A method and system for constructing a model of intestinal fibrosis and creeping fat animals

Abstract

The application belongs to the technical field of animal model construction, and discloses a method for constructing an intestinal fibrosis and creeping fat animal model, which comprises the following steps: shocking the colon of an animal, wherein the shocking comprises the following steps: placing the animal on an electrode plate, inserting a dual cutter head end into the colon of the animal from the anus of the animal, and shocking a predetermined position of the colon by generating a transient high-frequency current at the dual cutter head end; shocking the colon of the animal again after a period of time, and repeating the above steps multiple times. The existing methods for constructing an intestinal fibrosis model mainly include chemical agent induction, microbial induction, immune mediation, gene modification and the like, and all of the methods have some defects and cannot form creeping fat. The animal model constructed by the application solves the problem.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and in particular to a method and system for constructing an intestinal fibrosis and reptile fat animal model. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Intestinal fibrosis is a disease characterized by excessive deposition of extracellular matrix (primarily collagen fibers) in the mucosa, submucosa, and muscularis, replacing normal tissue. It often causes intestinal stenosis and structural damage and is a significant complication of Crohn's disease (CD). Ten years after initial diagnosis, nearly 70% of CD patients experience clinical symptoms such as abdominal pain, flatulence, nausea, vomiting, and constipation due to intestinal fibrosis and strictures, and over 30% require endoscopic or surgical treatment. Creeping fat is a typical extraintestinal manifestation unique to CD, defined as mesenteric adipose tissue surrounding an inflamed and fibrotic intestinal wall, and is closely associated with intestinal fibrosis and strictures in CD.

[0004] Currently, the mechanism of intestinal fibrosis in CD is still unclear and there is no effective clinical treatment, and the effect of drug treatment is still unsatisfactory. In order to study the pathogenesis and treatment methods, suitable animal models are indispensable. Existing intestinal fibrosis models mainly include chemical-induced models, microbial-induced models, immune-mediated models, genetic modification models, spontaneous colitis models, small intestinal heterotopic transplantation models, etc. These models have some disadvantages, such as high difficulty, poor stability, low controllability, inability to observe the development of the disease, long time, or susceptibility to microbial influences, and none of them can form creeping fat. The only existing animal model for the formation of creeping fat is to repeatedly biopsy fixed positions in the colon using a mouse colonoscope. This method is expensive and requires specialized technical training, which is not easy to promote. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the purpose of the embodiments of the present invention is to provide a method and system for constructing an animal model of intestinal fibrosis and creeping fat. By using high-frequency current to generate electric shocks to the animal intestine, collagen fiber proliferation and deposition occur in the colon mucosa, submucosa and corresponding muscular layer, thereby generating mesenteric fat surrounding the inflamed and fibrotic intestine, simulating the intestinal fibrosis and creeping fat of CD patients.

[0006] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A method for constructing an animal model of intestinal fibrosis and creeping fat, comprising: performing electric shock on the colon of the animal, wherein the electric shock comprises:

[0008] Place the animal on the electrode plate and insert the dual blade tip into the animal's colon through the animal's anus;

[0009] The dual blade head generates instantaneous high-frequency current to shock the predetermined position of the colon;

[0010] After a period of time, the animal's colon is shocked again and this is repeated several times.

[0011] The dual blade tip of the present invention generates a transient high-frequency current that shocks the animal's colon. Repeating this process repeatedly causes collagen fiber proliferation and deposition in the colonic mucosa, submucosa, and corresponding muscularis, while also generating mesenteric fat surrounding the inflamed and fibrotic intestine, simulating the intestinal fibrosis and creeping fat seen in patients with CD. The instantaneous high-frequency current generated by the dual blade tip allows for precise control of the shock, improving the controllability and stability of the model.

[0012] In some embodiments, the colon of the mouse is electroshocked.

[0013] In some embodiments, electric shocks are applied to multiple locations on the dorsal side of the colon, with adjacent locations spaced a certain distance apart. The dorsal side of the colon is the back of the colon lumen when the mouse lies flat.

[0014] In some embodiments, electric shocks are delivered to multiple locations on the dorsal side of the colon, namely 4 cm, 3.5 cm, 3 cm, 2.5 cm, and 2 cm from the anus.

[0015] In some embodiments, the shocks are administered once per week for a total of four cycles.

[0016] In some embodiments, the electrode plate is a neutral electrode plate.

[0017] In some embodiments, a preparation operation is further included, wherein the preparation operation includes selecting an electrocautery mode and adjusting the voltage value to 4-8V.

[0018] In some embodiments, the preparation operation is to connect the high-frequency electric knife power cord, connect the neutral electrode plate, dual knife and foot switch, turn on the power switch, select the electric cutting mode, and adjust the voltage value to 5V.

[0019] In some embodiments, the animal is a mouse, preferably a male mouse of 8-10 weeks old.

[0020] In some embodiments, the mouse is pretreated before electric shock, and the pretreatment includes anesthetizing the mouse and cleaning the mouse's colon of feces.

[0021] In some embodiments, the electric shock is performed using a high-frequency electrosurgery device in combination with a dual knife.

[0022] Another embodiment of the present invention provides a system for constructing an intestinal fibrosis and reptile fat animal model, including: a high-frequency electric knife device, a foot switch, a neutral electrode plate, a dual knife, a high-frequency electric knife device power cord, a high-frequency electric knife device and dual knife connection cable, and a high-frequency electric knife device and neutral electrode plate connection cable.

[0023] In some embodiments, the dual knife includes a handle, an outer sheath and a middle guide wire. The guide wire is retractable and can be fixed in the extended and retracted states. The guide wire includes a head end. The sheath diameter is smaller than the inner diameter of the mouse colon tissue. The guide wire head end is in a retracted state when inserted into the anus and colon. When the knife wire is extended, it exits the colon while delivering an electric shock, thereby not damaging the colon.

[0024] The high-frequency electric knife device is used to provide current, which is inserted into the colon of the animal through the dual knife. The head end emits current to shock the colon. The current is an instantaneous high-frequency current of 5V.

[0025] One or more technical solutions provided in the embodiments of the present invention have at least the following technical effects or advantages:

[0026] 1. The present invention uses a dual blade end to generate instantaneous high-frequency current to shock the dorsal side of the colon to prepare a model, which reduces the trauma of animal surgery and helps to improve the recovery speed of the animal.

[0027] 2. The present invention uses high-frequency electrosurgery technology to precisely control the electric shock to the intestinal tissue of mice. Compared with the intestinal fibrosis model caused by commonly used chemical inducers, the controllability and stability of the model establishment are improved.

[0028] 3. By simulating the changes in inflammation and fibrosis in the colon, the present invention can more realistically observe the development process of intestinal fibrosis and creeping fat in intestinal diseases.

[0029] 4. The present invention adopts high-frequency electric knife equipment and dual knife head for use, which has the advantages of small size, simple operation, easy use and low price. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 This is a typical Masson staining image of the colon section of the mouse model group of the present invention;

[0032] Figure 2 This is the gross appearance of the colon of mice in the model group of the present invention;

[0033] Figure 3This is a typical Masson staining image of the colon section of mice in the control group of the present invention;

[0034] Figure 4 This is the gross appearance of the colon of mice in the control group of the present invention;

[0035] Figure 5 Schematic diagram of the operation of extending the dual blade tip into the mouse colon tissue from the mouse anus.

[0036] In order to show the positions of various parts, the distances or sizes between them are exaggerated. The schematic diagram is for reference only. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0038] As introduced in the background technology, the construction of existing intestinal fibrosis models mainly includes chemical induction, microbial induction, immune mediation, gene modification, etc. The existing model construction methods have some defects and it is difficult to form creeping fat. In order to solve the above technical problems, the present invention proposes a method for constructing an intestinal fibrosis and creeping fat animal model.

[0039] A method for constructing an animal model of intestinal fibrosis and creeping fat, comprising: performing electric shock on the colon of the animal, wherein the electric shock comprises:

[0040] Place the animal on the electrode plate and insert the dual blade tip into the animal's colon through the animal's anus.

[0041] The dual blade head generates instantaneous high-frequency current to shock the predetermined position of the colon;

[0042] After a period of time, the animal's colon is shocked again and this is repeated several times.

[0043] To facilitate understanding of the present invention, the following examples are provided:

[0044] In the following examples, the instruments used include:

[0045] High-frequency electrosurgical unit manufactured by Shandong Yuhua Electric Co., Ltd., model number YH300;

[0046] Neutral electrode plate, model GPD-Ⅱ;

[0047] The dual knife is an Olympus dual knife, model KD-650L;

[0048] Foot switch.

[0049] The animals involved in the following examples are 8-10 week old male C57BL / 6J mice, from Beijing Vital River Laboratory Animal Technology Co., Ltd., with a body weight of about 20-25 g.

[0050] Example 1

[0051] The present embodiment provides a system for constructing an intestinal fibrosis and creeping fat animal model, comprising a high-frequency electrotome device, a foot switch, a neutral electrode plate and a dual knife, the foot switch, the neutral electrode plate and the dual knife are respectively connected to the high-frequency electrotome device through interfaces, and the high-frequency electrotome device is connected to a power supply through a power supply line.

[0052] The high-frequency electrotome device is also provided with a power switch for controlling the on-off of the current of the high-frequency electrotome device. The foot switch is used to control the on-off of the high-frequency current of the dual knife.

[0053] The dual knife is composed of a handle, an outer sheath tube and a middle guide wire, the knife wire is telescopic and can be fixed in the extended and retracted state. The sheath tube has a diameter of about 2 mm, which is smaller than the inner diameter of the colon tissue of the mouse, and the knife wire head end is in the retracted state when it is inserted into the anus and colon, and the knife wire is withdrawn from the colon while being electrified when it is extended, so as not to damage the colon.

[0054] Example 2

[0055] The present embodiment constructs an intestinal fibrosis and creeping fat animal model, and the method is as follows:

[0056] All mice are raised in the same environment, with a room temperature of 18-25℃ and a humidity of 40%-50%, and are fed with standard solid feed. The 24h natural light and dark cycle is used. The mice are evenly divided into two groups according to the body weight, i.e. a control group and a model group, with 20 mice in each group.

[0057] The dual knife, the neutral electrode plate and the foot switch are connected to the high-frequency electrotome device, the high-frequency electrotome power line is connected, the power switch is turned on, the cutting mode is selected, and the voltage value is adjusted to 5V.

[0058] All mice are fasted for 24h (without water restriction) before being electrified.

[0059] Then, the mice in the model group were subjected to the following operations: the mice were anesthetized and the colon feces of the mice were cleaned. After anesthesia, the mice in the model group were placed on the electrode plate, and the dual blade tip was inserted into the mouse colon from the mouse anus until it was 4 cm away from the anus. The foot switch was controlled to make the dual blade tip generate instantaneous high-frequency current, and the dorsal colon was shocked. The shock positions were 4 cm, 3.5 cm, 3 cm, 2.5 cm and 2 cm from the anus, a total of 5 points. The shock was once a week for a total of 4 cycles.

[0060] The mice in the control group were only subjected to the following operations: the mice were anesthetized and the feces in the mouse colon were cleaned. The anesthetized mice in the control group were placed on the electrode plate, and the dual blade tip was inserted into the mouse colon from the mouse anus until it was 4 cm away from the anus, and then directly withdrawn. No electric shock was performed during this process.

[0061] All mice had free access to food and water after treatment.

[0062] Evaluation test:

[0063] (1) Statistical indicators: Compared with the control group, the colon tissue of mice in the model group showed intestinal fibrosis and creeping fat ratio.

[0064] (2) Pathological evaluation: After the fourth week, the mice were killed and the samples were collected. After the mice were killed by cervical dislocation, the colon was immediately dissected and the presence of creeping fat on the outside of the colon was observed with the naked eye. If creeping fat was found, the colon was collected together with the sample. If no creeping fat was found, the entire colon was collected. All tissues were fixed with 4% paraformaldehyde, routinely dehydrated and paraffin-embedded, sectioned, and histologically stained. The severity of intestinal fibrosis was observed using an optical microscope.

[0065] Here are the results:

[0066] (1) Figure 2 As shown in Figure 2, all 20 model group mice had intestinal fibrosis, and 10 of them had creeping fat (mesenteric fat tissue surrounding the intestine) by gross observation; Figure 4 As shown, the control group showed no intestinal fibrosis and creeping fat. It can be seen that the treatment method of the present invention has a good effect in that the intestinal fibrosis model construction ratio is 100% and the creeping fat model construction ratio is about 50%.

[0067] (2) Pathological evaluation results: Figure 1 As shown in the figure, Masson staining of the colon of the model group mice showed that the whole layer of intestinal fibrosis occurred at the site of electric shock, and creeping fat appeared on the serosal side of the intestinal wall at the site of intestinal fibrosis, surrounding the intestinal wall at the site of intestinal fibrosis. Figure 3 As shown in the figure, the control group did not develop intestinal fibrosis and creeping fat. This shows that the electric shock method of the present invention has higher controllability and stability.

[0068] This method uses electric shock to induce collagen fiber proliferation and deposition in the colonic mucosa, submucosa, and corresponding muscularis, simulating the progression of intestinal fibrosis. We also found that mesenteric fat surrounding the intestine in areas of colonic fibrosis in mice mimics the formation of creeping fat in Crohn's disease. This animal model facilitates in-depth research into the mechanisms underlying intestinal fibrosis, the formation of creeping fat, and its relationship to the development and progression of intestinal fibrosis.

[0069] Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without any creative work are still within the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of intestinal fibrosis and reptilian fat, characterized in that: include: The animal's colon is subjected to electric shock, the electric shock comprising: Place the animal on the electrode plate and insert the dual blade tip into the animal's colon through the animal's anus; The dual blade head generates instantaneous high-frequency current to shock the predetermined position of the colon; After a certain period of time, the animal's colon is shocked again, and this is repeated several times; The animal is a mouse.

2. The method for constructing an intestinal fibrosis and reptilian fat animal model according to claim 1, wherein: Electric shocks are delivered to the dorsal area of ​​the colon, with adjacent areas spaced a certain distance apart.

3. The method for constructing an intestinal fibrosis and reptilian fat animal model according to claim 1, wherein: Electric shocks were applied to multiple locations on the dorsal side of the colon, namely 4 cm, 3.5 cm, 3 cm, 2.5 cm and 2 cm from the anus.

4. The method for constructing an animal model of intestinal fibrosis and reptilian fat according to claim 1, wherein: Electric shock was given once a week for a total of 4 cycles.

5. The method for constructing an intestinal fibrosis and reptilian fat animal model according to claim 1, wherein: The electrode plate is a neutral electrode plate.

6. The method for constructing an intestinal fibrosis and reptilian fat animal model according to claim 1, wherein: The method also includes a preparation operation, which includes selecting an electric cutting mode and adjusting a voltage value to 4-8V.

7. The method for constructing an intestinal fibrosis and reptilian fat animal model according to claim 6, wherein: The preparation operation is to connect the high-frequency electric knife power cord, connect the neutral electrode plate, dual knife and foot switch, turn on the power switch, select the electric cutting mode, and adjust the voltage value to 5V.

8. The method for constructing an intestinal fibrosis and reptilian fat animal model according to claim 1, wherein: The animals were male mice aged 8-10 weeks.

9. The method for constructing an intestinal fibrosis and reptilian fat animal model according to claim 1 or 8, wherein: The mice were pretreated before electric shock, which included anesthetizing the mice and cleaning the feces from their colons.

10. A system for constructing an animal model of intestinal fibrosis and reptilian fat, characterized in that: The system implements the method for constructing an intestinal fibrosis and reptilian fat animal model according to any one of claims 1 to 9; Including: high-frequency electrosurgical equipment, foot switch, neutral electrode plate, dual knife, high-frequency electrosurgical equipment power cord, dual knife connecting cable, neutral electrode plate connecting cable; The high-frequency electric knife device is used to provide current, and is inserted into the colon of the animal through the dual knife to emit current to shock the colon. The current is an instantaneous high-frequency current of 4-8V.

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