A method for constructing an animal model of systemic lupus erythematosus

Abstract

The application provides a method for constructing an animal model of systemic lupus erythematosus, and the animal model of systemic lupus erythematosus shows high autoantibody, low complement, high inflammatory factor and severe kidney damage. The animal model of systemic lupus erythematosus provided by the application has a clear cause, can cause abnormalities of innate immunity and acquired immunity systems, and has a short experimental cycle, thereby providing a new animal model for drug candidate screening, drug treatment effect evaluation and pathogenesis research of systemic lupus erythematosus.

Description

TECHNICAL FIELD

[0001] The application 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 a systemic lupus erythematosus animal model and application thereof. BACKGROUND

[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by abnormal production of autoantibody IgG, inflammatory factor storm and neutropenia. Although SLE has been an independent disease for more than a hundred years in clinic, the cause of the disease is still not fully clear. A large number of studies have shown that genetics, endocrine, infection, immune abnormalities and environmental factors are related to the onset of SLE. The existing medical means cannot completely cure SLE, and most patients can only control the disease condition by long-term application of hormones and immunosuppressive agents. Therefore, it is of great significance to study the pathogenesis of SLE for understanding and treating SLE.

[0003] Animal models of human diseases are animal experimental objects and materials with human disease mimetic performance established in biomedical scientific research. Animal models help to more conveniently and effectively understand the occurrence, development rules and research prevention and control measures of human diseases. Female SPF MRL / lpr mice are the most commonly used SLE model in the world at present, which were established by Murphy et al. in 1978. The mice are produced by complex crossbreeding of LG / J, AKR / J, C3H, HeDi and C57BL / 6J strains. Due to the Fas gene defect, the T cell death rate of the mice is reduced, the self-reactive lymphocytes cannot be cleared through the apoptosis pathway, the lymph nodes are enlarged, the spleen is enlarged, and the autoimmune disease symptoms are produced, characterized by a large number of anti-dsDNA, ANA antibodies, and severe glomerulonephritis, early onset, and no gender difference. The MRL / lpr mouse simulates the functional abnormalities caused by T cell apoptosis defects, which is characterized by lymph node and spleen enlargement. The two organs contain a large number of lymphocytes, so the model mainly simulates the abnormalities of the adaptive immune system. The innate immune system is normal, for example, IFN-alpha does not increase. Although the MRL / lpr mouse shows anti-dsDNA antibody elevation, proteinuria, and low complementemia, it does not fully meet the characteristics of human diseases.

[0004] NZB / NZW F1 mice are the first generation of mice after mating of NZB mice and NZW mice, and spontaneously appear the lupus phenotype, and neither of the parents develops the disease. It was discovered by Helyer in 1963. The model mice begin to develop symptoms at 4-5 months, and obvious symptoms of glomerulonephritis appear at 5-6 months, and progress to severe lupus from 10-12 months, and die due to renal failure. The symptoms of such mice are similar to those of humans, and sex hormones have a significant effect on the mice, and female mice develop earlier and more severely than male mice. The characteristics are high titer anti-dsDNA and hyperglobulinemia. However, the reason why NZB / NZW F1 mice exhibit the lupus phenotype is unknown, and the disease has a long development period (6 months), is easily affected by environmental factors, and is difficult to control during the experiment, which brings difficulties to the study of the pathogenesis of the disease.

[0005] Therefore, it is urgent to study a lupus model with a clear cause, which can cause abnormalities in the innate and acquired immune systems, and has a short experimental period. SUMMARY

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

[0007] The first aspect of the present application provides a method for constructing a systemic lupus erythematosus non-human animal model, the method comprising causing the non-human animal to express or lack activity of Fth1.

[0008] In some embodiments, the non-human animal lacks expression or activity of Fth1 in neutrophils.

[0009] In some embodiments, the method further comprises using a systemic lupus erythematosus inducing drug to induce the non-human animal to express or lack activity of Fth1.

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

[0011] In the present application, the expression of Fth1 refers to a decrease or loss in the expression level of Fth1, and the activity of Fth1 refers to a decrease or loss in the functional activity of Fth1. The expression or activity of Fth1 can be achieved by knocking out and / or knocking down Fth1, or by administering an Fth1 inhibitor. Regardless of the method used, as long as the expression or activity of Fth1 is achieved, it falls within the scope of the present application. In one embodiment of the present application, the expression or activity of Fth1 is knocking out and / or knocking down Fth1 of the non-human animal.

[0012] In some embodiments, the knockout and / or knockdown can use one or several of the following technologies 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 nucleases technology, interfering RNA.

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

[0014] In the present application, there are 9 transcription variants of the Fth1 gene, and the knockout and / or knockdown of the Fth1 gene can reduce or lose the expression level or functional activity of any one or several transcription variants, preferably reduce or lose the expression level or functional activity of all transcription variants.

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

[0016] The non-human animal FTH1 fl / fl Crossing with neutrophil conditional knockout Cre non-human animals.

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

[0018] In some embodiments, the neutrophil conditional knockout Cre non-human animals include Mrp8 Cre non-human animals, LysM Cre non-human animals.

[0019] In some embodiments, the neutrophil conditional knockout Cre non-human animals are selected from Mrp8 Cre non-human animals.

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

[0021] (1) Introducing a targeting vector containing homologous arms and loxp sequences into the zygote of the parent non-human animal 1 together with sgRNA and Cas9-mRNA;

[0022] (2) Transplanting the zygote into a surrogate recipient to obtain F0 generation non-human animals by surrogacy;

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

[0024] (4) crossing F1 generation non-human animals FTH1 fl / fl with neutrophil conditional knockout Cre non-human animals to obtain FTH1 fl / fl Mrp8cre + non-human animals.

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

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

[0027] In one embodiment, the way is selected from the group consisting of microinjection.

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

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

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

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

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

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

[0034] In one embodiment, the frequency of imiquimod used is 3 times per week.

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

[0036] In one embodiment, the time of imiquimod used lasts for 6 weeks, and the non-human animal has or shows the characteristics of systemic lupus erythematosus disease or condition within 6 weeks.

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

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

[0039] In some embodiments, the non-human animal has or shows features of systemic lupus erythematosus disease or condition within 1-6 months after using Pristane.

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

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

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

[0043] In some embodiments, the mammal includes but is not limited to rodents, carnivores, chiropterans, shrews, insectivores.

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

[0045] In some embodiments, the rodent includes but is not limited to Cricetidae, Cricetidae, Muridae, Muriidae, Thryonomyidae, Myospalacidae, Echimyidae, Petromyidae, Geomyidae.

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

[0047] In the present invention, the Muridae includes but is not limited to Mus, Rattus, Meriones, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus, Rattus,

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

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

[0050] In some embodiments, the C57BL / 6 subline mice are selected from the group consisting of C57BL / 6J Gpt subline mice.

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

[0052] (1) A method of screening a drug candidate for treating systemic lupus erythematosus, the method comprising:

[0053] a) administering a test agent to be screened to a non-human animal with systemic lupus erythematosus prepared by the construction method of the first aspect of the present application;

[0054] b) detecting the therapeutic effect of the test agent to be screened on systemic lupus erythematosus;

[0055] (2) A method of evaluating the therapeutic effect of a drug for treating 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 of the first aspect of the present application;

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

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

[0059] In the present application, the term "treatment" refers to the administration of a compound or composition to control the progression of a disease. The control of disease progression is understood to achieve a beneficial or desired clinical outcome, including but not limited to alleviating symptoms, reducing disease duration, stabilizing pathological state (especially avoiding additional deterioration), delaying disease progression, improving pathological state, and remission (partial and total). The control of disease progression also involves prolonging survival compared to expected survival without treatment.

[0060] In the present application, drug candidates can be obtained from a wide 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, libraries of compounds can be generated through random and directed synthesis of a variety of organic compounds and biological molecules, or libraries of natural products in the form of extracts of bacteria, fungi, plants or animals, or naturally occurring or synthetic libraries and compounds modified by conventional chemical, physical or biochemical means, or directed or random chemical modifications made to known pharmacological agents, such as acylation, alkylation, esterification, amidification, etc. to generate structural analogs.

[0061] The third aspect of the present application provides the use of any one of the following:

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

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

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

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

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

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

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

[0069] In some embodiments, the mammal includes, but is not limited to, rodents, carnivores, chiropterans, erinaceans, insectivores.

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

[0071] In some embodiments, the rodent includes, but is not limited to, Cricetidae, Cricetidae, Muridae, Muridae, Thryonomyidae, Myospalacidae, Spalacidae, Petromyidae.

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

[0073] In the present invention, the murine species include, but are not limited to, black rat, brown rat, sand rat, New World rat, Old World rat, SD rat, Polynesian rat, tree rat, wood rat, stick rat, rice rat, pouched rat, climbing rat, spiny mouse, Sri Lankan mouse, Sikkimese mouse, Javanese mouse, Indian mouse, field mouse, brown mouse, Korean mouse, Cypriot mouse, South Indian mouse, Thai mouse, Yugoslavian mouse, Mus musculus, Burmese mouse, Hungarian mouse, Mediterranean mouse, earth color mouse, Ivorian mouse, toad mouse, Angolan mouse, plateau mouse, Botswana mouse, cloth mouse, Somali mouse, Ghanaian mouse, South African mouse, desirable mouse, spiny mouse, valley mouse, Central African mouse, hirsute mouse, Zambian mouse, Ugandan mouse, delicate mouse, sea mouse, FVB mouse, Fisher 344 mouse, flat mouse, cave mouse, Shaw's mouse.

[0074] In some embodiments, the murine species is selected from Mus musculus.

[0075] In some embodiments, the Mus musculus includes C57BL / 6 subline mice.

[0076] In some embodiments, the C57BL / 6 subline mice are selected from C57BL / 6J Gpt subline mice.

[0077] The present invention has the advantages and beneficial effects:

[0078] The present invention provides a method for constructing an animal model of systemic lupus erythematosus, which exhibits high autoantibodies, low complement, high inflammatory factors, and severe kidney damage. The systemic lupus erythematosus animal model provided by the present invention has a clear etiology, can cause abnormalities in the innate and acquired immune systems, and has a short experimental period, thereby 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 DRAWINGS

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

[0080] Figure 2 is a hybrid 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 (FTH1 fl / fl );

[0082] Figure 4 FTH1 fl / fl Mrp8cre + ) and control mice Wild Type (FTH1 fl / fl ) after 6 weeks of Imiquimod induction;

[0083] Figure 5 FTH1 fl / fl Mrp8cre + ) and control mice Wild Type (FTH1 fl / fl ) after 6 weeks of Imiquimod induction;

[0084] Figure 6 FTH1 fl / fl Mrp8cre + ) and control mice Wild Type (FTH1 fl / fl ) after 6 weeks of Imiquimod induction. Red represents IgG, yellow represents IgM, green represents Clq, blue represents DNA, and Merge represents all colors overlaid together;

[0085] Figure 7 FTH1 fl / fl Mrp8cre + ) and control mice Wild Type (FTH1 fl / fl ) after 6 weeks of Imiquimod induction;

[0086] Figure 8 FTH1 fl / fl Mrp8cre + ) and control mice Wild Type (FTH1 fl / fl ) after 6 weeks of Imiquimod induction. Red represents IgG, yellow represents IgM, green represents Clq, blue represents DNA, and Merge represents all colors overlaid together;

[0087] Figure 9 FTH1 fl / fl Mrp8cre +) and control mice without knockdown of FTH1, Wild Type (FTH1 fl / fl Comparison of skin lesions of mice after 6 months of Pristane stimulation

[0088] Figure 10 Ferritin protein expression in neutrophils of SLE patients fl / fl Mrp8cre + ) and control mice without knockdown of FTH1, Wild Type (FTH1 fl / fl Comparison of inflammatory factor content in plasma of mice after 6 months of Pristane stimulation DETAILED DESCRIPTION

[0089] The application is further described below in connection with the embodiments. The following description is only preferred embodiments of the application, and is not intended to limit the application in other forms. Any skilled person in the art can modify the above disclosed technical content to equivalent embodiments. Any simple modification or equivalent change of the following embodiments according to the technical essence of the application, without departing from the scheme of the application, falls within the protection scope of the application.

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

[0091] Isolation of neutrophils: K2E (EDTA) tubes (BD Vacutainer Company) were used to collect peripheral blood of 5 healthy people and 4 SLE patients. To isolate neutrophils, the peripheral blood was diluted with phosphate buffered saline solution, and the diluted peripheral blood was poured into the upper layer of Ficoll-Hypaqu separation liquid in a 15 ml centrifuge tube. The interface of the separation liquid was not broken, and then centrifugal separation was performed. The rotation speed was 500G, the maximum ascending speed and the minimum descending speed, and the centrifugation was performed at room temperature for 20 minutes. After centrifugation, the white precipitate on the upper layer of red blood cells was sucked with a pipette, which was neutrophils. The next step was to lyse the red blood cells with lysis buffer (BD), and then centrifugation was performed at a rotation speed of 500G for 10 minutes, and the neutrophils at the bottom of the centrifuge tube were reserved. The purity of neutrophils was more than 98% by flow cytometry using CD16 (Biosciences Company) and CD11b (Biolegend Company) antibodies. The isolated neutrophils were immediately subjected to flow cytometry staining analysis, or stored in a-80 degree refrigerator.

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

[0093] Protein spectrum: Protein extraction and digestion: The sample was added with 300 μL of 8M urea lysate containing 10% protease inhibitor, centrifuged (14,100 x g, 20 min) 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 times, and then trypsin was added (1:25, 37°C overnight digestion). Reverse phase separation: after digestion termination, the C18 column was washed with acetonitrile and 0.1% formic acid in sequence, and then eluted with pH 10 aqueous solution gradient (6%-50% acetonitrile), and freeze-dried into three groups. LC-MS / MS analysis: the freeze-dried sample was dissolved in 2% methanol / 0.1% formic acid, centrifuged and then loaded, and separated by gradient elution of mobile phase A (0.1% formic acid water) and B (0.1% formic acid acetonitrile) (4%-99% B, 120 min). Mass spectrometry parameters include full scan (250-1450 m / z, resolution 120,000), CID fragmentation (collision energy 30%), dynamic exclusion 18 seconds, and data processed by Maxquant (Uniprot_HUMAN database). Identification criteria: parent ion / fragment ion mass error ±15 ppm / ±0.5 Da, cysteine alkylation fixed modification, methionine oxidation variable modification.

[0094] Result analysis: After quantile normalization of the data using the R language Limma package, differential protein expression analysis was performed. P value < 0.05 and |log 2 fold change| ≥ 1.1 were used as the screening criteria for differentially expressed proteins. FerrDb iron death database was used to screen cell iron death related pathway molecules, which were sorted according to the protein expression amount. It can be seen that FTH1 in SLE neutrophils is the most obvious decrease Figure 1 ).

[0095] Example 2 Ferritin gene conditional knockdown in neutrophil mouse experiment scheme

[0096] I. Gene target design

[0097] There are 9 transcription variants of Fth1 gene, and the following knockout region is selected through structure analysis:

[0098] Fth1-203 transcript (ENSMUST00000235196.1): Exon 1-2 segment (this region contains the start codon ATG coding sequence, which will completely destroy the protein function after knockout)

[0099] II. Gene editing process

[0100] 1) CRISPR / Cas9 system construction: including gene editing system (including sgRNA, Cas9-mRNA) and donor vector (including homologous arm and loxp sequence).

[0101] 2) Microinjection: inject CRISPR / Cas9 system into C57BL / 6JGpt strain mouse zygotes.

[0102] 3) Embryo transfer: obtain F0 generation mice by surrogate pregnancy.

[0103] 4) Gene verification: use PCR amplification combined with sequencing to screen positive F0 individuals.

[0104] 5) Strain breeding: backcross positive F0 with C57BL / 6JGpt to establish stable genetically F1 generation model FTH1 fl / fl .

[0105] The present application uses CRISPR / Cas9 technology to knock in two loxp elements into the upstream of exon 1 and the downstream of exon 2 of Fth1 gene. According to the selected sgRNA (sequences are shown in Table 1 SEQ ID NO. 1-2), a targeting vector containing a homologous arm and a loxp sequence is designed, and after enzyme digestion and purification, it is co-injected with sgRNA and Cas9-mRNA into C57BL / 6JGpt mouse zygotes. After injection, the zygotes are transplanted into the oviduct of surrogate recipient mice.

[0106] Table 1 sgRNA sequences

[0107]

[0108] III. Breeding of Fth1 specific knockout model

[0109] For neutrophil conditional knockout, cre mice are Mrp8Cre and LysMcre, in this experiment, Mrp8Cre (also known as S100A8-iCre) mice were selected, purchased from Jisui Yaoke. The S100A8 gene (MRP8) of this mouse has a nuclear-localized Cre recombinase, the mouse is live and fertile, normal size, and does 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. FTH1 fl / fl Mice were crossed with Mrp8Cre mice, and the crossing strategy is shown in Figure 2 A. By identifying the mice after crossing (B), FTH1 -deficient mice derived from myeloid neutrophils were finally obtained. This mouse knocked down the expression of FTH1 in neutrophils derived from myeloid cells, which led to iron death of neutrophils. Figure 2

[0110] Example 3 Phenotype identification of neutrophil conditional knockout mice

[0111] FTH1 fl / fl and FTH1 fl / fl Mrp8cre + Mice need to be given intervention. Select 8-week-old female mice for systemic lupus erythematosus induction.

[0112] Induction scheme one: use imiquimod cream (Mingxinlidi, Sichuan Mingxin Pharmaceutical Products), 25 mg / ear dose to smear the mouse ear skin, 3 times a week, and euthanize the mice after 6 weeks. The control group uses an equal amount of Vaseline instead of IMQ.

[0113] Induction scheme two (Pristane): Pristane (sigma company) gives intraperitoneal injection of Pristane 0.5ml, and the mice are euthanized for sampling after 6 months.

[0114] ​ELISA: Mouse sera were diluted 1:100 for detection of anti-dsDNA antibodies, 1:100 for detection of anti-ANA antibodies, and 1:25000 for detection of complement C3 with assay buffer. Assays were performed according to the manufacturer’s instructions using Mouse Anti-dsDNA IgG ELISA Kit (5120, Alpha Diagnostic International), Anti-ANA Antibody ELISA Kit (5210, Alpha Diagnostic International), and Complement 3 ELISA Kit (6270, Alpha Diagnostic International).

[0115] Immunofluorescence: Kidneys of mice were frozen in OCT compound (Tissue-Tek) at -80°C for evaluation of immune complex deposition. Frozen kidneys were cut into 6 pm thick longitudinal sections and 6 pm snap-frozen kidney tissue sections were treated with blocking buffer (100 mM Tris-HCl, pH 8.0, 0.3% Triton X-100, 2% BSA, and 50 pg / ml goat non-specific IgG) for 1 h. Then, tissues were 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. DAPI reagent (Solarbio) containing anti-quenching agent was added onto the sections, and coverslips were mounted for use. All slides were observed with LSM 980 / Axio Observer 7 Zeiss confocal laser microscope. Images were optimized using ZEN (Zeiss).

[0116] Multifactor assay: Cytokines in mouse plasma were measured using LEGENDplex™ Mouse Th Cytokine Panel (Biolegend) according to the manufacturer’s instructions. Data analysis was performed using LEGENDplex™ Data Analysis Software (version 8.0).

[0117] After imiquimod induction, mice developed skin lesions, FTH1 fl / fl Mrp8cre + developed larger and more severe skin lesions (FTH1 Figure 3 ); mice had enlarged spleens, FTH1 fl / fl Mrp8cre + had larger spleens and more severe SLE disease response compared to Wild Type (FTH1 Figure 4 ); mice had enlarged submandibular and axillary lymph nodes, and FTH1 fl / flMrp8cre + Compared with Wild Type lymph nodes are larger, showing more severe SLE disease response ( Figure 5 ) ; FTH1 fl / fl Mrp8cre + Mice appear kidney immune complex IgG, IgM, C1q deposition ( Figure 6 ) ; FTH1 fl / fl Mrp8cre + Mice inflammatory factor higher than control mice after modeling ( Figure 7 ) ; FTH1 fl / fl Mrp8cre + Mice anti-dsDNA, anti-ANA significantly increased, complement 3 decreased significantly. Suggests that systemic lupus erythematosus phenotype aggravation ( Figure 8 ).

[0118] After Pristane induction, wild-type mice back hair slightly sparse, FTH1 fl / fl Mrp8cre + Back head of the larger area of skin lesions generated ( Figure 9 ) ; FTH1 fl / fl Mrp8cre + Mice inflammatory factor higher than control mice after modeling ( Figure 10 ).

[0119] In summary, FTH1 fl / fl Mrp8cre + Mice in systemic lupus erythematosus modeling induced ( Imiquimod IMQ / Pristane induction) showed higher autoantibodies, lower complement, higher inflammatory factors, more severe kidney damage. At the same time, the mouse neutrophil Ferritin decreased similar to the neutrophil expression characteristics of human SLE. In summary, these results suggest that the absence of Ferritin in neutrophils can induce a significant aggravation of systemic lupus erythematosus phenotype, and thus is a new type of systemic lupus erythematosus mouse model.

[0120] The above description of the embodiments is only for the purpose of understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. A method for constructing a mouse model of systemic lupus erythematosus, characterized in that, The construction method includes inducing Fth1 expression or activity deficiency in the neutrophils of the mice; and then inducing mice with Fth1 expression or activity deficiency using a systemic lupus erythematosus inducing drug. Inducing the loss of Fth1 expression or activity in the mice includes knocking out Fth1 in mice; The knockout techniques used include CRISPR-Cas9 and Cre-LoxP. The knockout method includes the following steps: removing FTH1 containing the loxp sequence... fl / fl Mice were crossed with Cre mice that had neutrophil conditionally knocked out. The FTH1 containing the loxp sequence fl / fl Mice include FTH1, which was constructed using the following method. fl / fl Mouse: Using CRISPR-Cas9 technology, loxp sequences were inserted upstream of exon 1 and downstream of exon 2 of the Fth1-203 transcript to obtain FTH1. fl / fl Mouse; the sgRNA sequence used in the CRISPR-Cas9 technology is shown in SEQ ID NO.1-2; The neutrophil conditionally knocked-out Cre mice are Mrp8 Cre mice; The systemic lupus erythematosus inducing drugs are imiquimod or pristane.

2. The construction method according to claim 1, characterized in that, The knockout method includes the following steps: (1) The targeting vector containing the homologous arm and loxp sequence was introduced into the parent mouse zygote along with sgRNA and Cas9-mRNA; (2) The fertilized eggs were transferred to the surrogate recipient to obtain F0 generation mice through surrogacy; (3) Backcrossing F0 generation mice with parent mice to obtain stably inherited F1 generation FTH1 fl / fl Mice; (4) Replace F1 with FTH1 fl / fl FTH1 mice were obtained by crossing mice with Cre mice that had neutrophil conditionally knocked out. fl / fl Mrp8cre + Mice; The sgRNA sequence is shown in SEQ ID NO.1-2.

3. The construction method according to any one of claims 1-2, characterized in that, The dosage of imiquimod is 10-50 mg / ear.

4. The construction method according to claim 3, characterized in that, The recommended dosage of imiquimod is 25 mg per ear.

5. The construction method according to any one of claims 1-2, characterized in that, The recommended frequency of use for imiquimod is 1-5 times per week.

6. The construction method according to claim 5, characterized in that, The recommended frequency of use for imiquimod is 3 times per week.

7. The construction method according to any one of claims 1-2, characterized in that, The duration of imiquimod use is 2-10 weeks.

8. The construction method according to claim 7, characterized in that, The imiquimod was used for 6 weeks.

9. The construction method according to any one of claims 1-2, characterized in that, The method of administration for Pristane is a single injection of 0.2-1 ml.

10. The construction method according to claim 9, characterized in that, The method of administration for Pristane is a single injection of 0.5 ml.

11. The construction method according to any one of claims 1-2, characterized in that, The mice included the C57BL / 6 subspecies.

12. The construction method according to claim 11, characterized in that, The C57BL / 6 subspecies mice are C57BL / 6JGpt strain mice.

13. A method for screening drug candidates for the treatment of systemic lupus erythematosus, characterized in that, The method includes: a) Apply the screening reagent to systemic lupus erythematosus mice prepared by the construction method according to any one of claims 1-10; b) To test the therapeutic effect of the reagent to be screened on systemic lupus erythematosus.

14. A method for evaluating the therapeutic efficacy of a drug for treating systemic lupus erythematosus, characterized in that, The method includes: a) Administering the drug to systemic lupus erythematosus mice prepared by the construction method according to any one of claims 1-10; b) To test the therapeutic effect of the drug on the systemic lupus erythematosus.

15. A method for studying the pathogenesis of systemic lupus erythematosus, characterized in that, The method described herein is to study the pathogenesis of systemic lupus erythematosus (SLE) using systemic lupus erythematosus mice prepared by the construction method according to any one of claims 1-10.

16. The use of a systemic lupus erythematosus mouse model prepared by the construction method according to any one of claims 1-10 in screening drug candidates for the treatment of systemic lupus erythematosus.

17. The use of a systemic lupus erythematosus mouse model prepared by the construction method according to any one of claims 1-10 in evaluating the therapeutic effect of drugs for treating systemic lupus erythematosus.

18. The application of the systemic lupus erythematosus mouse model prepared by the construction method according to any one of claims 1-10 in the study of the pathogenesis of systemic lupus erythematosus.

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