A method for constructing an animal model of systemic lupus erythematosus

Abstract

The present invention provides a method for constructing an animal model of systemic lupus erythematosus, belonging to the technical field of methods for constructing disease animal models. In the present invention, mice GPX4<supgt;fl / wt< / supgt;VilCre+ containing heterozygous GPX4 gene and Cre gene are first obtained, and then SLE induction drugs are used to further induce the mice to develop the disease. In view of the specific gene knockdown in specific cells in the present invention, the etiology of the disease development in the model is clear, that is, the abnormal intestinal barrier system triggers adaptive immune changes, recognizes autoantigens, and ultimately leads to the development of lupus. At the same time, the finally obtained SLE model in the present invention can exhibit higher autoantibodies, lower complement, higher inflammatory factors, and more severe lung damage, and the decrease of intestinal GPX4 in these mice is similar to the intestinal expression characteristics of human SLE.

Description

Technical Field

[0001] The present invention relates to the technical field of disease animal model construction methods, and in particular to a method for constructing an animal model of systemic lupus erythematosus. Background Art

[0002] Systemic lupus erythematosus (SLE) is an autoimmune inflammatory connective tissue disease that affects multiple organs and is common in young women. Although SLE has been an independent disease for more than a hundred years, its cause is still not fully understood. A large number of studies have shown that genetic, endocrine, infection, immune abnormalities and environmental factors are related to the onset of SLE. Existing medical methods cannot completely cure SLE, and most patients can only control the disease through long-term use of hormones and immunosuppressants. Therefore, studying the pathogenesis of SLE is of great significance to understanding and treating SLE.

[0003] Animal models of human diseases are animal experimental subjects and materials established in biomedical scientific research that have human disease-like performance. Animal models help to more conveniently and effectively understand the occurrence and development of human diseases and study prevention and control measures. Female SPF-grade MRL / lpr mice are currently the most commonly used SLE model in the world. This model is Fas gene defective and is characterized by a large amount of anti-dsDNA, ANA antibodies, and severe glomerulonephritis. The symptoms of the NZB / NZW F1 model are similar to those of humans. Sex hormones have a significant effect on the mice. Female mice develop earlier and more severely than male mice; they are characterized by high titers of anti-dsDNA, anti-ssDNA antibodies, and hypergammaglobulinemia. The MRL / lpr model mainly simulates abnormalities in the adaptive immune system, and its innate immune system is normal, which does not fully conform to the characteristics of human diseases. Although the existing animal models induce SLE from different pathogenesis, and the characteristics and symptoms are also different, they still cannot meet the needs of research on the mechanism of SLE.

[0004] Intestinal homeostasis is closely related to the immune balance of the human body, and the disruption of intestinal homeostasis may be involved in the occurrence of autoimmunity. It has been confirmed that the intestinal flora of SLE patients is significantly different from that of healthy people. However, there are few studies reporting how the permeability of the intestinal barrier in SLE affects the onset of SLE, and no studies have reported how SLE intestinal ferroptosis is involved in the onset of SLE. Therefore, it is urgent to study SLE animal models with symptoms of intestinal homeostasis. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for constructing an animal model of systemic lupus erythematosus. The animal model provided by the present invention is SLE caused by abnormalities in the intestinal barrier system, which is of great significance for studying the occurrence, development laws and prevention and treatment measures of SLE.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for constructing an animal model of systemic lupus erythematosus, comprising the following steps:

[0008] S1. Using the Crispr / Cas9 technology, loxP sites are inserted upstream of exon 2 and downstream of exon 4 of the glutathione peroxidase 4 (GPX4) gene in mice to construct parental mouse 1;

[0009] S2. Parental mouse 2 is a Cre mouse specific to intestinal epithelial cells;

[0010] S3. Parental mouse 1 and parental mouse 2 are hybridized, and the offspring mouse GPX4 containing the GPX4 gene and the Cre gene is screened. fl / wt VilCre+;

[0011] S4. An SLE induction drug is introduced into the abdominal cavity of GPX4 fl / wt VilCre+;

[0012] S5. Monitor the onset of systemic lupus erythematosus in mice.

[0013] In certain embodiments, both parental mouse 1 and parental mouse 2 are C57 / B6. The backgrounds of the two parental mice of the present invention are both C57 / B6. After hybridization and backcrossing, the level of type I interferon in the plasma of the obtained GPX4 fl / wt VilCre+ mice increases, and a more severe lupus phenotype appears after Pristane induction.

[0014] In certain embodiments, the specific construction steps of parental mouse 1 are as follows:

[0015] Using sgRNA-Vector as a template, a DNA fragment of Gpx4-sgRNA is amplified by PCR, recovered by gel electrophoresis, and used as a template for in vitro transcription of sgRNA, followed by purification and recovery;

[0016] According to the selected sgRNA, a targeting vector is designed, which includes homologous arms and loxp sequences. After construction, it is digested with enzymes, purified, and co-microinjected into C57 mouse embryos together with sgRNA and Cas9-mRNA. The injected embryos are transplanted into the fallopian tubes of surrogate recipient mice.

[0017] Specifically, GPX4 encodes a member of the glutathione peroxidase family, whose function is to reduce hydroperoxides in membrane lipids and lipoproteins, thereby protecting mitochondrial function and inhibiting ferroptosis. Heterozygous and homozygous loxp mice are alive and fertile, with no reported abnormalities. When bred with Cre recombinase mice, the offspring can produce inducible GPX4 gene knockout, homozygous knockout is lethal, and heterozygous knockdown is viable.

[0018] In certain embodiments, the parental mouse 2 is a Vilcre mouse, which has a nuclear-localized Cre recombinase on the Villin gene.

[0019] Specifically, the Vilcre mouse, namely the Villin-Cre mouse, is alive and fertile, of normal size, and does not exhibit any obvious physical or behavioral abnormalities, and can be used for Cre-loxp studies of intestinal epithelial cell lines and innate immune responses.

[0020] In certain embodiments, the GPX4 fl / wt VilCre+ exhibits a systemic lupus erythematosus phenotype characterized by intestinal damage.

[0021] In certain embodiments, the SLE-inducing drug is one or more of pristane, imiquimod, peptides, lipopolysaccharide, Campylobacter jejuni and Freund's complete adjuvant, lymphocyte-activated chromatin.

[0022] In certain embodiments, when the SLE-inducing drug is pristane, the import volume is preferably 0.4 mL to 0.6 mL, more preferably 0.5 mL.

[0023] In certain embodiments, when the SLE-inducing drug is lipopolysaccharide, the import volume is 50 μg, twice a week for 2 to 4 weeks.

[0024] In certain embodiments, when the SLE-inducing drug is Campylobacter jejuni and Freund's complete adjuvant, the import volume is 0.2 mL, injected into the toes on days 0 to 15.

[0025] In certain embodiments, the import timing of the SLE-inducing drug is when the GPX4 fl / wt VilCre+ mice reach sexual maturity at eight weeks of age.

[0026] In certain embodiments, monitoring the onset of systemic lupus erythematosus in mice is to monitor the autoantibodies, complement, inflammatory factors, lung injury, and intestinal permeability of the mice.

[0027] In certain embodiments, when the SLE-inducing drug is pristane, the time for monitoring the onset of systemic lupus erythematosus in mice is 1 to 6 months after the import of pristane.

[0028] Specifically, the mice finally obtained in the present invention start to exhibit phenotypes 1 to 6 months after induction, including autoantibodies, low complement, high inflammatory factors, and lung injury, which are in line with the disease characteristics of humans and more in line with the characteristics of low expression of GPX4 in the intestine of SLE patients.

[0029] Beneficial technical effects: The present invention provides a method for constructing an animal model of systemic lupus erythematosus. In the present invention, mice GPX4 containing heterozygous GPX4 gene and Cre gene are first obtained fl / wt VilCre +, and then SLE induction drugs are used to further induce the mice to develop the disease. In view of the fact that the present invention is the knockdown of a specific gene in specific cells, the cause of the disease in the model is clear. Abnormal intestinal barrier system triggers adaptive immune changes, recognizes autoantigens, and ultimately leads to lupus onset. At the same time, the SLE model finally obtained in the present invention can exhibit higher autoantibodies, lower complement, higher inflammatory factors, and more severe lung injury, and the decrease of intestinal GPX4 in these mice is similar to the intestinal expression characteristics of human SLE. Description of the Drawings

[0030] Figure 1 A is an immunohistochemical staining map of GPX4 in intestinal epithelial cells of healthy (HC) and lupus patients (SLE); Figure 1 B is the quantitative score of the immunohistochemical results of GPX4 in the left figure; Figure 1 C is the quantitative score of the immunohistochemical results of GPX4 in lupus patients with intestinal manifestations in the left figure; where, "*" indicates P < 0.05, and "****" indicates P < 0.0001;

[0031] Figure 2 A is a schematic diagram of gene targeting and conditional knockout of Gpx4 gene in mice with conditional knockout of Gpx4; Figure 2 B is the hybridization strategy;

[0032] Figure 3 A is GPX4 fl / wt Immunohistochemical staining map of the intestine of Vilcre + mice and WT mice, Figure 3 B The left figure of GPX4 fl / wt Calprotectin content in the feces of Vilcre + mice and WT mice; Figure 3 B The right figure of GPX4 fl / wt LPS content in the plasma of Vilcre + mice and WT mice; where, "*" indicates P < 0.05;

[0033] Figure 4 For GPX4 fl / wt Vilcre + and GPX4 fl / flChanges in the contents of dsDNA antibody (4A), complement C3 (4B), IFN-α (4C right), and IL-1b (4C left) after Pristane induction; where "*" indicates P < 0.05 and "**" indicates P < 0.01.

[0034] Figure 5 The left and middle figures are GPX4 respectively fl / wt HE staining images of the lungs of Vilcre+ mice and WT mice before and after Pristane stimulation, and the right figure is the histological injury score based on HE staining of the lungs; where "**" indicates P < 0.01. Specific implementation manners

[0035] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments. However, the content of the present invention is not limited to the following embodiments. The materials, reagents, etc. used in the embodiments and experimental examples of the present invention can be obtained from commercial channels without special instructions; the methods used in the embodiments and experimental examples of the present invention are conventional methods without special instructions.

[0036] Example 1 Identification of the expression content of GPX4 protein in intestinal epithelial cells of lupus patients

[0037] Isolation of intestinal epithelial cells: Select polyp patients with age and gender matching in the Department of Gastroenterology of Beijing Hospital, and screen those without autoimmune, inflammatory or infectious diseases and no other underlying diseases as healthy controls; collect biopsy specimens obtained from previous colonoscopy examinations of patients and control populations in the Department of Pathology, that is, intestinal tissue paraffin blocks, and obtain the informed consent of the patients. The collection of clinical specimens involved in this patent has been approved by the Ethics Committee of our hospital.

[0038] Immediately add the biopsied intestinal epithelial cells to formalin for fixation, embed them in paraffin, and perform immunohistochemical staining of intestinal biopsy specimens of SLE patients and healthy samples (HC) ( Figure 1 A), and the protocol is that the anti-GPX4 (1:200) antibody is incubated overnight at 4°C. And incubate with anti-rabbit IgG-HRP (1:1000) for 1 h. The quantitative scoring results of the immunohistochemical staining of intestinal biopsy specimens are as Figure 1 shown in B and 1C. It can be clearly seen from the quantitative scoring results that the expression level of GPX4 is significantly lower than that of normal people ( Figure 1 B); and in SLE patients with gastrointestinal manifestations, the expression level of intestinal GPX4 further decreases ( Figure 1 C).

[0039] The immunohistochemical results show that the expression of GPX4 in lupus intestinal epithelial cells decreases, and the decrease is more obvious when the patient has clinical manifestations of gastrointestinal involvement.

[0040] Example 2 Experimental Protocol for Generating Neutrophil-Conditional Knockdown Mice of GPX4 Gene

[0041] Gene Information: The GPX4 gene is located on mouse chromosome 10. The protein encoded by the GPX4 gene mainly has two splice variants: GPX4-201 and GPX4-202. Both of these splice variants have 7 exons, and they differ only in exon 1.

[0042] Principle of Generation: Using the cas9 / gRNA system, two loxp elements are respectively knocked into the upstream of exon 2 and the downstream of exon 4 of the Gpx4 gene, that is, the 2-4 exons of the Gpx4 gene are anchored by two loxp. This mouse is crossed with a mouse with tissue-specific expression of cre, and the 2-4 exons of the Gpx4 gene can be specifically deleted in the tissues and organs expressing the cre enzyme. The total length of these 3 exons is 392bp, which is not an integer multiple of 3. After deletion, frameshift mutations will occur in both splice variants of the Gpx4 gene, resulting in knockout of the Gpx4 gene, thus obtaining mice with conditional knockout of the Gpx4 gene.

[0043] Gene targeting of Gpx4 gene conditional knockout mice, and the schematic diagram of conditional knockout is as Figure 2 shown in A.

[0044] Design, in vitro transcription and purification of gRNA: Select sgRNA with a low probability of off-target. Design 2-3 gRNAs near the preset loxp target site by sequence alignment. After measuring the cleavage efficiency, select the gRNA with the highest cleavage activity for subsequent experiments. PCR amplify the DNA fragment of Gpx4-sgRNA using sgRNA-Vector as a template, and then recover it by gel electrophoresis as a template for in vitro transcription of sgRNA. After in vitro transcription and purification and recovery of sgRNA, it is aliquoted and stored in a -80°C refrigerator for later use.

[0045] Design and construction of the targeting vector: According to the selected sgRNA, design the targeting vector, including homologous arms and loxp sequences. After construction, it is digested with enzymes, purified, and co-microinjected into fertilized eggs with sgRNA and Cas9-mRNA.

[0046] Microinjection: Co-inject the purified sgRNA and Cas9-mRNA into C57 mouse embryos, and transplant the embryos into the oviduct of surrogate recipient mice after injection.

[0047] Mouse Identification: Mice are born 21 days after embryo transfer, and genotype identification (mouse tail DNA identification) is completed about 2 weeks after birth to identify GPX4 mutants.

[0048] Intestinal epithelial cell Cre: In this experiment, healthy 8-week-old Vilcre mice were purchased from Cyagen Biosciences. These mice have a Cre recombinase with nuclear localization in the Villin gene (Vil). These Vilcre mice can be used for Cre-lox studies of intestinal epithelial cells.

[0049] The hybridization strategy is as Figure 2 shown in B.

[0050] Example 3 Identification of lupus phenotype in intestinal epithelial cell conditional knockdown mice

[0051] Control mice without knockdown of GPX4, GPX4 fl / fl (purchased from Cyagen) and mice with conditional knockdown of GPX4 in intestinal epithelial cells, Gpx4 fl / wt VilCre + mice were all intervened with Pristane administration. 8-week-old mice were given an intraperitoneal injection of 0.5 mL of Pristane, and the mice were sacrificed and sampled 6 months later.

[0052] Enzyme-linked immunosorbent assay: Blood was collected from the inner canthus of the eyes of mice 6 months after Pristane stimulation, and plasma was extracted. The changes in the contents of anti-double-stranded DNA antibody (dsDNA antibody), complement C3, interferon (IFN-α), and interleukin 1b (IL-1b) in the plasma were detected by ELISA. Mouse plasma was diluted 1:100 with assay buffer for detecting anti-dsDNA antibody and 1:25000 for detecting complement C3. According to the instructions, a mouse anti-dsDNA IgG ELISA kit (5120, Alpha Diagnostic) and a complement 3 ELISA kit (6270, Alpha Diagnostic) were used, and interferon and interleukin were directly detected using a multi-factor kit (740622, Biolegend), and their contents were measured by flow cytometry. Among them, multi-factor detection was performed according to the manufacturer's instructions, and a LEGENDplexTM mouse Th cytokine detector (Biolegend) was used to measure cytokines in mouse plasma. LEGENDplexTM data analysis software (version 8.0) was used for data analysis.

[0053] GPX4 fl / wt Vilcre+ and control mice (GPX4 fl / fl , WT) The changes in the contents of dsDNA antibody, complement C3, IFN-α, and IL-1b in the plasma before and after Pristane induction are as Figure 4 shown. It can be seen from Figure 4 that GPX4 fl / wtAfter stimulation with pristane, the autoantibodies in Vilcre+ mice increased, the content of complement C3 decreased significantly, the content of IFN-α increased significantly, and the content of IL-1b increased significantly. This indicates an exacerbation of the lupus phenotype.

[0054] Immunohistochemistry: Intestines were taken from GPX4 fl / wt Vilcre+ and control mice (WT, GPX4 fl / fl ) were fixed in 4% paraformaldehyde and embedded in paraffin. Among them, the intestines were stained by immunohistochemistry with GPX4 antibody.

[0055] The content of GPX4 in intestinal epithelial cells was detected by immunohistochemistry, Gpx4 fl / wt VilCre + The expression of GPX4 in the intestinal epithelium of VilCre Figure 3 mice decreased ( Figure 3 A), indicating that this model meets the design requirements. At the same time, there was a higher level of LPS in the blood and a higher level of calprotectin in the feces of this model (

[0056] B), suggesting a change in intestinal wall permeability. fl / wt VilCre + mice and control mice (WT, GPX4 fl / fl ) were collected, fixed in formalin, and the lung injury, alveolar hemorrhage and immune cell infiltration of the mice were detected by HE staining.

[0057] The results showed that after stimulation with pristane, the mice had more alveolar hemorrhage, pulmonary immune cell infiltration, and a higher lung injury score ( Figure 5 ).

[0058] In summary, Gpx4 fl / wt VilCre + mice showed higher autoantibodies, lower complement, higher inflammatory factors, and more severe lung injury after induction with pristane. It is worth noting that Gpx4 fl / wt VilCre + mice had higher intestinal permeability, and all the above lupus phenotypes were changes based on this; at the same time, the decrease in intestinal GPX4 in this mouse was similar to the intestinal expression characteristics of human SLE. Therefore, the mouse model obtained in the present invention can be used as a new model for studying the pathogenesis of lupus intestinal related diseases.

[0059] The above are only the preferred embodiments 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 refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing an animal model of systemic lupus erythematosus, characterized in that, It includes the following steps: S1. Using the Crispr / Cas9 technology, loxP sites are inserted upstream of exon 2 and downstream of exon 4 of the glutathione peroxidase 4 (GPX4) gene in mice to construct parental mouse 1; S2. Parental mouse 2 is an intestinal epithelial cell-specific Cre mouse; S3. Parental mouse 1 and parental mouse 2 are crossed, and the offspring mouse GPX4 containing the GPX4 gene and the Cre gene is screened out. fl / wt VilCre+; S4. Introduce the SLE-inducing drug into GPX4 fl / wt The peritoneal cavity of VilCre+; the SLE-inducing drug is pristane; S5. Monitor the onset of systemic lupus erythematosus in mice.

2. The method for constructing an animal model of systemic lupus erythematosus according to claim 1, wherein The backgrounds of both parental mouse 1 and parental mouse 2 are C57 / B6.

3. The method for constructing an animal model of systemic lupus erythematosus according to claim 1 or 2, characterized in that, The specific construction steps of parental mouse 1 are as follows: PCR amplify the DNA fragment of Gpx4-sgRNA using sgRNA-Vector as a template. After gel recovery, it is used as a template for in vitro transcription of sgRNA, and then purified and recovered; According to the selected sgRNA, design a targeting vector containing homologous arms and loxp sequences. After construction, it is digested with enzymes, purified, and co-microinjected into C57 mouse embryos together with sgRNA and Cas9-mRNA. The injected embryos are transplanted into the oviducts of surrogate recipient mice.

4. The method for constructing an animal model of systemic lupus erythematosus according to claim 1, wherein, Parental mouse 2 is a Vilcre mouse, which has a nuclear-localized Cre recombinase on the Villin gene.

5. The method for constructing an animal model of systemic lupus erythematosus according to claim 1 or 4, characterized in that, The GPX4 fl / wt VilCre+ presents a systemic lupus erythematosus phenotype characterized by intestinal injury.

6. The method for constructing an animal model of systemic lupus erythematosus according to claim 1, wherein, When the SLE inducer is Pristane, the import volume is 0.4 mL to 0.6 mL.

7. The method for constructing an animal model of systemic lupus erythematosus according to claim 1, wherein The introduction timing of the SLE-inducing drug is GPX4 fl / wt When VilCre+ mice reach sexual maturity at eight weeks of age.

8. The method for constructing an animal model of systemic lupus erythematosus according to claim 1, wherein, Monitoring the onset of systemic lupus erythematosus in mice is to monitor the autoantibodies, complements, inflammatory factors, lung damage, intestinal permeability, hair loss, kidney damage, and spleen enlargement of the mice.

9. The method for constructing an animal model of systemic lupus erythematosus according to claim 1 or 8, characterized in that, When the SLE inducer is Pristane, the time for monitoring the onset of systemic lupus erythematosus in mice is 1 to 6 months after the import of Pristane.

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