Modeling method of Crohn disease mouse model

Abstract

The invention relates to the technical field of animal disease models, in particular to a modeling method of a Crohn disease mouse model, which comprises the following steps: step 1, drinking a mouse to be injured with 0.5-2% dextran sodium sulfate aqueous solution, and continuously feeding the mouse with animal feed for 3-10 days as an injury period; step 2, drinking the mouse subjected to DSS injury with purified water, and continuously feeding the mouse with animal feed for 6-20 days as a recovery period; and step 3, sequentially adopting the step 1 and the step 2 as a DSS injury cycle on the mouse in the step 2, and circularly treating for 1-4 times to obtain the Crohn disease mouse model. According to the invention, an animal model of high-protein diet combined with low-concentration DSS enteritis injury of young mice is established, and clinical phenotypes of Crohn's disease are more fully simulated, including the characteristics that intestinal inflammation which is difficult to repair still continuously exists after DSS administration is stopped, penetrating fibrosis repair is accompanied by slow and acute inflammatory cell infiltration, and'beaded 'lymphatic follicular hyperplasia and the like.

Description

Technical Field

[0001] The invention relates to the technical field of animal disease models, and in particular to a method for establishing a Crohn's disease mouse model. Background Art

[0002] Crohn's disease is common in the terminal ileum and adjacent colon, with abdominal pain, diarrhea and weight loss as the main symptoms. The patient's condition is usually chronic, and may also present with acute abdominal symptoms, which can be easily confused with other intestinal diseases. The onset of the disease is related to multiple factors such as environmental factors, genetics, intestinal flora and immunity, and there are obvious regional and racial differences. The high-risk age group is 15-40 years old, with no significant gender differences.

[0003] There are currently a variety of preclinical mouse models used in IBD research, and disease models obtained based on different induction methods have different uses. Common IBD mouse models include chemical induction, spontaneous mutation, adoptive T cell transfer, gene editing, and microbial induction.

[0004] The chemically induced IBD model is the most widely used preclinical mouse model of IBD, which is generally induced using dextran sodium sulfate (DSS), trinitrobenzene sulfonic acid (TNBS), oxazolidinone, etc. Its main advantages are relatively low cost and easy development.

[0005] However, in the prior art, the DSS-induced intestinal inflammation injury model phenotype is not sufficient to simulate Crohn's disease and the self-limiting chronic intestinal inflammation process. Model mice usually die due to excessive inflammation after stopping DSS administration, or the intestinal inflammation recovers and is completely repaired. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a method for establishing a Crohn's disease mouse model to solve the problems in the prior art.

[0007] To achieve the above purposes and other related purposes,

[0008] In a first aspect of the present invention, a method for establishing a Crohn's disease mouse model is provided, comprising the following steps:

[0009] Step 1: The mice to be injured are given 0.5-2% sodium dextran sulfate aqueous solution for drinking and animal feed for 3-10 days as the injury period;

[0010] Step 2: The mice injured by DSS were fed with purified water and animal feed for 6-20 days as a recovery period;

[0011] Step 3: The mice in step 2 are subjected to step 1 and step 2 in sequence as a DSS injury cycle, and the cycle treatment is repeated 1-4 times to obtain a Crohn's disease mouse model.

[0012] In an embodiment of the present invention, the treatment process of the mice to be injured in step 1 is: feeding the mice with animal feed for 3-10 days to obtain the mice to be injured.

[0013] In an embodiment of the present invention, the protein content in the animal feed is 20-60%.

[0014] In an embodiment of the present invention, the protein content in the animal feed is 35-45%.

[0015] In an embodiment of the present invention, the content of sodium dextran sulfate in the sodium dextran sulfate aqueous solution is 0.5-1.5%.

[0016] In an embodiment of the present invention, the content of sodium dextran sulfate in the sodium dextran sulfate aqueous solution is 0.8-1.2%.

[0017] In an embodiment of the present invention, the method for establishing a Crohn's disease mouse model comprises the following steps:

[0018] Step 1: The mice to be injured are given 0.8-1.2% dextran sulfate sodium aqueous solution for drinking and animal feed for 6-8 days as the injury period;

[0019] Step 2: The mice injured by DSS were fed with purified water and animal feed for 12 to 16 days as a recovery period;

[0020] Step 3: The mice in step 2 are subjected to step 1 and step 2 in sequence as a DSS injury cycle, and the cycle treatment is repeated 1-3 times to obtain a Crohn's disease mouse model.

[0021] In an embodiment of the present invention, the method for establishing a Crohn's disease mouse model comprises the following steps:

[0022] Step 1: The mice to be injured were given 1% dextran sulfate sodium aqueous solution to drink and animal feed to continue feeding for 7 days as the injury period;

[0023] Step 2: The mice injured by DSS were fed with purified water and animal feed for 14 days as a recovery period;

[0024] Step 3: The mice in step 2 are subjected to step 1 and step 2 in sequence as a DSS injury cycle, and the cycle treatment is repeated twice to obtain a Crohn's disease mouse model.

[0025] As described above, the method for establishing a Crohn's disease mouse model of the present invention has the following beneficial effects:

[0026] 1. The phenotype of the previously known mouse DSS enteritis injury model is not sufficient to simulate the chronic intestinal inflammation process of Crohn's disease that is difficult to limit by itself. The model mice usually die due to excessive inflammation after stopping DSS administration, or the intestinal inflammation recovers and is completely repaired. The present invention is based on the common transcriptome sequencing of biopsy samples of macaque disease animals and human disease patients, and it is known that high nitrogen metabolism and high protein diet are related to the onset of Crohn's disease.

[0027] 2. Crohn's disease usually develops in childhood. In order to establish a mouse animal model that better simulates the disease phenotype, the present invention established an animal model of high-protein diet combined with low-concentration DSS enteritis injury in young mice. The HPD+DSS injury model of young mice more fully simulates the clinical phenotype of Crohn's disease than previous models, including persistent intestinal inflammation that is difficult to repair after stopping DSS administration, penetrating fibrosis repair accompanied by chronic-acute inflammatory cell infiltration, and "beaded" lymphoid follicle hyperplasia. It is closer to the clinical characteristics of Crohn's disease than the previous simple DSS enteritis injury model, and proposes a new modeling method for basic research on Crohn's disease from the perspective of animal models. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the experimental feeding scheme for each group of mice.

[0029] Figure 2 The following are microscopic images of the intestines of mice in each group on Day 7.

[0030] Figure 3 The colon HE staining images of mice in each group on Day 7.

[0031] Figure 4 The colon HE staining images of mice in each group on Day 52.

[0032] Figure 5 The AB-PAS staining images of the colon of each group of mice on Day 52.

[0033] Figure 6 The colon SR staining images of mice in each group on Day 63.

[0034] Figure 7 The colon HE staining images of mice in each group on Day 63.

[0035] Figure 8 Immunofluorescence images of colon tissues in the HPD+DSS group.

[0036] Fig. 9 This is the immunofluorescence image of the colon tissue of the HPD+DSS group, which shows widespread "beaded" lymphoid follicle hyperplasia.

[0037] Fig.10This is the nutritional composition table of animal feed used in the experiment of simulating clinical Crohn's disease phenotype in young mice with high-protein diet combined with low-concentration DSS injury. DETAILED DESCRIPTION

[0038] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0039] High-protein diet combined with low-concentration DSS injury in young mice simulates clinical Crohn's disease phenotype

[0040] 1. Animals and Materials

[0041] The feeds in this experiment were provided by Chengdu Biopark Technology Co., Ltd. (Moldiets: 20% protein energy supply ratio, item number M10002; 40% protein energy supply ratio, item number M21091501).

[0042] Male C57BL / 6J mice were purchased from Chengdu Dashuo Animal Company.

[0043] 2. Experimental Grouping

[0044] Young male C57BL / 6J mice (3 weeks old, 12-15 g) were housed in the SPF animal room of Xinchuan Experimental Animal Center of Sichuan University and randomly divided into enteritis group and control group (as Figure 1 Under normal dietary conditions, protein energy supply accounts for about 20% of mice, so a normal protein diet group (20% protein energy supply ratio, standard diet, SD) and a high-protein diet group (40% protein energy supply ratio, High-protein diet, HPD) were established.

[0045] 3. Experimental plan

[0046] The control group was only fed with SD and HPD diets and had free access to normal drinking water throughout the whole process.

[0047] The enteritis group was fed adaptively with SD and HPD diets for one week, and then three rounds of low-concentration DSS damage were given on this basis. As shown in Figure 1, the red line indicates free drinking of DSS aqueous solution, and the blue line indicates free drinking of normal drinking water. The 7-day free drinking of DSS aqueous solution is the injury period, and the 14-day normal drinking water is the repair period, totaling 21 days as one cycle. This model is a chronic inflammation model that requires a total of 3 cycles, each cycle is 1% DSS concentration, in order to establish a low-concentration chronic injury background.

[0048] 4. Model experimental data

[0049] 4.1Day7 intestinal test (7 days after DSS use, corresponding to Figure 1 Day 7)

[0050] ①、 Figure 2 The following are microscopic images of the intestines of mice in each group on Day 7.

[0051] After the first round of DSS injury in young mice (samples were taken on day 7), an acute inflammatory state was observed, and intestinal inflammation was more severe in the HPD+DSS group. Figure 2 This is a gross image under a stereomicroscope. There is no significant change in the HPD diet alone compared with the SD diet. SD+DSS can show enteritis damage and ulcer formation, mainly in the distal colon, while HPD+DSS can show inflammatory damage and extensive ulcer formation in the entire colon (proximal + distal).

[0052] ②、 Figure 3 The colon HE staining images of mice in each group on Day 7.

[0053] The whole colon of each group of mice was fixed and pathologically sectioned after Swiss roll, and then HE (Hematoxylin-eosintaining, HE) staining was performed. The specific steps are summarized as follows: tissue fixation for 48 hours → section dewaxing and rehydration → hematoxylin staining → eosin staining → dehydration and transparency → sealing and image collection. The center of the Swiss roll is the direction of the distal colon.

[0054] from Figure 3 It can be seen that obvious inflammation was observed in both the SD+DSS and HPD+DSS groups. The SD+DSS group had obvious distal colon inflammation, epithelial layer destruction, and inflammatory cell infiltration, while the HPD+DSS group had more severe inflammation, with inflammation of the entire colon, intestinal epithelial cell damage, goblet cell loss, crypt destruction, and inflammatory cell infiltration in both the mucosa and submucosa.

[0055] 4.2Day52 intestinal micrograph (corresponding to Figure 1 Day 52 in

[0056] ①. Colon HE staining

[0057] Figure 4 The colon HE staining images of mice in each group on Day 52. ​​After the third round of DSS injury in young mice (samples were collected on Day 52), the distal and proximal colons were sampled, fixed, embedded and sectioned, and then HE staining was performed. The HE procedure was the same as above.

[0058] from Figure 4 It can be seen that the HPD diet alone did not significantly change the SD diet. The SD+DSS group still had mild inflammation, local epithelial destruction, and inflammatory cell infiltration. However, the HPD+DSS group had intestinal inflammation that was difficult to repair, extensive epithelial destruction, goblet cell loss, crypt destruction, inflammatory cell infiltration, and multiple mucosal and submucosal lymphoid follicle hyperplasia.

[0059] ②, Colon AB-PAS staining

[0060] Figure 5 The AB-PAS staining of the colon of each group of mice on Day 52. ​​After the third round of DSS injury in young mice (samples were collected on day 52), the distal and proximal colons were fixed, embedded and sliced, and then AB-PAS staining (Alcian blue-Periodic acid-schiffstain, AB-PAS) was performed using the Solebol kit (G1285). The specific steps are summarized as follows: dewaxing and rehydration of the slices → staining with Alcian blue staining solution for 15 minutes → washing with distilled water 3 times, 2 minutes each time → oxidation with iodic acid solution for 5 minutes → Schiff Reagent staining for 15 minutes → washing with running water for 10 minutes → staining with hematoxylin staining solution for 1 minute → differentiation with acid differentiation solution for 5 seconds and then washing with water → blueing with Scott blue solution → washing with water for 3 minutes → dehydration and transparency → sealing and image collection.

[0061] from Figure 5 AB-PAS staining showed that the mucus layer was destroyed in the HPD group compared with the SD group, and the number of goblet cells decreased. The mucus layer was obviously destroyed in the SD+DSS and HPD+DSS groups, and the mucus layer destruction was more serious in the HPD+DSS group. The epithelial layer was almost infiltrated by inflammatory cells, with only a small number of residual goblet cells.

[0062] 4.3Day63 intestinal micrograph (corresponding to Figure 1 Day 63 of

[0063] ① Colon SR staining

[0064] Figure 6 The following are the SR staining images of the colon of each group of mice on Day 63. After the third round of DSS injury in young mice (samples were collected on day 63), in order to further observe the changes in intestinal fibrosis, the colon tissues of each group of mice were stained with SR (Sirius Red) using the Solebow kit (G1472). The specific steps are summarized as follows: tissue fixation → dewaxing and rehydration of sections → dripping with Sirius Red staining solution for 10 minutes → rinsing with running water → dehydration and transparency → sealing and image collection.

[0065] SR staining showed chronic penetrating fibrosis in the colon tissue of the HPD+DSS group, which was similar to the phenotype of Crohn's disease, while the other groups did not have such significant transmural fibrosis.

[0066] ②Hematoxylin and eosin staining of colon

[0067] Figure 7The colon HE staining images of mice in each group on Day 63. HE staining was performed on young mice after the third round of DSS injury repair (samples were collected on Day 63). The HE steps were the same as above.

[0068] from Figure 7 As can be seen, HE staining of the HPD+DSS group showed that the colon tissue still had irreparable epithelial damage and inflammatory cell infiltration. Pathology showed chronic-onset penetrating fibrosis changes (HE, left column) and extensive "beaded" lymphoid follicle hyperplasia (HE, right column), while the other interventions did not show such manifestations.

[0069] 4.4 Immunofluorescence images of colon tissues in the HPD+DSS group

[0070] ① Immunofluorescence experiment of colon tissue in HPD+DSS group

[0071] Figure 8 This is the immunofluorescence image of colon tissue in the HPD+DSS group. The specific steps of immunofluorescence co-localization are summarized as follows: dewaxing and rehydrating the slices → antigen repair → washing the slides → blocking with donkey serum → preparing the co-staining target indicator antibodies as mixed primary antibodies in proportion → incubating the primary antibodies at 4°C overnight → washing the slides → preparing mixed secondary antibodies in proportion → incubating the secondary antibodies at room temperature for 30 minutes → washing the slides → DAPI staining the nuclei → washing the slides → sealing the slides with anti-fluorescence quencher and collecting images.

[0072] Figure 8 It was further described by immunofluorescence Figure 7 The penetrating lesion area of ​​colon tissue in the HPD+DSS group is shown in the middle HE. The upper picture shows the chronic inflammation field, and the lower picture shows the acute inflammation field: F4 / 80 shows macrophages, which reflect chronic inflammation in enteritis; MPO shows neutrophils, which reflect acute inflammation in enteritis; CD3 shows T cells, which are the cell types that affect the degree of inflammation in enteritis and are widely studied; Collagen1 shows fibroblasts, which are the outcome of fibrosis repair.

[0073] like Figure 8 As shown in the data, during the remission period, the intestinal damage in the HPD+DSS group was still difficult to completely repair, and was repaired by fibrosis accompanied by infiltration of neutrophils, macrophages, and T cells in the area of ​​fibrosis repair. The chronic-acute inflammation with transmural fibrosis changes was the difficult-to-repair intestinal inflammation outcome caused by HPD+DSS, which is also the most typical clinical feature of Crohn's disease patients.

[0074] ②. Immunofluorescence experiment of extensive lymphoid follicle hyperplasia in the colon tissue of the HPD+DSS group showed "beaded" appearance.

[0075] Fig. 9 This is an immunofluorescence image of extensive "beaded" lymphoid follicle hyperplasia in the colon tissue of the HPD+DSS group. Fig. 9 It was further described by immunofluorescence Figure 7 The HE images of the HPD+DSS group showed extensive and multiple lymphoid follicle proliferation in the colon tissue: CD19 indicated B cells, which are the main components of lymphoid follicles; CD3 indicated T cells, which surround B cells and provide co-stimulatory signals to form intestinal lymphoid structure tissue; Collagen1 indicated fibroblasts. The immunofluorescence co-localization steps were the same as above.

[0076] like Fig. 9 The extensive multiple proliferative lymphoid follicles formed in the colon are the phenotype of Crohn's disease simulated by HPD+DSS intervention. Clinically, it is believed that it is related to the occurrence and development of chronic intestinal inflammation.

[0077] In summary, the present invention has established an animal model of high-protein diet combined with low-concentration DSS enteritis injury in young mice, wherein the HPD+DSS injury model of young mice more fully simulates the clinical phenotype of Crohn's disease than previous models, including persistent intestinal inflammation that is difficult to repair after stopping DSS administration, penetrating fibrosis repair accompanied by chronic acute inflammatory cell infiltration, and "beaded" lymph follicle hyperplasia. It is closer to the clinical characteristics of Crohn's disease than the previous simple DSS enteritis injury model, and proposes a new modeling method for basic research on Crohn's disease from the perspective of animal models. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has a high industrial utilization value.

[0078] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for establishing a Crohn's disease mouse model, characterized in that: The steps include: Step 1: The mice to be injured are given 0.5-2% sodium dextran sulfate aqueous solution for drinking and animal feed for 3-10 days as the injury period; Step 2: The mice injured by DSS were fed with purified water and animal feed for 6-20 days as a recovery period; Step 3: The mice in step 2 are subjected to step 1 and step 2 in sequence as a DSS injury cycle, and the cycle treatment is repeated 1-4 times to obtain a Crohn's disease mouse model.

2. The method for establishing a Crohn's disease mouse model according to claim 1, characterized in that: The process of treating the mice to be injured in step 1 is as follows: feeding the mice with animal feed for 3-10 days to obtain the mice to be injured.

3. The method for establishing a Crohn's disease mouse model according to claim 1 or 2, characterized in that: The protein content in the animal feed is 20-60%.

4. The method for establishing a Crohn's disease mouse model according to claim 3, characterized in that: The protein content in the animal feed is 35-45%.

5. The method for establishing a Crohn's disease mouse model according to claim 1, characterized in that: The content of sodium dextran sulfate in the sodium dextran sulfate aqueous solution is 0.5-1.5%.

6. The method for establishing a Crohn's disease mouse model according to claim 5, characterized in that: The content of sodium dextran sulfate in the sodium dextran sulfate aqueous solution is 0.8-1.2%.

7. The method for establishing a Crohn's disease mouse model according to claim 4, characterized in that: The steps include: Step 1: The mice to be injured are given 0.8-1.2% dextran sulfate sodium aqueous solution for drinking and animal feed for 6-8 days as the injury period; Step 2: The mice injured by DSS were fed with purified water and animal feed for 12 to 16 days as a recovery period; Step 3: The mice in step 2 are subjected to step 1 and step 2 in sequence as a DSS injury cycle, and the cycle treatment is repeated 1-3 times to obtain a Crohn's disease mouse model.

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