Construction method of atherosclerotic plaque thrombosis model

Abstract

The invention relates to the technical field of animal model construction, and particularly discloses a construction method of an atherosclerotic plaque thrombosis model. Comprising the following steps: 1) selecting carotid artery constricted ApoE- / -mice fed with high-fat feed; and 2) injecting LPS (Lipopolysaccharide) into the abdominal cavity to obtain the atherosclerotic plaque thrombosis animal model. The construction method provided by the invention is simple to operate and high in repeatability, atherosclerotic plaque thrombus formation can be induced under the stimulation of low-dose LPS, the thrombus occurrence rate equivalent to the thrombus occurrence rate in the prior art can be obtained, the construction method can be closer to the formation of atherosclerotic thrombus related to clinical endotoxemia, and the clinical pathological process can be better simulated.

Description

Technical Field

[0001] The invention relates to the technical field of animal model construction, in particular to a method for constructing an atherosclerotic plaque thrombosis model. Background Art

[0002] Atherosclerosis is one of the most common cardiovascular diseases in humans. Atherosclerotic thrombosis can lead to cardiovascular and cerebrovascular events such as myocardial infarction and cerebral infarction. Inflammation is the main factor leading to atherosclerotic thrombosis. Among them, lipopolysaccharide (LPS) can induce endotoxemia and is one of the main causes of inflammation in the body. However, due to species differences, ApoE - / - Mice cannot spontaneously form atherosclerotic thrombi, and even a high dose of LPS (3 mg / kg) injected intraperitoneally cannot induce atherosclerotic plaque thrombosis. There are few reports on animal models of atherosclerotic plaque thrombosis. Jen Erh Jaw et al. reported in 2016 that LPS (3 mg / kg) administered via the lungs of mice using a spray device can induce ApoE - / - Atherosclerotic plaque thrombosis in the brachiocephalic region of mice (Lung exposure to lipopolysaccharide causes atherosclerotic plaque destabilisation. Eur Respir J. 2016). However, the atherosclerotic thrombosis shown in the article does not have a typical atherosclerotic plaque, and there are few research papers citing their experimental methods when searching on the Web of Science website. In addition, the use of a spray device for pulmonary administration requires special devices and operations, which is difficult to promote and apply. This laboratory has also failed to replicate the reported results. Moreover, the 3 mg / kg dose of LPS is also relatively high and not very close to clinical endotoxemia. Therefore, there is currently a lack of an endotoxemia-related atherosclerotic thrombosis model that is simple to operate, highly reproducible, and close to clinical practice. Summary of the invention

[0003] The purpose of the present invention is to provide a method for constructing an atherosclerotic plaque thrombosis model that is simple to operate, highly repeatable, and closer to clinical practice, in view of the deficiencies in the prior art.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] A method for constructing an atherosclerotic plaque thrombosis model comprises the following steps:

[0006] 1) Select ApoE with carotid artery constriction fed a high-fat diet - / - Mice;

[0007] 2) Intraperitoneal injection of LPS to obtain an animal model of atherosclerotic plaque thrombosis.

[0008] In the method for constructing an atherosclerotic plaque thrombosis model as described above, preferably, male mice or female mice are used in step 1).

[0009] In the method for constructing an atherosclerotic plaque thrombosis model as described above, preferably, the mice in step 1) are 15-20 weeks old.

[0010] In the method for constructing the atherosclerotic plaque thrombosis model as described above, the high-fat feed mass ratio is preferably: casein 22.18%, L-cystine 0.334%, corn starch 23.51%, maltodextrin 7.87%, sucrose 13.794%, cellulose 5.54%, soybean oil 2.774%, cocoa butter 17.19%, sodium amide 0.29%, mixed mineral S10020 0.55%, calcium hydrogen phosphate 1.44%, calcium carbonate 0.61%, potassium citrate monohydrate 1.834%, vitamin mixture 0.110%, choline bitartrate 0.224%, cholesterol 1.25%, sodium cholate 0.50%. The high-fat feed calorie ratio is: protein 19.97%, fat 39.85%, carbohydrate 40.18%, totaling 4.51 kcal / g.

[0011] In the method for constructing an atherosclerotic plaque thrombosis model as described above, preferably, the high-fat diet is fed for 8-12 weeks in step 1).

[0012] In the method for constructing an atherosclerotic plaque thrombosis model as described above, it is preferred that the ApoE of carotid artery stenosis in step 1) - / - Mice with ApoE carotid artery constriction for 3-4 weeks - / - Mouse.

[0013] In the method for constructing an atherosclerotic plaque thrombosis model as described above, preferably, the step 1) is right carotid artery constriction or left carotid artery constriction.

[0014] In the method for constructing an atherosclerotic plaque thrombosis model as described above, the preferred dose of LPS for intraperitoneal injection is 0.5-1 mg / kg.

[0015] The present invention also provides ApoE in carotid artery constriction in patients fed with a high-fat diet. - / - The use of mice in preparing animal models is used to prepare atherosclerotic plaque thrombosis models. - / -The mice were intraperitoneally injected with LPS to obtain an animal model of atherosclerotic plaque thrombosis.

[0016] The present invention has the advantages that:

[0017] The present invention selects ApoE with carotid artery stenosis fed with high-fat diet - / - Mice were stimulated by intraperitoneal injection of LPS to successfully construct an animal model of atherosclerotic plaque thrombosis. Compared with the prior art method of using a spray device to spray 3mg / kg LPS through the mouse lungs, the construction method provided by the present invention is not only simple to operate and highly repeatable, but can also induce atherosclerotic plaque thrombosis under low-dose 1mg / kg LPS stimulation, and can obtain a thrombosis incidence rate comparable to that of the prior art, which can be closer to the formation of atherosclerotic thrombosis associated with clinical endotoxemia and better simulate clinical pathological processes. The method of the present invention can significantly reduce the cost of modeling experiments, shorten the modeling time, has strong repeatability, and is more practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Attached Figure 1 Results of the detection of atherosclerotic plaque thrombosis at the carotid artery constriction induced by LPS. (A) Schematic diagram of the structure of the carotid artery constriction; (B) HE staining and immunohistochemical staining of platelet markers (vWF and CD41) showed atherosclerotic thrombosis at segment I; (C) The incidence of atherosclerotic thrombosis in different experimental groups. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0020] The present invention provides a method for constructing an atherosclerotic plaque thrombosis model, wherein an ApoE-positive rat with carotid artery stenosis fed with a high-fat diet is selected. - / -Mice were intraperitoneally injected with LPS to obtain an animal model of atherosclerotic plaque thrombosis. The experimental results show that the construction method of the present invention can successfully induce atherosclerotic plaque thrombosis at an extremely low dose of 0.5 mg / kg LPS stimulation, and increasing the LPS stimulation dose to 1 mg / kg can increase the incidence of atherosclerotic thrombosis to more than 60%. Compared with the prior art method of using a spray device to perform 3 mg / kg LPS spray stimulation through the mouse lungs, the present invention is not only simple to operate and highly repeatable, but also can obtain a thrombosis incidence rate comparable to the prior art under a low dose of 1 mg / kg LPS stimulation, which can be closer to the formation of atherosclerotic thrombosis associated with clinical endotoxemia and better simulate the clinical pathological process.

[0021] ApoE for carotid artery stenosis described in the specific embodiments of the present invention - / - ApoE-mediated carotid artery constriction in mice - / - Model animal, ApoE in carotid artery constriction - / - Model mice can be purchased commercially, for example, they can be 15-20 week old male right carotid artery constricted apolipoprotein E knockout mice fed a high-fat diet constructed by Shanghai South Model Organisms Technology Co., Ltd. - / - Based on the mouse model, ApoE was physically - / - Carotid artery constriction in mice, obtaining ApoE in carotid artery constriction - / - Mice, including physical methods such as ligation, thread plug, U-shaped silver clip and balloon constriction of ApoE - / - The left and right carotid arteries of mice. Or ApoE - / - Carotid artery constriction of mice: use isoflurane to perform gas anesthesia on mice, and fix the mice in a supine position after anesthesia; use depilatory cream to remove the hair on the mouse neck, and wipe with 75% alcohol for disinfection; cut the skin of the mouse neck along the midline of the neck to expose and separate the right common carotid artery; use 6-0 surgical sutures to tie the right common carotid artery with a fine needle with a diameter of 150μm about 3mm below the bifurcation of the internal and external carotid arteries, then withdraw the fine needle, suture the skin, and complete the right carotid artery constriction. According to the principle of the present invention, those skilled in the art can also select ApoE from different sources for carotid artery constriction that are fed with a high-fat diet about 6 weeks before and about 4 weeks after constriction. - / - The mice were modeled and stimulated by intraperitoneal injection of 0.5-1 mg / kg LPS to obtain an animal model of atherosclerotic plaque thrombosis. - / - Methods for constructing atherosclerotic plaque thrombosis models in mice are all within the protection scope of the present invention.

[0022] Example 1 Construction and identification of lipopolysaccharide (LPS)-induced atherosclerotic plaque thrombosis model

[0023] 1. Methods

[0024] Model construction method: 16-week-old male apolipoprotein E knockout mice (ApoE knockout mice) that had been fed a high-fat diet for 10 weeks and had right carotid artery constriction for 4 weeks were selected. - / - ) was used to model carotid artery atherosclerosis and thrombosis were induced by intraperitoneal injection of different concentrations of LPS. After 24 hours, the mice were anesthetized and samples were collected for pathological examination.

[0025] Basic feed: The mass ratio is casein 14%, corn starch 49.6%, maltodextrin 12.5%, sucrose 10%, cellulose 5%, soybean oil 4%, L-cystine 0.18%, mixed minerals S10022M 3.5%, mixed vitamins V100371%, choline bitartrate 0.25%. The caloric ratio of the basic feed is protein 14.7%, fat 9.4%, carbohydrates 75.9%, a total of 3.9kcal / g.

[0026] High-fat feed: The mass ratio is casein 22.18%, L-cystine 0.334%, corn starch 23.51%, maltodextrin 7.87%, sucrose 13.794%, cellulose 5.54%, soybean oil 2.774%, cocoa butter 17.19%, sodium amide 0.29%, mixed mineral S10020 0.55%, calcium hydrogen phosphate 1.44%, calcium carbonate 0.61%, potassium citrate monohydrate 1.834%, vitamin mixture 0.110%, choline bitartrate 0.224%, cholesterol 1.25%, sodium cholate 0.50%. The caloric ratio of high-fat feed is protein 19.97%, fat 39.85%, carbohydrate 40.18%, totaling 4.51kcal / g.

[0027] Lipopolysaccharide: purchased from Sigma, product number L2630.

[0028] ApoE in right carotid artery coarctation - / - Mice with ApoE-positive right carotid artery constriction of 60-80% - / - For mice, the constriction rate = (DCC-DMC) / DCC×100%, where DMS represents the diameter of the carotid artery lumen at the narrowest area, and DCC represents the normal carotid artery lumen diameter.

[0029] 2. Results

[0030] According to the above method, we set up the non-carotid artery stenosis + LPS (3 mg / kg) group: ApoE without carotid artery stenosis - / - Mice were fed a high-fat diet for 10 weeks and then intraperitoneally injected with LPS (3 mg / kg);

[0031] Carotid artery stenosis 30 days group: ApoE - / - Mice;

[0032] Carotid artery constriction 30 days + LPS (0.5 mg / kg) group: ApoE mice with right carotid artery constriction were fed a high-fat diet for 10 weeks. - / - Mice, intraperitoneal injection of LPS (0.5 mg / kg);

[0033] Carotid artery constriction 30 days + LPS (1 mg / kg) group: ApoE mice with right carotid artery constriction were fed a high-fat diet for 10 weeks. - / - Mice were intraperitoneally injected with LPS (1 mg / kg).

[0034] There were 20 mice in each group. HE staining, immunohistochemical staining and other pathological methods were used to detect the formation of atherosclerotic plaques and thrombi in the carotid artery segment I, brachiocephalic trunk and aorta root. HE staining showed that after intraperitoneal injection of LPS, the formation of atherosclerotic plaques and thrombi in the segment I of mice in the carotid artery constriction 30 days + LPS (0.5 mg / kg) group and the carotid artery constriction 30 days + LPS (1 mg / kg) group was significantly higher than that in the control group. Figure 1 Immunohistochemical staining of vWF (platelet marker) and CD41 (platelet marker) further demonstrated the thrombosis of atherosclerotic plaque in segment I ( Figure 1 ), while no atherosclerotic thrombosis was found in the brachiocephalic trunk and aortic root. Figure 1 As shown in C, when the intraperitoneal LPS dose was increased from 0.5 mg / kg to 1 mg / kg, the incidence of atherosclerotic thrombosis increased significantly. In the carotid artery constriction 30 days + LPS (1 mg / kg) group, the incidence of atherosclerotic thrombosis was greater than 60%.

[0035] In summary, in ApoE - / - In the mouse atherosclerotic carotid artery constriction model, intraperitoneal injection of LPS successfully induced thrombosis of segment I atherosclerotic plaques, and different doses of LPS model animals had different incidences of atherosclerotic thrombosis. - / - The mouse atherosclerosis model cannot form atherosclerotic plaque thrombus even after injection of high doses of LPS.

[0036] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing an atherosclerotic plaque thrombosis model, characterized in that: The following steps are involved: 1) Select ApoE with carotid artery constriction fed a high-fat diet - / - Mice; 2) Intraperitoneal injection of LPS to obtain an animal model of atherosclerotic plaque thrombosis.

2. The method for constructing an atherosclerotic plaque thrombosis model according to claim 1, characterized in that: In the step 1), male mice or female mice are used.

3. The method for constructing an atherosclerotic plaque thrombosis model according to claim 1, characterized in that: In the step 1), the mice are 15-20 weeks old.

4. The method for constructing an atherosclerotic plaque thrombosis model according to claim 3, characterized in that: In the step 1), the mice are fed with a high-fat diet for 8-12 weeks.

5. The method for constructing an atherosclerotic plaque thrombosis model according to claim 4, characterized in that: ApoE in carotid artery stenosis in step 1) - / - Mice with ApoE carotid artery constriction for 3-4 weeks - / - Mouse.

6. The method for constructing an atherosclerotic plaque thrombosis model according to claim 1, characterized in that: In the step 1), the right carotid artery is constricted or the left carotid artery is constricted.

7. The method for constructing an atherosclerotic plaque thrombosis model according to claim 1, characterized in that: The dosage of LPS injected intraperitoneally in step 2) is 0.5-1 mg / kg.

8. ApoE in carotid artery constriction in subjects fed a high-fat diet - / - Use of mice in preparing animal models of atherosclerotic plaque thrombosis.

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