Method for constructing systemic lupus erythematosus animal model and application
Systemic lupus erythematosus model mice were constructed through hybridization and drug-induced methods, which solved the problems of high prices, slow disease and susceptibility to environmental impact in the prior art model mice, and provided low-cost and fast drug screening tools.
Patent Information
- Application Number
- CN202510835974.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-20
- Publication Date
- 2025-08-29
AI Technical Summary
Existing lupus models mice are expensive, have late onset, long cycles and are susceptible to environmental factors, and are not suitable as drug screening tools.
By hybridizing TMEM187-loxp homozygous mice and LysM-cre tool mice, F1 generation mice were produced, and the genotype of F2 generation mice was identified. TMEM187+/+LysM+/+ homozygous mice were selected as the target mice, and systemic lupus erythematosus model could be spontaneously formed, and IMQ or phytane-lowering drugs could be used to induce it.
Systemic lupus erythematosus model mice with low prices, fast onset, short cycles and are not susceptible to environmental factors were constructed, providing efficient drug screening tools for lupus treatment, shortening modeling time and workload.
Smart Images
Figure CN120549040A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of animal models, and specifically relates to a method and application of constructing an animal model of systemic lupus erythematosus based on human gene-overexpressing mice. Background Art
[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease influenced by multiple factors, including genetics, environmental factors, and hormone levels. It is more common in women. Its main characteristics are the loss of tolerance to self-antigens, abnormal T and B cell responses, and the production of antinuclear antibodies, resulting from the disruption of immune system homeostasis.
[0003] Systemic lupus erythematosus (SLE) can affect multiple systems and organs, including the skin, joints, central nervous system, heart, kidneys, and cardiovascular system. The pathogenesis of SLE is complex and includes immune complex deposition, impaired clearance of cellular debris, Toll-like receptors, and abnormal lymphocyte activation. Despite extensive research, the etiology of SLE remains elusive, leading to diverse treatment options, including glucocorticoids, immunosuppressants, and targeted therapies. However, existing SLE treatments suffer from poor efficacy and significant side effects, necessitating the development of novel therapeutics.
[0004] The establishment of lupus mouse models provides an important tool for the prevention, treatment, and pathogenesis of related diseases. Currently, there are multiple methods for establishing lupus mouse models, which can be divided into two categories: hybridization of lupus model mice, such as MRL / lpr, BXSB / MpJ, and NZBWE1 / J mice; and induced lupus model mice, typically using 8-10 week-old female mice. A single intraperitoneal injection of 0.5 mL of pristane is then administered, followed by normal maintenance for seven months to induce lupus-like disease.
[0005] Mice, as a classic model organism, share a high degree of homology with the human genome, offering advantages such as ease of care, high reproductive rates, high genetic purity, and similarities in tissue, organ structure, and cellular function to humans. However, lupus mouse models are expensive, exhibit a late onset, a long cycle, and are susceptible to environmental factors, making them unsuitable tools for drug screening. Therefore, establishing a stable, early-onset, and easy-to-care, spontaneous lupus mouse model holds great promise for research. Summary of the Invention
[0006] The purpose of the present invention is to construct a systemic lupus erythematosus model mouse and provide a tool for drug screening for the treatment of lupus.
[0007] The purpose of the present invention is achieved through the following technical solutions: A method for constructing an animal model of systemic lupus erythematosus comprises the following steps: (1) Carry TMEM187 -loxp homozygous mice and LysM -cre tool mice were hybridized to produce F1 generation mice; (2) pairing the F1 generation mice to produce F2 generation mice; (3) Identify the genotype of F2 mice and select the genotype TMEM187 + / + LysM + / + Homozygous mice are used as target mice, and when they grow to 10 to 12 weeks of age, the systemic lupus erythematosus animal model is obtained.
[0008] Genotype is TMEM187 + / + LysM + / + Mice homozygous for myeloid overexpression TMEM187 Mice that are fed normally will spontaneously develop systemic lupus erythematosus when they are 10 to 12 weeks old.
[0009] Preferably, the method further comprises: The target mice are induced with drugs to obtain the animal model; the drug is imiquimod cream (IMQ) or pristane.
[0010] Carry as described in step (1) TMEM187 -loxP homozygous mice and methods for obtaining them are prior art, as described in GHOSH K, VAN DUYNE G D. Cre-loxP biochemistry [J]. Methods, 2002, 28(3): 374-83.
[0011] As described in step (1) LysM -cre tool mice are existing technologies and can be purchased through commercial channels.
[0012] Preferably, the target mouse is a female mouse.
[0013] Preferably, the method of inducing with IMQ cream is: applying IMQ cream to the ears of the target mice. More preferably, the inducing method is: applying 1.25 mg of IMQ cream to the target mice aged 6 to 8 weeks, three times a week, for 10 consecutive weeks.
[0014] Preferably, the pristane induction method comprises: intraperitoneally injecting pristane into the target mice. More preferably, the induction method comprises: injecting 0.5 mL of pristane into the target mice at 8 to 10 weeks of age once, followed by conventional feeding for 12 weeks.
[0015] The present invention also provides applications of the animal model constructed by the above method.
[0016] Preferably, the application includes application in systemic lupus erythematosus drug screening or systemic lupus erythematosus disease progression research.
[0017] The beneficial effects of the present invention are: The present invention constructs a systemic lupus erythematosus model mouse, providing a tool for drug screening for the treatment of lupus. The construction method is low in price, has a rapid onset, a short cycle, and is not easily affected by environmental factors.
[0018] The present invention can be spontaneous without drug induction. Compared with the induction means in the prior art, the method of the present invention has lower cost.
[0019] At the same time, the systemic lupus erythematosus model mice of the present invention can also be induced at the same time, which can greatly shorten the modeling time and workload.
[0020] Compared with the lupus mice obtained by hybridization in the prior art, the model mice constructed in the present invention have a simple hybrid background and a longer lifespan. The average lifespan of MRL / lpr female mice is 17 weeks of age, while the lifespan of the female mice constructed in the present invention is much greater than 17 weeks of age. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a comparison chart of the anti-dsDNA antibody content in the serum of 12-week-old female mice in Example 1 (1).
[0022] Figure 2 This is a comparison chart of the anti-dsDNA antibody content in the serum of 16-18 week old female mice in Example 1 (2).
[0023] Figure 3 This is a comparison chart of immune complex deposition in the kidneys of 16-18 week old female mice in Example 1 (3).
[0024] Figure 4 This is a comparison chart of the antinuclear antibody levels in the serum of female mice induced with IMQ in Example 2 (1-2).
[0025] Figure 5 This is a comparison of immune complex deposition in the kidneys of female mice induced with IMQ in Example 2 (1-3).
[0026] Figure 6 This is a comparison of abdominal fat granulomas in female mice induced in Example 3 (2-2) after pristane induction.
[0027] Figure 7This is a comparison of immune complex deposition in the kidneys of female mice induced in Example 3 (2-3) after pristane induction.
[0028] Figure 8 This is a comparison chart of the anti-dsDNA content in the serum of each group of mice before and after CTX treatment in the application example. DETAILED DESCRIPTION
[0029] Example 1 (1) Human source TMEM187 Construction of myeloid overexpression mice: (1-1) Get Carry TMEM187 -loxP homozygous mice: Using CRISPR / Cas9 technology to construct human TMEM187 Gene knock-in mice were developed with the assistance of Saiye Biotech.
[0030] Specifically, the donor vector carrying the "CAGpromoter-loxP-3*SV40 pA-loxP-Kozak-human TMEM187CDS-3×Myc-rBG pA" element and Cas9 mRNA were co-injected into fertilized eggs to obtain knock-in offspring. The offspring were genotyped by PCR to obtain the carriers. TMEM187 -loxp homozygous mice, named TMEM187 loxP / loxP .
[0031] (1-2) Purchased from Saiye Bio LysM -cre tool mouse.
[0032] (1-3) TMEM187 loxP / loxP Mice and LysM -cre tool mice were hybridized to produce F1 mice.
[0033] (1-4) Pair the F1 generation mice to produce F2 generation mice.
[0034] Identify the genotype of F2 generation mice and select the genotype TMEM187 + / + LysM + / + The homozygous target mice were named TMEM187 loxP / loxP LysM -cre.
[0035] (1-5) Construction of spontaneous lupus mouse model: Will TMEM187 loxP / loxP LysM-cre female mice were raised normally until 12 weeks of age. Blood was collected from the heart after deep anesthesia, and the serum was obtained by centrifugation after standing. The anti-dsDNA antibody content in the serum was detected using an Elisa kit.
[0036] The results are as follows Figure 1 Shown: 12 weeks old TMEM187 loxP / loxP LysM -cre female mice compared with female mice of the same age TMEM187 loxP / loxP Mice with the same dsDNA mutation had significantly higher levels of anti-dsDNA antibodies in their serum, indicating that mice can spontaneously develop lupus at as young as 12 weeks of age.
[0037] Will TMEM187 loxP / loxP LysM -cre female mice were raised normally until 16-18 weeks of age and used as the experimental group for comparative experiments.
[0038] (1-6) TMEM187 loxP / loxP Female mice were raised normally until 16-18 weeks of age and served as the control group.
[0039] (2) Detection of anti-double-stranded DNA (dsDNA) antibody content in mouse serum: 16-18 weeks old TMEM187 loxP / loxP LysM -cre female mice and TMEM187 loxP / loxP After deep anesthesia, blood was collected from the heart of female mice, and the serum was obtained by centrifugation after standing. The anti-dsDNA antibody content in the serum was detected using an Elisa kit.
[0040] The results are as follows Figure 2 As shown: TMEM187 loxP / loxP LysM -cre female mice compared with female mice of the same age TMEM187 loxP / loxP Mice had significantly higher levels of anti-dsDNA antibodies in their serum.
[0041] (3) Human sources TMEM187 Detection of kidney damage in myeloid overexpression mice: (3-1) Obtaining mouse kidney tissue: 16-18 weeks old TMEM187 loxP / loxP LysM -cre female mice and TMEM187 loxP / loxPAfter deep anesthesia, female mice were perfused with approximately 25 mL of pre-cooled 1× PBS, which was then punctured from the left ventricle with an injection needle and the right atrial appendage was cut to bleed. After perfusion, the desired tissues were separated and fixed in paraformaldehyde fixative, then embedded in paraffin and sectioned.
[0042] (3-2) Immune complex deposition in mouse kidney tissue: After dewaxing, antigen retrieval, blocking, secondary antibody application, and nucleus staining, the kidney tissue sections were placed under a laser confocal microscope or bright field to observe the deposition of immune complexes in the glomeruli.
[0043] The results are as follows Figure 3 As shown: TMEM187 loxP / loxP LysM -cre female mice compared with female mice of the same age TMEM187 loxP / loxP Mice show immune complex deposition in their kidneys.
[0044] Example 2 (1) Construction of IMQ-induced systemic lupus erythematosus animal model: (1-1) Drug-induced method: Take the 6-8 week old ones obtained in Example 1 TMEM187 loxP / loxP LysM -cre female mice and TMEM187 loxP / loxP Female mice (control) were treated with IMQ cream (1.25 mg / time) applied to the ears of each mouse three times a week for ten consecutive weeks to induce lupus.
[0045] (1-2) Detection of antinuclear antibody content in mouse serum: After deep anesthesia, the female mice induced by (1-1) were blooded from the heart. After standing, the blood was centrifuged to obtain serum, and the antinuclear antibody content in the serum was detected using an Elisa kit.
[0046] The results are as follows Figure 4 As shown: After IMQ induction, TMEM187 loxP / loxP LysM -cre female mice compared with female mice of the same age TMEM187 loxP / loxP Mice had higher levels of antinuclear antibodies in their serum.
[0047] (1-3) Immune complex deposition in mouse kidney tissue: Take (1-1) after induction TMEM187 loxP / loxP LysM -cre female mice and TMEM187 loxP / loxP Kidney tissue sections of female mice were subjected to the same steps as (3-1) and (3-2) of Example 1, including dewaxing, antigen retrieval, blocking, secondary antibody application, and nuclear staining. The sections were then placed under a laser confocal microscope or bright field to observe the deposition of immune complexes in the glomeruli.
[0048] The results are as follows Figure 5 Show: After IMQ induction, TMEM187 loxP / loxP LysM -cre female mice compared with female mice of the same age TMEM187 loxP / loxP Mice had more immune complex deposits in their kidneys.
[0049] Example 3 (1) The same method as in Example 1 (1) was used to obtain the target mice, namely TMEM187 loxP / loxP LysM -cre.
[0050] (2) Construction of pristane-induced systemic lupus erythematosus animal model: (2-1) Drug-induced method: Take 8 to 10 weeks old TMEM187 loxP / loxP LysM -cre female mice and TMEM187 loxP / loxP Female mice were injected intraperitoneally with 0.5 mL of pristane once and housed normally for 12 weeks to induce lupus.
[0051] (2-2) Gross diagram of mouse abdominal cavity: After drug induction, the mice were anesthetized and dissected to observe their abdominal cavity grossly.
[0052] The results are as follows Figure 6 As shown: After induction with pristane, TMEM187 loxP / loxP LysM -cre female mice compared with female mice of the same age TMEM187 loxP / loxP The mice developed more fat granulomas in their abdominal cavities.
[0053] (2-3) Immune complex deposition in mouse kidney tissue: Take (2-1) after induction TMEM187 loxP / loxP LysM -cre female mice and TMEM187 loxP / loxPKidney tissue sections of female mice were prepared in the same manner as in steps (3-1) and (3-2) of Example 1. After dewaxing, antigen retrieval, blocking, secondary antibody application, and nuclear staining, the sections were placed under a laser confocal microscope to observe the deposition of immune complexes in the glomeruli.
[0054] The results are as follows Figure 7 As shown: After induction with pristane, TMEM187 loxP / loxP LysM -cre female mice compared with female mice of the same age TMEM187 loxP / loxP Mice had more immune complex deposits in their kidneys.
[0055] Application example: Human TMEM187 Myeloid overexpression mice can be used as an animal model for evaluating and screening drugs for treating SLE.
[0056] (1) Cyclophosphamide (CTX) treatment: The same method as in Example 1 (1) was used to obtain the target mice, namely TMEM187 loxP / loxP LysM -cre, and were raised normally until 10 weeks of age to spontaneously develop systemic lupus erythematosus, after which CTX 20 mg / kg was injected intraperitoneally every other day for 6 weeks. TMEM187 loxP / loxP LysM -cre+Ctx).
[0057] by TMEM187 loxP / loxP LysM -cre as a model group, with TMEM187 loxP / loxP As a control group.
[0058] (2) Detection of anti-dsDNA antibody content in mouse serum: Female mice in each group aged 14 to 16 weeks were deeply anesthetized and blood was collected from the heart. The blood was allowed to stand and then centrifuged to obtain serum. The anti-dsDNA antibody content in the serum was detected using an Elisa kit.
[0059] The results are as follows Figure 8 Shown: After CTX treatment, TMEM187 loxP / loxP LysM -cre female mice showed decreased levels of anti-dsDNA antibodies in their serum.
Claims
1. A method for constructing an animal model of systemic lupus erythematosus, characterized in that: The steps include: (1) Carry TMEM187 -loxp homozygous mice and LysM -cre tool mice were hybridized to produce F1 generation mice; (2) pairing the F1 generation mice to produce F2 generation mice; (3) Identify the genotype of F2 mice and select the genotype TMEM187 + / + LysM + / + Homozygous mice are used as target mice, and when they grow to 10 to 12 weeks of age, the systemic lupus erythematosus animal model is obtained.
2. The method according to claim 1, characterized in that The target mice are female mice.
3. The method according to claim 1, characterized in that The method further comprises: The target mice are induced with drugs to obtain the animal model; the drugs are IMQ cream or pristane.
4. The method according to claim 3, characterized in that The method of inducing with IMQ cream is as follows: applying IMQ cream on the ears of the target mice.
5. The method according to claim 4, wherein The induction method is as follows: 1.25 mg is applied to the target mice aged 6 to 8 weeks, three times a week, for 10 consecutive weeks.
6. The method according to claim 3, characterized in that The method of inducing with pristane is as follows: injecting pristane into the abdominal cavity of the target mice.
7. The method according to claim 6, wherein The induction method is as follows: a single intraperitoneal injection of 0.5 mL of pristane is performed on the target mice aged 8 to 10 weeks, followed by conventional feeding for 12 weeks.
8. Use of the animal model constructed by the method according to any one of claims 1 to 7.
9. The use according to claim 8, characterized in that This includes applications in systemic lupus erythematosus drug screening or systemic lupus erythematosus disease progression research.