CN102090373A - Human Crohn disease-simulating murine colitis model, and preparation method and use thereof - Google Patents

Abstract

The invention discloses a mouse colitis model simulating human Crohn's disease and its preparation method and application, (1) establishing the mouse colitis model by using TNBS enema method; (2) observing the mouse through gross physical signs and real-time observation (3) After the mouse was killed, the detection of clinical indicators was carried out, and the whole colon of the mouse was taken for length measurement, disease scoring, and histological examination to evaluate the establishment of the mouse model; ( 4) Spleen cells from mice were taken to examine the distribution of T cell subsets to establish the changes of immune cells in this model. The invention provides a research carrier for the clinical detection of colitis medication, and lays a foundation for the research and development of effective therapeutic drugs.

Description

Mouse colitis model simulating human Crohn's disease and its preparation method and application

technical field

The invention relates to the field of animal models, in particular to a mouse colitis model simulating human Crohn's disease, a preparation method and application thereof.

Background technique

Inflammatory bowel disease is a common chronic gastrointestinal disease, including ulcerative colitis and Crohn's colitis, whose etiology and exact pathogenesis are still unclear. At present, it is generally believed that the pathogenesis of IBD is a chronic autoimmune disease mainly manifested by intestinal inflammation due to abnormal immune response of genetically susceptible hosts to intestinal flora due to genetic defects and environmental factors. In recent years, with the improvement of people's living standards, this disease, which is traditionally believed to have a relatively high incidence only in Europe and the United States, is gradually increasing in Asia, and refractory IBD has also increased significantly. In the intestinal tract with inflammation, intestinal myofibroblasts are activated and upregulate a series of cytokines, chemokines, growth factors and adhesion molecules, increasing the secretion of soluble mediators and extracellular matrix factors that promote inflammation. This is quite different from the response of the normal gut, where the intestinal myofibroblasts are quiescent and inactive. The pathogenesis of IBD may be related to the dysfunction of the intestinal flora and the intestinal mucosal immune system. The abnormal function of T cells or the imbalance of cytokines will lead to the abnormality of the intestinal mucosal immune system. In the normal gut, effector and regulatory immune cells are kept stable through complex cytokine networks that regulate each other. In the inflamed gut, an exaggerated effector immune response or a lack of regulatory immune response to innocuous antigens in the intestinal mucosal system microflora can lead to intestinal mucosal inflammation and make the internal environment of the intestinal mucosal immune system Homeostasis favors inflammation.

Inflammatory bowel disease is closely related to autoimmunity. T lymphocytes are the central link in immune regulation, and CD4+ T cells play an extremely important role in experimental mucosal inflammation. CD4+ T cells are the main effector cells of intestinal mucosal inflammation. Effector CD4+ T cells rapidly divide and proliferate to form sensitized lymphocytes, which can also activate granulocytes, enhance the lethality of NK cells and LAK cells, and induce the expression of adhesion molecules and degranulation of mast cells to enhance the inflammatory response. CD4+ T cells are traditionally divided into two subgroups according to the production of cytokines and biological functions: Th1 cells and Th2 cells. Under the action of IL-12, CD4+ T cells are polarized into Th1 cells, forming Th1-type inflammation with elevated IFN-γ and TNF-α; under the action of IL-13, they are polarized into Th2 cells, and IL-4, Th2-type inflammation with elevated IL-5. Crohn's colitis is characterized by transmural granulomatous inflammation in the gastrointestinal tract. The site of inflammation is usually located in the terminal ileum and ascending colon. Crohn's colitis is a type of Th1 cell polarization-dominated disease. Naive T Under the induction of IL-12, IL-18 or IL-23, cells produce a series of pro-inflammatory cytokines, including IL-2 and IFN-γ. IFN-γ can promote the high expression of adhesion molecules in endothelial cells, thereby promoting the recruitment of inflammatory cytokines and the activation of macrophages. Ulcerative colitis is different from Crohn's colitis in that it is caused by the destruction of endothelial cells due to superficial inflammation, that is, ulcers, and the main affected parts are the rectum and colon. Ulcerative colitis is a typical disease dominated by the polarization of Th2 cells, and Th2 cells mainly produce cytokines such as IL-4, IL-5 and IL-10. When natural killer T cells are activated, a large amount of IL-13 is secreted, thereby mediating the inflammatory response, by producing free radicals and a large amount of pro-inflammatory factors TNF-α, IL-1, IL-6, IL-8, IL-12 and IL-18, thus producing a series of inflammation and joint reactions. Although both diseases are abnormal immune responses to antigens in the mucosal system, they lead to disease through distinct physiological pathways. Crohn's colitis is induced by IL-12 or IL-23, and the immune function is abnormal mediated by Th1 cells, while ulcerative colitis is under the action of IL-13 secreted by activated natural killer T cells. Abnormal immune function mediated by Th2 cells.

Contents of the invention

The technical problem to be solved by the invention is: to induce a stable colitis model through TNBS, and make it exhibit characteristics similar to human Crohn's disease, providing a research basis for drug screening of Crohn's disease.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a preparation method of a mouse colitis model simulating human Crohn's disease, comprising the following steps:

(1) Inject TNBS into mice by rectal perfusion; (2) Determine whether the mouse model is successfully established by detecting the general signs and clinical indicators of mice; (3) Detect mouse splenocytes by flow cytometry Distribution of T cell subsets to establish models of immune cell changes.

The general indications of the detected mice refer to: total colonic damage of the mice, weight loss, mental fatigue, loss of luster in coat color, diarrhea and blood in the stool.

The detection of clinical indicators refers to: intestinal tissue sampling and disease scoring, colon histopathological examination, including membrane congestion and edema, erosion, necrosis, multiple ulcers, deep ulcers, some ulcers reaching the serosa layer, severe necrosis, intestinal lumen Stenosis, the lesion reaches the end of the small intestine.

The examination of the distribution of T cell subsets by taking spleen cells from mice means that acute enteritis induced by TNBS exhibits Th1 type inflammation, and the inflammation characteristics of the Th1 model are similar to CD disease at the macroscopic and light microscope levels.

A mouse colitis model simulating human Crohn's disease is characterized in that it is obtained by the above method.

A mouse model of colitis simulating human Crohn's disease, and its application in screening drugs for the treatment of human Crohn's disease.

Beneficial effects of the present invention: the present invention provides a research carrier for the clinical detection of colitis medication, and lays the foundation for the development of effective therapeutic drugs.

Description of drawings

Figure 1 is the body weight change: the mice were weighed every day, and the body weight change was the percentage of the daily body weight divided by the initial day's body weight, and all data were expressed as mean ± standard deviation (*p<0.05).

Figure 2. Colon length. After the mice were sacrificed, the length of the whole colon was measured, and all data were expressed as mean ± standard deviation.

Figure 3. Histochemical scoring of HE-stained intestinal slices: Histochemical scoring of intestinal slices was performed under a light microscope, and all data were expressed as mean ± standard deviation.

Detailed ways

The preparation method of the mouse colitis model simulating human Crohn's disease of the present invention comprises the following steps:

(1) Inject TNBS into mice by rectal perfusion; (2) Determine whether the mouse model is successfully established by detecting the general signs and clinical indicators of mice; (3) Detect mouse splenocytes by flow cytometry Distribution of T cell subsets to establish models of immune cell changes.

The general indications of the detected mice refer to: total colonic damage of the mice, weight loss, mental fatigue, loss of luster in coat color, diarrhea and blood in the stool.

The detection of clinical indicators refers to: intestinal tissue sampling and disease scoring, colon histopathological examination, including membrane congestion and edema, erosion, necrosis, multiple ulcers, deep ulcers, some ulcers reaching the serosa layer, severe necrosis, intestinal lumen Stenosis, the lesion reaches the end of the small intestine.

The examination of the distribution of T cell subsets by taking spleen cells from mice means that acute enteritis induced by TNBS exhibits Th1 type inflammation, and the inflammation characteristics of the Th1 model are similar to CD disease at the macroscopic and light microscope levels.

A mouse colitis model simulating human Crohn's disease is characterized in that it is obtained by the above method.

A mouse model of colitis simulating human Crohn's disease, and its application in screening drugs for the treatment of human Crohn's disease.

The present invention will be described in further detail below in conjunction with accompanying drawing and specific embodiment: (seeing Fig. 1,2,3)

1. Establishment of TNBS mouse model: TNBS was injected into mice by rectal perfusion method to establish TNBS model.

1. The experimental animals are 8-week-old BALB/c mice with a weight of about 20 g, all purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences, and kept in SPF clean animal rooms. The animals were kept in an air-conditioned room with SPF clean grade, room temperature (22±2)°C, relative humidity 50%-60%, automatic regulation day and night for 12 hours, pellet feed and free water.

2. Fast for 12 hours and drink water freely.

3. The administration method refers to the internationally recognized Morris method 81. During the experiment, the mice were anesthetized with pentobarbital sodium at 30 mg/kg.

4. Insert a silicone tube with a diameter of 2 mm into the anus to a depth of about 4 cm, and perfuse a 50% ethanol solution (1:1) of trinitrobenzenesulfonic acid (150 mg/kg) to induce inflammation; animals in the control group are perfused with 50% ethanol solution.

5. Lift the animal upside down for 1 minute, so that the reagent can fully enter the entire colon.

6. Keep the animal lying flat and awake naturally.

2. Detection of clinical indicators of enteritis: the establishment of the mouse model was identified through the observation of general signs and anatomy.

1. Observation of general signs

Rats were observed daily for defecation, eating, diarrhea, and activities.

Body weight changes of the mice were measured daily.

2. Intestinal tissue sampling and disease scoring

(1) Five animals were sacrificed in each group on day 1, 3 and 5.

(2) The mice were sacrificed, the abdominal cavity was opened to separate the whole colon, and placed in D-hank's solution.

(3) The whole colon was spread naturally on a plate, and the length of the intestine was measured.

(4) Observe the intestinal appearance and make a score.

Gut Specimen Scoring

0 points: roughly normal,

1 point: no ulcer, local congestion,

2 points: Visible ulceration, but no hyperemia,

3 points: only 1 ulcer and inflammation,

4 points: 2 or more ulcers and inflammation,

5 points: the ulcer is longer than 2cm,

6 to 10 points: For ulcers > 2 cm, add 1 point for every 1 cm increase.

(5) Cut the intestinal wall along the attachment of the mesentery, rinse the intestinal contents,

(6) Spread it flat on tinfoil paper, observe the changes of intestinal mucosa, roll it up, place it in 4% paraformaldehyde solution for fixation, embed, section, and observe histopathological changes with HE staining light microscope.

3. Histopathological examination of the colon

Group scoring criteria:

0 points without obvious inflammation;

1 point A small amount of lymphocyte infiltration (≤10% high power field) without structural changes;

2 points Moderate lymphocyte infiltration (10%-25% high-power field), crypts become longer, intestinal wall thickens, but does not penetrate the mucosal layer, and no ulceration;

3 points Obvious lymphocyte infiltration (25%-50% high-power field), increased blood vessel density, thickened intestinal wall, and penetrated the mucosal layer;

4 points: Massive lymphocyte infiltration (≥50% high-power field), increased blood vessel density, elongated and distorted crypts, full-thickness intestinal wall, and visible ulceration.

3. Detection of the distribution of T cell subsets in mouse splenocytes

1. Spleen lymphocyte extraction

The mouse spleen lymphocyte extraction method is carried out according to the traditional gradient centrifugation method. Proceed as follows:

(1) The mice were sacrificed, and the spleen was removed.

(2) Cut off the spleen, squeeze out the cells in the spleen with the inner tube of the syringe, and discard the capsule.

(3) Resuspend the cells with PBS.

(4) Spleen lymphocytes were separated with mouse lymphocyte separation medium.

(5) Centrifuge at 2,000 rpm for 20 minutes.

(6) Absorb the buffy film, add PBS, and repeatedly blow and beat.

(7) Centrifuge at 1,500 rpm for 15 minutes.

(8) Discard the supernatant, add PBS, and pipette repeatedly.

(9) Centrifuge at 1,000 rpm for 10 minutes.

(10) Discard the supernatant and resuspend with RP-MI1640 culture medium to obtain spleen lymphocyte suspension with a yield of about (2-4)×107/monkey.

2. Detection of T cell subsets

(1) Collect mouse spleen lymphocytes and centrifuge at 1000g (about 1,000-2,000rpm) for 5 minutes, discard the supernatant, gently resuspend the cells in PBS and count them.

(2) Divide 2×105 resuspended cells into each tube, centrifuge at 1000 g for 5 minutes, discard the supernatant, add 3 μl of rat anti-mouse-CD3-PreCP antibody, and 3 μl of rat anti-mouse-CD4/CD8-PE antibody. Rat IgG1 was used as an isotype control.

(3) Incubate at room temperature (20-25°C) in the dark for 20 minutes.

(4) Add 1ml of PBS containing sodium azide and pipette repeatedly.

(5) Centrifuge at 1000 g for 5 minutes, discard the supernatant, and add 350 μl of 2% paraformaldehyde to resuspend the cells.

(6) Immediately carry out flow cytometry detection.

Table 1 Comparison of mouse spleen T cell subsets

Mononuclear cells CD3+CD4+(%) CD3+CD8+(%) CD4+/CD8+ control group 36.01±5.25 16.89±3.48 2.15±0.17

model group 48.39±10.15 10.76±4.23 4.69±2.22 therapy group 35.5±0.98 13.1±3.29 2.83±0.73

In summary, the content of the present invention is not limited to the above-mentioned embodiments, people of insight in the same field can easily propose other embodiments within the technical guidance of the present invention, but this embodiment All are included within the scope of the present invention.

Claims (6)

1. a preparation method of a mouse colitis model simulating human Crohn's disease, comprising the following steps: (1) Inject TNBS into mice by rectal perfusion; (2) Determine whether the mouse model is successfully established by detecting the general signs and clinical indicators of mice; (3) Detect mouse splenocytes by flow cytometry Distribution of T cell subsets to establish models of immune cell changes. 2. the preparation method of the mouse colitis model of simulating human Crohn's disease according to claim 1, is characterized in that, the general indication of described detection mouse refers to: mouse whole colon injury, weight loss, mental Lassitude, loss of luster, diarrhea and blood in the stool. 3. the preparation method of the mouse colitis model of simulating human Crohn's disease according to claim 1, is characterized in that, the detection of described carrying out clinical index refers to: intestinal tissue drawing and disease score, colon histopathological examination , including membrane congestion and edema, erosion, necrosis, multiple ulcers, deep ulcers, some ulcers reaching the serosal layer, severe necrosis, intestinal lumen narrowing, and lesions reaching the end of the small intestine. 4. the preparation method of the mouse colitis model of simulating human Crohn's disease according to claim 1, is characterized in that, described getting mouse splenocyte and carrying out T cell subpopulation distribution inspection refers to: acute enteritis induced by TNBS It manifests as Th1-type inflammation, and the inflammatory features of Th1 model are similar to CD disease under the naked eye and light microscope. A mouse colitis model simulating human Crohn's disease, characterized in that it is obtained by the method according to any one of claims 1-4. 6. A mouse colitis model simulating human Crohn's disease as defined in claim 5, the application in screening the medicine for treating human Crohn's disease.

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