A method for constructing and applying a mouse model of systemic lupus erythematosus.

Abstract

This invention discloses a method for constructing and applying a mouse model of systemic lupus erythematosus (SLE). The SLE mouse model constructed by this invention utilizes intraperitoneal injection of pristane combined with topical application of imiquimod cream. Specifically, C57 BL / 6J mice are injected intraperitoneally with pristane, followed by continuous application of an appropriate dose of imiquimod to a single ear. The SLE mouse model provided by this invention effectively simulates the immune response induced by pristane and imiquimod in mice. This model can be used to explore the pathogenesis, pathophysiology, immunology, and therapeutic drug development of SLE, and is of great significance for accelerating the translation of scientific research into clinical practice.

Description

Technical Field

[0001] The invention belongs to the field of animal experimental models and their preparation technology, specifically involving a systemic lupus erythematosus mouse model and its construction method and application. Background Technology

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by T and B lymphocyte disorders, the production of various autoantibodies, and multi-system and multi-organ involvement. When large amounts of autoantibodies, such as anti-dsDNA, accumulate in the kidneys, they lead to systemic or localized proliferative glomerular lesions, resulting in varying degrees of kidney damage and the formation of immune-mediated complex nephritis. This nephritis has an insidious onset and is easily overlooked. Its incidence rate in SLE patients is as high as 50-60%, significantly impacting their work capacity and quality of life, and even threatening their lives. It places a heavy burden on patients, their families, and society, and is a leading cause of death in SLE patients, representing the most severe characteristic of the disease.

[0003] Currently, two main types of mice are used to study the pathogenesis of SLE and novel treatment methods. These mouse models primarily exhibit lupus-like nephritis and the production of autoantibodies, including spontaneous and induced models. Spontaneous models include New Zealand Black / White F1 mice (NZB / W), Murphy Roths Large / Lpr mice (MRL / LPR), and BXSB / Yaa mice, while induced models are Pristane or Imiquimod induced mouse models. In spontaneous models, the onset time of disease is inconsistent, and some mice may not develop the disease at all; the onset time of the same batch of mice may differ by up to two months. The induction model also suffers from inconsistent and delayed onset, with symptoms typically appearing more than 5 months after Pristane induction. Overdose of Pristane induces widespread fatty granulomas in the peritoneum, spleen, and liver within a short period. Pathological examination reveals mononuclear phagocytes phagocytizing Pristane droplets, and T and B lymphocyte proliferation and aggregation forming ectopic lymphoid tissue, which can induce acute death in mice. Small doses of Pristane reduce mortality but are insufficient to induce disease, thus failing to successfully establish the model. The Imiquimod induction model requires continuous application of Imiquimod three times a week for more than 12 weeks to induce the lupus phenotype. Because it is an external application, mice will rub off some of the medication, making it difficult to ensure consistency between medication administration and disease onset. Therefore, to better explore the pathogenesis of SLE disease progression and its application in the development of drugs targeting lupus nephritis, a rapidly and stably established mouse model of systemic lupus erythematosus is urgently needed. Summary of the Invention

[0004] Purpose of the invention: To address the issues of delayed onset and inconsistent disease progression in traditional SLE modeling methods, this invention provides a mouse model of systemic lupus erythematosus (SLE). By immunizing C57BL / 6J mice with a combination of Pristane and Imiquimod, SLE symptoms can be stably developed in a short period of time. These symptoms are highly similar to those of clinical SLE patients, showing positive urine protein, positive anti-dsDNA, and renal IgG immune deposition. This model can effectively simulate the occurrence and development of human SLE, presenting typical SLE kidney damage. Moreover, it is simple to operate, has a rapid onset, strong disease consistency, and a short modeling time, making it an ideal, rapid, and stable modeling method.

[0005] To address the aforementioned technical problems, this invention discloses a method for constructing a systemic lupus erythematosus (SLE) mouse model. The method involves intraperitoneal injection of Pristane into healthy mice, combined with application of Imiquimod. The intraperitoneal injection is administered twice, with 270-330 μL each time. Imiquimod is applied as a single-ear smear once a week until the SLE mouse model is obtained. Successful model establishment can typically be determined by examining urinary protein, microurinary protein, plasma anti-dsDNA levels, and urinary protein content.

[0006] Preferably, mice are given an intraperitoneal injection of 270-330 μL of Pristane once in week 1 and week 2, and 2-2.5 mg / cm² of 5% imiquimod cream is applied to the inner side of each ear weekly. 2 A systemic lupus erythematosus mouse model was obtained after 65 days. More preferably, mice were intraperitoneally injected with 270-330 μL Pristane on day 0 and day 7, respectively. Starting from day 0, 5% Imiquimod 2-2.5 mg / cm² was applied to the inner side of one ear of the mice every 7 days. 2 The coating covers the entire inner ear of the mice, thereby triggering an autoimmune response until the SLE model is established on day 65. Pristane requires two injections, while Imiquimod is applied once a week.

[0007] The injection dose of pristane affects the survival time of mice. In the attempt to build this model, 500 μL of pristane was injected into the peritoneum at once, which caused the mice to lose weight rapidly within a month and one-third of them died. Therefore, the injection dose of pristane should be controlled within an appropriate range.

[0008] Preferably, the mouse is a C57BL / 6J mouse.

[0009] Preferably, after injecting the pristane, press for 10–40 seconds or seal the injection site with glue. Ensuring that the pristane does not overflow after injection effectively prevents mold failure. Pressing or sealing the injection site with glue after injecting the pristane can prevent this from happening.

[0010] Preferably, the intraperitoneal injection site is located 0.5cm ± 0.05cm to the side of the midline of the lower abdomen.

[0011] Preferably, during intraperitoneal injection, the mouse is restrained with its head tilted back, causing the abdominal organs to shift upwards, thereby avoiding damage to the organs. More preferably, the injection site and depth of Pristane should be such that they do not damage the organs, thus preventing the mouse from dying.

[0012] The systemic lupus erythematosus mouse model prepared by the method of this invention is also within the scope of protection of this application.

[0013] This application further proposes the application of the above-mentioned systemic lupus erythematosus mouse model in the study of SLE disease progression.

[0014] This invention also proposes the application of the above-mentioned systemic lupus erythematosus mouse model in screening drugs targeting lupus nephritis.

[0015] Beneficial effects: This invention, by immunizing C57 mice with a combination of Pristane and Imiquimod, can stably induce SLE symptoms in a short period of time. These symptoms are highly similar to those of clinical lupus patients, who show positive urine protein, positive anti-dsDNA, and renal IgG immune deposition. This is an ideal, rapid, and stable modeling method. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the treatment of C57 mice with Pristane (P) and / or Imiquimod (m) and the experimental process.

[0017] Figure 2 Routine monitoring of body weight and urinary protein in C57 mice after treatment with Pristane and / or Imiquimod;

[0018] Figure 3 The study included monitoring the anti-dsDNA antibody levels in the plasma of C57 mice after treatment with Pristane and / or Imiquimod, and analyzing the differences within each treatment group.

[0019] Figure 4 The pathological analysis of kidney tissue and immunofluorescence analysis of IgG1 / 2a antibody in each group of C57 mice after treatment with Pristane and / or Imiquimod;

[0020] Figure 5 The difference in IL17 secretion in plasma of C57 mice was observed between different treatments of Pristane and / or Imiquimod.

[0021] Figure 6 This is a schematic diagram of the gate logic for flow cytometry analysis of the spleen of C57 mice;

[0022] Figure 7 The changes in spleen and lymph nodes in C57 mice in each group after treatment with Pristane and / or Imiquimod, as well as the changes in the proportion of various T cells and B cells in the spleen. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0024] Laboratory animals

[0025] C57BL / 6J mice (also referred to as C57 mice), approximately 8 weeks old and weighing 17-19g, were provided by Beijing Speford Biotechnology Co., Ltd. The outer packaging was kept intact and sprayed with disinfectant. After sterilization under UV light for 30 minutes in a UV transfer window, they were introduced into an SPF-grade barrier environment. Husbandry conditions: room temperature 18-20℃, humidity 50-60%, alternating light and dark (12h), moderate light intensity, and good ventilation and cleanliness. All experiments were approved and conducted in accordance with the guidelines of the Ethics Committee of the Institute of Dermatology, Chinese Academy of Medical Sciences (Chinese Academy of Medical Sciences Institute of Dermatology).

[0026] Example: Construction of a mouse model of systemic lupus erythematosus.

[0027] Treatment method for mice:

[0028] All C57 mice were weighed and recorded, then arranged in ascending order of weight. Using a random number table, the C57 mice were randomly divided into four groups of eight mice each. Figure 1 ):

[0029] Group 1 (Blank): Mice received no treatment;

[0030] Group 2 (2P): On days 0 and 7, mice were intraperitoneally injected with 270-330 μL of Pristane (Sigma, P2870);

[0031] Group 3 (2P+1m / w): Mice were intraperitoneally injected with 270-330 μL of Pristane on days 0 and 7. Starting from day 0, 5% Imiquimod was applied to the inner side of one ear of each mouse every 7 days at a dose of 2-2.5 mg / cm². 2 ;

[0032] Group 4 (2m): Apply 5% Imiquimod at a concentration of 2-2.5 mg / cm² to the inner side of each ear of mice twice a week. 2 Each time, the interval is 3-4 days.

[0033] The intraperitoneal injection method is as follows: 0.5 cm to the side of the midline of the lower abdomen (level with the root of the thigh). In order to avoid damaging the organs, when fixing the mouse, its head should be tilted back slightly so that the organs in the lower abdomen are moved upward. After the needle enters the skin, it is lifted upward to ensure that no organs are punctured. Pristane is injected into the abdominal cavity in one go.

[0034] Imiquimod is administered once weekly at a dose of 2-2.5 mg / cm³. 2 Apply evenly to the entire inner ear; to avoid rubbing it off, apply it a little deeper.

[0035] Strict control is required for the intraperitoneal injection method and dosage. In our previous model construction attempts, a single injection of 500 μL Pristane into the peritoneal cavity resulted in rapid weight loss in mice within a month, with one-third of the mice dying. It is also crucial to ensure that the injection site and depth of Pristane are insufficient to damage organs and prevent mouse death. To avoid model failure, ensuring no leakage of Pristane after injection is also paramount; therefore, apply pressure for 20 seconds after injection or seal the injection site with glue.

[0036] 1) Detection of kidney damage in C57 mice by Pristane and Imiquimod treatment, either individually or in combination.

[0037] According to the needs of this study, we divided C57 mice into four groups and treated them according to the above-mentioned drug administration method. The mice were given the drug at 8 weeks of age. During the drug administration period, the mice's body weight, urine protein (Nanjing Jiancheng Biotechnology, C035-2-1), urine creatinine (Nanjing Jiancheng Biotechnology, C011-2-1), and micro-urinary protein (Elabscience, E-EL-M0792c) were monitored.

[0038] The results are as follows Figure 2 As shown, in the 6th week after drug administration, mice in the third group (2P+1m / w) began to develop symptoms. Compared with the blank control group, different treatments had no significant effect on the body weight of the mice. Figure 2A); In the third group, the combined treatment of Pristane and Imiquimod with C57 mice significantly reduced urinary protein levels (P<0.01). Figure 2 B) Inhibit the decrease in the urine protein / creatinine ratio in mice (P<0.01) Figure 2 C), and the level of microurinary protein (P<0.001) Figure 2 D), and the incidence was consistent, with little difference within the group. Figure 2 E); while neither Pristane nor Imiquimod showed any pathogenic effects when used alone or in combination, the second group, which received Pristane injection alone, showed minimal proteinuria at the end stage. Figure 2 Group D) was significantly higher than the blank control group (P<0.01), while there was no significant difference in urinary protein and the urinary protein / creatinine ratio (P>0.05); Group IV, using Imiquimod alone, showed significantly lower urinary protein levels at the end of the treatment period. Figure 2 B) and microurinary protein ( Figure 2 D) was significantly higher than that of the blank control group (P<0.01). This indicates that the combined use of Pristane and Imiquimod can accelerate the onset of lupus nephritis symptoms in mice and expedite disease progression.

[0039] Meanwhile, combined treatment with Pristane and Imiquimod increased the level of anti-dsDNA autoantibodies in the plasma of C57 mice. Figure 3 As shown, starting 4 weeks after administration, the plasma anti-dsDNA antibody levels in mice in the combined drug group began to increase significantly and rapidly compared to the blank control group (P<0.001). Figure 3 A) showed more stable pathogenicity than Pristane or Imiquimod alone, with smaller intragroup differences. Figure 3 B). This indicates that combined drug therapy can accelerate the production of autoantibodies and kidney damage in mice, thereby accelerating the onset and progression of SLE.

[0040] 2) Histopathological and immunofluorescence analysis of kidney tissue from C57 mice

[0041] At week 9 post-modeling, the levels of urinary protein, micro-urinary protein, and anti-dsDNA autoantibodies in plasma were significantly increased in mice, with urinary protein exceeding 100 mg / dL, indicating successful modeling in the third group of mice. To analyze renal inflammatory cell infiltration and pathological damage, mice were euthanized after anesthesia at week 9 post-administration. The kidneys were removed and cut open from the coronal plane. A portion was rapidly fixed in 4% paraformaldehyde and stained with hematoxylin and eosin (HE) to analyze the inflammatory cell infiltration in the kidneys. Another portion was placed in OCT tissue embedding medium, prepared into frozen sections, and subjected to immunofluorescence experiments to analyze the secretion of IgG1 (Abclonal, AS066) and IgG2a (Abclonal, AS065) antibodies in the kidneys of mice in different treatment groups.

[0042] The combined use of Pristane and Imiquimod increased the severity of kidney lesions and the deposition of IgG21 / 2a immune complexes in the kidneys of C57 mice. Figure 4 As shown, the histopathological results of mouse kidney tissue hematologic staining (HE staining and immunofluorescence staining) indicated that the third group, which combined the use of Pristane and Imiquimod, resulted in a significant increase in lymphocyte infiltration in the kidneys of C57 mice, enhanced glomerular swelling and proliferation, and a significantly higher pathological score compared to other groups. Figure 4 A), significantly higher than the blank control group (P<0.01), renal IgG1 (P<0.01) ( Figure 4 B) and IgG2a (P<0.05) Figure 4 C) A consistent increase in immune complex deposition was observed, while neither Pristane nor Imiquimod alone resulted in consistent pathological damage and immune complex deposition. This also indicates that the combined use of Pristane and Imiquimod can exacerbate lupus-induced kidney damage in mice.

[0043] 3) Effects of Pristane and / or Imiquimod treatment on serum IL17 secretion in C57 mice

[0044] After anesthetizing C57 mice, peripheral blood was collected from terminally ill mice and administered via LEGENDplex. TM The levels of cytokines IL-17A and IL-17F in the plasma of mice in each group were detected by flow cytometry using the BioLegend kit (740637).

[0045] Treatment of C57 mice with a combination of Pristane and Imiquimod increased plasma IL-17 levels. Figure 5As shown, peripheral blood was collected from mice in the terminal stage after anesthesia. The results showed that combined drug administration could increase the levels of IL-17A and IL-17F in peripheral blood. This indicates that combined treatment may further induce SLE by increasing the release of IL-17A and IL-17F cytokines from Tfh cells, while single drug administration could not increase the secretion of IL-17F cytokines.

[0046] 4) Regulation of T and B cells in the spleen of C57 mice by Pristane and / or Imiquimod treatment

[0047] C57 mice were anesthetized and sacrificed. The spleens were removed under aseptic conditions, ground, filtered, and used to prepare single-cell suspensions. Two × 10⁻⁶ cells were then collected from each suspension. 6 Splenic cells were subjected to flow cytometry staining for T cells and B cells, and then analyzed using FlowJo software (version 10.8.1). The results are shown in Figure 1.

[0048] Table 1

[0049]

[0050] like Figure 7 As shown, the spleen and lymph nodes of mice in the combined treatment group showed significantly increased swelling and weight compared to the blank control group, while there was no significant difference between the drug-only treatment and the blank control group; further flow cytometry analysis was performed on the mouse spleens, according to... Figure 6 The workflow analyzes various types of T / B cells, and the analysis results are as follows: Figure 7 As shown, compared with the blank control group, the combined treatment with Pristane and Imiquimod significantly increased the proportions of CD3+ T cells (P<0.01), Tfh cells (P<0.05), and Treg cells in the spleen of mice (P<0.05). Figure 7 A); Combined treatment also significantly increased the proportion of plasma cells (PC) (P<0.001) and germinal center B cells (GC B) (P<0.05) in the spleen of mice. Figure 7 B) This result indicates that the combined drug treatment enhances the inflammatory response of the immune organs, spleen and lymph nodes, increases the proportion of Tfh cells in the spleen, thereby promoting the development and function of PC and GC B cells and promoting the occurrence and development of SLE.

[0051] The systemic lupus erythematosus (SLE) mouse model provided by this invention accelerates the progression of SLE and effectively simulates the typical immune response and kidney damage associated with SLE. Many methods can induce SLE mouse models; the above-described method is merely a preferred embodiment of this invention, which can stably accelerate disease progression in mice. This model can be used to explore the mechanisms of SLE disease progression and to develop targeted SLE drugs, which is of great significance for accelerating the translation of scientific research into clinical practice. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention.

[0052] This invention provides a method and approach for constructing a systemic lupus erythematosus (SLE) diagnosis. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing a mouse model of systemic lupus erythematosus, characterized in that, Mice were administered intraperitoneal injections of 270-330 μL of Pristane on days 0 and 7, respectively. Starting from day 0, 5% imiquimod cream at a concentration of 2-2.5 mg / cm² was applied to the inner side of each ear every 7 days. 2 The coating was applied to cover the entire inner ear of the mice, thereby triggering an autoimmune response until the systemic lupus erythematosus model was established on day 65.

2. The construction method according to claim 1, characterized in that, The mice in question were C57BL / 6J mice.

3. The method according to claim 1, characterized in that, After injecting Pristane, press for 10-40 seconds or seal the injection site with glue.

4. The construction method according to claim 1, characterized in that, The intraperitoneal injection site is located 0.5cm ± 0.05cm to the sides of the midline of the lower abdomen.

5. The construction method according to claim 1, characterized in that, When administering an intraperitoneal injection, fix the mouse so that its head is tilted back and its lower abdominal organs are moved upward to avoid accidental damage to the organs.

6. The application of the systemic lupus erythematosus mouse model prepared by the construction method according to any one of claims 1-5 in the study of SLE disease progression.

7. The application function of the systemic lupus erythematosus mouse model prepared by the construction method according to any one of claims 1-5 in screening drugs targeting lupus nephritis.

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