sea god mice, Freund's mice, Phe's mice, flat-haired mice, cave mice, Shaw's mice.

[0074] In some embodiments, the Muridae is selected from house mice.

[0075] In some embodiments, the house mice include C57BL / 6 sub-line mice.

[0076] In some embodiments, the C57BL / 6 sub-line mice are selected from C57BL / 6JGpt sub-line mice.

[0077] Advantages and beneficial effects of the present invention:

[0078] The present invention provides a method for constructing an animal model of systemic lupus erythematosus, and the animal model of systemic lupus erythematosus exhibits high autoantibodies, low complement, high inflammatory factors, and severe kidney damage. The animal model of systemic lupus erythematosus provided by the present invention has a clear etiology, can simultaneously cause abnormalities in the innate and adaptive immune systems, and has a short experimental period, providing a new animal model for the screening of drug candidates for systemic lupus erythematosus, the evaluation of drug treatment effects, and the study of pathogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 is a heat map of the protein profile of neutrophils in patients with systemic lupus erythematosus;

[0080] Figure 2 is a hybridization strategy diagram;

[0081] Figure 3 is a comparison diagram of skin lesions after 6 weeks of imiquimod induction in mice with conditional knockdown of FTH1 in neutrophils (FTH1 fl / fl Mrp8cre + ) and control mice without knockdown of FTH1, Wild Type (FTH1 fl / fl );

[0082] Figure 4 are schematic diagrams of the spleen sizes of mice with conditional knockdown of FTH1 in neutrophils (FTH1 fl / fl Mrp8cre + ) and control mice without knockdown of FTH1, Wild Type (FTH1 fl / fl ) after 6 weeks of imiquimod induction;

[0083] Figure 5 are schematic diagrams of the lymph nodes of mice with conditional knockdown of FTH1 in neutrophils (FTH1 fl / fl Mrp8cre + ) and control mice without knockdown of FTH1, Wild Type (FTH1 fl / fl ) after 6 weeks of imiquimod induction;

[0084] Figure 6 are diagrams showing the deposition of immune complexes in the kidneys of mice with conditional knockdown of FTH1 in neutrophils (FTH1 fl / fl Mrp8cre + ) and control mice without knockdown of FTH1, Wild Type (FTH1 fl / fl ) after 6 weeks of imiquimod induction. Red represents IgG, yellow represents IgM, green represents C1q, blue represents DNA, and Merge represents the overlapping of all colors;

[0085] Figure 7 are comparison charts of the levels of inflammatory factors in the plasma of mice with conditional knockdown of FTH1 in neutrophils (FTH1 fl / fl Mrp8cre + ) and control mice without knockdown of FTH1, Wild Type (FTH1 fl / fl ) after 6 weeks of imiquimod induction;

[0086] Figure 8 are comparison charts of the levels of anti - double - stranded DNA antibodies (anti - dsDNA), anti - nuclear antibodies (anti - ANA), and complement C3 in the plasma of mice with conditional knockdown of FTH1 in neutrophils (FTH1 fl / fl Mrp8cre + ) and control mice without knockdown of FTH1, Wild Type (FTH1 fl / fl ) after 6 weeks of imiquimod induction;

[0087] Figure 9 are mice with conditional knockdown of FTH1 in neutrophils (FTH1 fl / fl Mrp8cre +), and the control mice without knocking down FTH1, Wild Type (FTH1 fl / fl ), the comparison diagram of skin lesions of mice after 6 months of Pristane stimulation;

[0088] Figure 10 are the mice with conditional knockdown of FTH1 in neutrophils (FTH1 fl / fl Mrp8cre + ), and the control mice without knocking down FTH1, Wild Type (FTH1 fl / fl ), the comparison diagram of the content of inflammatory factors in the plasma of mice after 6 months of Pristane stimulation. Detailed implementation manners

[0089] The present invention will be further described below in conjunction with embodiments. The following description is only for the preferred embodiments of the present invention, and does not limit the present invention in other forms. Any person skilled in the art may use the disclosed technical content to change it into an equivalent embodiment with the same change. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention's solution falls within the protection scope of the present invention.

[0090] Example 1 Identification of the expression content of Ferritin protein in neutrophils of patients with systemic lupus erythematosus

[0091] Isolation of neutrophils: Collect the peripheral blood of 5 healthy people and 4 SLE patients using K2E (EDTA) tubes (BD Vacutainer). To isolate neutrophils, first dilute the peripheral blood in equal proportion with phosphate buffered saline for standby. Pour an appropriate amount of Ficoll-Hypaqu separation solution into a 15 ml centrifuge tube, and then lay the diluted peripheral blood flat on the upper layer of the separation solution, taking care not to damage the interface of the separation solution. Then perform centrifugal separation. The rotation speed is 500G, the acceleration is the maximum, the deceleration is the minimum, and the centrifugation is for 20 minutes at room temperature. After centrifugation, use a pipette to aspirate the white precipitate above the red blood cells, which is the neutrophils. Next, lyse the red blood cells with lysis buffer (BD), and centrifuge again at a rotation speed of 500G for 10 minutes, and retain the neutrophils at the bottom of the centrifuge tube. Verify this method by flow cytometry using CD16 (Biosciences) and CD11b (Biolegend) antibodies, and the purity of neutrophils is greater than 98%. Immediately perform flow cytometry staining analysis on the isolated neutrophils, or store them in a -80 °C refrigerator.

[0092] Western blot: Neutrophils were lysed on ice using RIPA buffer (Huaxingbio) in combination with a protease inhibitor and phosphatase inhibitor mixture (Thermo Fisher Scientific). The protein concentration of the lysate was determined using a BCA protein assay kit (Thermo Fisher Scientific). The RIPA lysate was boiled with loading buffer at 95°C for 10 minutes, followed by electrophoresis and then transferred to a PVDF membrane (Biorad). The membrane was blocked and then incubated with anti-Ferritin (abcam 1:1000) antibody overnight at 4°C. The membrane was washed and incubated with anti-rabbit IgG-HRP (1:5000) for 1 hour. Protein bands were visualized using a Western blot detection system Tanon-5200 (Bio-Tanon, Inc.). Gray value analysis was performed using ImageJ (1.50g, NIH) software. The results showed that the expression of Ferritin heavy chain in neutrophils of systemic lupus erythematosus decreased and recovered after treatment ( Figure 2 and 3 ).

[0093] Proteomics: Protein extraction and digestion: Samples were added with 300 μL of 8M urea lysis buffer (containing 10% protease inhibitor), centrifuged (14,100×g, 20 minutes), and the supernatant was taken. The concentration was determined by Bradford method and stored at -80°C. 50 μg of protein was reduced with 200 mM DTT (37°C, 1 hour), diluted 8-fold, and then trypsin (1:25) was added for overnight digestion at 37°C. Reversed-phase separation: After digestion was terminated, the C18 column was washed with acetonitrile and 0.1% formic acid in sequence. After sample loading, it was eluted with an aqueous solution of pH 10 gradient (6%-50% acetonitrile), and combined into three groups for lyophilization. LC-MS / MS analysis: The lyophilized sample was redissolved in 2% methanol / 0.1% formic acid, centrifuged and loaded onto the column. Mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) were used for gradient separation (4%-99% B, 120 minutes). Mass spectrometry parameters included full scan (250-1450 m / z, resolution 120,000), CID fragmentation (collision energy 30%), and dynamic exclusion for 18 seconds. The data was processed by Maxquant (Uniprot_HUMAN database). Identification criteria: Parent ion / fragment ion mass error ±15 ppm / ±0.5 Da, fixed modification of cysteine alkylation, variable modification of methionine oxidation.

[0094] Result analysis: After performing quantile normalization on the data using the Limma software package in R language, differential protein expression analysis was carried out. The screening criteria for differentially expressed proteins were set as P value < 0.05 and |log2 fold change| ≥ 1.1. Molecules related to the ferroptosis pathway in cells were screened through the FerrDb ferroptosis database and sorted according to the protein expression level. It can be seen that the decrease in FTH1 in neutrophils of SLE patients is the most obvious ( Figure 1 ).

[0095] Example 2 Experimental protocol for generating mice with conditional knockdown of Ferritin gene in neutrophils

[0096] I. Gene target design

[0097] There are 9 transcriptional variants of the Fth1 gene. The following knockout regions were selected through structural analysis:

[0098] Fth1-203 transcript (ENSMUST00000235196.1): Exon 1-2 segment (this region contains the coding sequence of the start codon ATG, and knocking it out will completely disrupt the protein function)

[0099] II. Gene editing process

[0100] 1) Construction of the CRISPR / Cas9 system: It includes a gene editing system (including sgRNA, Cas9-mRNA) and a donor vector (including homologous arms and loxp sequences).

[0101] 2) Microinjection: Inject the CRISPR / Cas9 system into the fertilized eggs of C57BL / 6JGpt strain mice.

[0102] 3) Embryo transfer: Obtain F0 generation mice through surrogacy.

[0103] 4) Gene verification: Screen positive F0 individuals by PCR amplification combined with sequencing.

[0104] 5) Strain cultivation: Backcross positive F0 with C57BL / 6JGpt to establish a stably inherited F1 generation model FTH1 fl / fl .

[0105] In the present invention, using the CRISPR / Cas9 technology, two loxp elements were respectively knocked into the upstream of exon 1 and the downstream of exon 2 of the Fth1 gene. According to the selected sgRNA (sequences are shown in Table 1 SEQ ID NO.1-2), a targeting vector was designed, including homologous arms and loxp sequences. After construction, it was digested with enzymes, purified, and co-microinjected into the fertilized eggs of C57BL / 6JGpt mice together with sgRNA and Cas9-mRNA. After injection, the fertilized eggs were transplanted into the fallopian tubes of surrogate recipient mice.

[0106] Table 1 sgRNA sequences

[0107]

[0108] III. Breeding of Fth1 specific knockout model

[0109] The cre mice for neutrophil conditional knockout are Mrp8Cre and LysMcre. In this experiment, Mrp8Cre (also known as S100A8-iCre) mice were purchased from Jicuiyaokang. The S100A8 gene (MRP8) of these mice has a nuclear-localized Cre recombinase. These mice are alive, fertile, of normal size, and do not show any obvious physical or behavioral abnormalities. These Mrp8Cre mice can be used in the Cre-loxp system to study neutrophils and innate immune responses. Cross the FTH1 fl / fl mice with Mrp8Cre mice, and the crossing strategy is as Figure 2 shown in A. By identifying the hybridized mice ( Figure 2 B), mice with defective FTH1 in myeloid-derived neutrophils were finally obtained. The expression of FTH1 was knocked down in the neutrophils of these mice, promoting ferroptosis of neutrophils.

[0110] Example 3 Phenotypic identification of neutrophil conditional knockdown mice

[0111] FTH1 fl / fl and FTH1 fl / fl Mrp8cre + mice need to be intervened with drugs. Female 8-week-old mice were selected for systemic lupus erythematosus induction.

[0112] Induction protocol 1: Use imiquimod cream (Mingxin Lidi, a product of Sichuan Mingxin Pharmaceutical Co., Ltd.), apply it to the ear skin of mice at a dose of 25 mg / ear, 3 times a week, and euthanize the mice after 6 weeks. The control group used an equal amount of vaseline instead of IMQ.

[0113] Induction protocol 2 (Pristane): Inject Pristane (sigma company) intraperitoneally at a dose of 0.5 ml, and euthanize the mice and collect samples after 6 months.

[0114] Enzyme-linked immunosorbent assay: Mouse serum was diluted 1:100 with assay buffer for detection of anti-dsDNA antibody, 1:100 for detection of anti-ANA antibody, and 1:25,000 for detection of complement C3. Assays were performed using a mouse anti-dsDNA IgG ELISA kit (5120, Alpha Diagnostic), an anti-ANA antibody ELISA kit (5210, Alpha Diagnostic), and a complement 3 ELISA kit (6270, Alpha Diagnostic) according to the manufacturer's instructions.

[0115] Immunofluorescence: Kidneys from mice were frozen at -80 °C in OCT compound (Tissue-Tek) for evaluation of immune complex deposition. Frozen kidneys were sectioned longitudinally at 6 μm thickness, and 6-μm cryosections of kidney tissue were treated with blocking buffer (100 mM Tris-HCl, pH 8.0, 0.3% Triton X-100, 2% BSA, and 50 μg / ml goat non-specific IgG) for 1 hour. The tissue was then stained with Alexa Fluor 594 goat anti-mouse IgG (1:200), goat anti-mouse IgM Alexa Fluor 647 (1:200), or rabbit anti-C1q antibody (1:200) plus goat anti-rabbit IgG Alexa Fluor 488 (1:500), washed three times, and air-dried. A drop of DAPI reagent containing anti-quencher (Solarbio) was added to the sections, and finally, the coverslips were sealed for later use. All slides were observed using an LSM 980 / Axio Observer 7 Zeiss confocal laser microscope. Images were optimized using ZEN (Zeiss).

[0116] Multiplex detection: Cytokines in mouse plasma were measured using a LEGENDplexTM mouse Th cytokine panel (Biolegend) according to the manufacturer's instructions. Data analysis was performed using LEGENDplexTM data analysis software (version 8.0).

[0117] After imiquimod induction, skin lesions appeared on the heads of mice, and FTH1 fl / fl Mrp8cre + produced larger and more severe skin lesions ( Figure 3 ); the spleens of mice were enlarged, and FTH1 fl / fl Mrp8cre + had a larger spleen and a more severe SLE disease response compared to Wild Type ( Figure 4 ); the submandibular and axillary lymph nodes of mice were enlarged, and FTH1 fl / flMrp8cre + Compared with WildType, the lymph nodes are larger and show a more severe SLE disease response ( Figure 5 ); FTH1 fl / fl Mrp8cre + deposition of renal immune complexes IgG, IgM, and C1q occurred in Mrp8cre mice ( Figure 6 ); FTH1 fl / fl Mrp8cre + After modeling, the inflammatory factors in Mrp8cre mice were higher than those in the control mice ( Figure 7 ); FTH1 fl / fl Mrp8cre + In Mrp8cre mice, anti-dsDNA and anti-ANA were significantly increased, and Complement 3 was significantly decreased. This indicates an exacerbation of the systemic lupus erythematosus phenotype ( Figure 8 ).

[0118] After Pristane induction, the back hair of wild-type mice was slightly sparse, and in FTH1 fl / fl Mrp8cre + Mrp8cre mice, the skin lesion area on the back and head was larger ( Figure 9 ); FTH1 fl / fl Mrp8cre + After modeling, the inflammatory factors in Mrp8cre mice were higher than those in the control mice ( Figure 10 ).

[0119] In summary, FTH1 fl / fl Mrp8cre + After induction of systemic lupus erythematosus (induced by imiquimod IMQ / Pristane), Mrp8cre mice showed higher autoantibodies, lower complement, higher inflammatory factors, and more severe kidney damage. At the same time, the decrease in neutrophil Ferritin in these mice was similar to the neutrophil expression characteristics in human SLE. In conclusion, these results indicate that the absence of Ferritin in neutrophils can induce a significant exacerbation of the systemic lupus erythematosus phenotype, and thus it is a new type of systemic lupus erythematosus mouse model.

[0120] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a non-human animal model of systemic lupus erythematosus, characterized in that, The construction method includes making the expression or activity of Fth1 in the non-human animal absent; Preferably, the expression or activity of Fth1 in neutrophils of the non-human animal is absent; Preferably, the construction method further includes using a systemic lupus erythematosus inducing drug to induce a non-human animal with absent Fth1 expression or activity.

2. The construction method according to claim 1, characterized in that, Making the expression or activity of Fth1 in the non-human animal absent includes knocking out and / or knocking down Fth1 in the non-human animal; Preferably, one or several of the following techniques can be used for the knocking out and / or knocking down: Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS, phiC31, CRISPR-Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology, interfering RNA; Preferably, the technique used for the knocking out and / or knocking down is selected from CRISPR-Cas9 and Cre-LoxP.

3. The construction method according to claim 2, characterized in that, The method for the knocking out and / or knocking down includes the following steps: Cross a non-human animal FTH1 containing loxp sequences fl / fl with a Cre non-human animal with conditional knockout of neutrophils; Preferably, using the CRISPR-Cas9 technology, loxp sequences are inserted upstream of exon 1 and downstream of exon 2 of the Fth1-203 transcript to obtain the non-human animal FTH1 fl / fl .

4. The construction method according to claim 3, wherein The Cre non-human animals with conditional knockout of neutrophils include Mrp8Cre non-human animals and LysMcre non-human animals; Preferably, the Cre non-human animals with conditional knockout of neutrophils are selected from Mrp8Cre non-human animals.

5. The construction method according to claim 4, wherein The method for the knocking out and / or knocking down includes the following steps: (1) Co-introduce a targeting vector containing homologous arms and loxp sequences, sgRNA, and Cas9-mRNA into the fertilized eggs of parental non-human animals 1; (2) Transplant the fertilized eggs into a surrogate recipient to obtain F0 generation non-human animals through surrogacy; (3) Backcross the F0 generation of non-human animals with the parental non-human animal 1 to obtain the stably inherited F1 generation of non-human animals FTH1 fl / fl ; (4) Cross the F1 generation of non-human animals with FTH1 fl / fl with Cre non-human animals with conditional knockout of neutrophils to obtain FTH1 fl / fl Mrp8cre + non-human animals; Preferably, the sgRNA sequence is as shown in SEQ ID NO.1-2.

6. The construction method according to claim 1, characterized in that The systemic lupus erythematosus inducing drug includes one or several of imiquimod, Pristane, peptides, lipopolysaccharides, Campylobacter jejuni, Freund's complete adjuvant, and lymphocyte active chromatin; Preferably, the systemic lupus erythematosus inducing drug is selected from imiquimod and Pristane; Preferably, the dosage of imiquimod is 10-50 mg / ear; Preferably, the dosage of imiquimod is 25 mg / ear; Preferably, the frequency of using imiquimod is 1-5 times per week; Preferably, the frequency of using imiquimod is 3 times per week; Preferably, the duration of using imiquimod lasts for 2-10 weeks; Preferably, the duration of using imiquimod lasts for 6 weeks; Preferably, the usage method of Pristane is a single injection of 0.2-1 ml; Preferably, the usage method of Pristane is a single injection of 0.5 ml.

7. The construction method according to claim 1, characterized in that The non-human animal model refers to a non-human animal having or showing the characteristics of a disease or medical condition; Preferably, the non-human animal is a mammal; Preferably, the mammals include rodents, carnivores, chiropterans, erinaceomorphs, and insectivores; Preferably, the mammals are selected from rodents; Preferably, the rodents include Calomyscidae, Cricetidae, Muridae, Nesomyidae, Platacanthomyidae, Talpidae, Echimyidae, and Petromuridae; Preferably, the rodent is selected from Muridae; Preferably, the Muridae includes black rat, brown rat, gerbil, New World rat, Old World rat, SD rat, Polynesian rat, tree rat, wood rat, stick rat, rice rat, kangaroo rat, climbing mouse, shrew mouse, Sri Lankan mouse, Sikkim mouse, Javan mouse, Indian mouse, field mouse, brown mouse, Kuhl's mouse, Cypriot mouse, South Indian mouse, Thai mouse, Yugoslavian mouse, house mouse, Burmese mouse, Hungarian mouse, Mediterranean mouse, earth-colored mouse, Ivory Coast mouse, toad mouse, Angolan mouse, upland mouse, Botswana mouse, dressed mouse, Somali mouse, Ghanaian mouse, South African mouse, pleasing mouse, shrew-shaped mouse, valley mouse, Central African mouse, bristly mouse, Zambian mouse, Ugandan mouse, delicate mouse, Poseidon mouse, Freund's mouse, Philip's mouse, flat-haired mouse, cave mouse, Shaw's mouse; Preferably, the Muridae is selected from house mouse; Preferably, the house mouse includes C57BL / 6 sub-line mice; Preferably, the C57BL / 6 sub-line mice are selected from C57BL / 6JGpt sub-line mice.

8. Any one of the following methods: (1)A method for screening drug candidates for the treatment of systemic lupus erythematosus, characterized in that, The method includes: a) Administering the reagent to be screened to a systemic lupus erythematosus non-human animal prepared by the construction method according to any one of claims 1-7; b) Detecting the therapeutic effect of the reagent to be screened on systemic lupus erythematosus; (2) A method for evaluating the therapeutic effect of a drug for treating systemic lupus erythematosus, characterized in that the method includes: a) Administering the drug to a systemic lupus erythematosus non-human animal prepared by the construction method according to any one of claims 1-7; b) Detecting the therapeutic effect of the drug on the systemic lupus erythematosus; (3) A method for studying the pathogenesis of systemic lupus erythematosus, characterized in that the method is to use a systemic lupus erythematosus non-human animal prepared by the construction method according to any one of claims 1-7 to study the pathogenesis of systemic lupus erythematosus.

9. Any one of the following applications: (1) Application of a systemic lupus erythematosus non-human animal model prepared by the construction method according to any one of claims 1-7 in screening drug candidates for treating systemic lupus erythematosus; (2) Application of a systemic lupus erythematosus non-human animal model prepared by the construction method according to any one of claims 1-7 in evaluating the therapeutic effect of a drug for treating systemic lupus erythematosus; (3) Application of a systemic lupus erythematosus non-human animal model prepared by the construction method according to any one of claims 1-7 in studying the pathogenesis of systemic lupus erythematosus.

10. Application of Fth1 in constructing a systemic lupus erythematosus non-human animal model; Preferably, a systemic lupus erythematosus non-human animal model is constructed by deleting the expression or activity of Fth1 in a non-human animal; Preferably, a systemic lupus erythematosus non-human animal model is constructed by deleting the expression or activity of Fth1 in neutrophils of a non-human animal; Preferably, the non-human animal is a mammal; Preferably, the mammal includes rodents, carnivores, chiropterans, erinaceomorphs, and insectivores; Preferably, the mammal is selected from rodents; Preferably, the rodents include Calomyscidae, Cricetidae, Muridae, Nesomyidae, Platacanthomyidae, Talpidae, Echimyidae, and Petromuridae; Preferably, the rodent is selected from Muridae; Preferably, the Muridae includes black rats, brown rats, gerbils, New World rats, Old World rats, SD rats, Polynesian rats, tree rats, wood rats, stick rats, rice rats, kangaroo rats, climbing mice, shrew mice, Sri Lankan mice, Sikkim mice, Javan mice, Indian mice, field mice, brown mice, Kuhl's mice, Cypriot mice, South Indian mice, Thai mice, Yugoslavian mice, house mice, Burmese field mice, Hungarian mice, Mediterranean mice, earth-colored mice, Ivory Coast mice, toad mice, Angolan mice, upland mice, Botswana mice, dressed mice, Somali mice, Ghanaian mice, South African mice, gratifying mice, shrew-shaped mice, valley mice, Central African mice, bristly mice, Zambian mice, Ugandan mice, delicate mice, sea god mice, Freund's mice, Phayre's mice, flat-haired mice, cave mice, and Shaw's mice; Preferably, the Muridae is selected from house mice; Preferably, the house mouse includes C57BL / 6 sub-line mice; Preferably, the C57BL / 6 sub-line mice are selected from C57BL / 6JGpt sub-line mice.

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