CN113424795B - Construction method and use of an acute pancreatitis animal model - Google Patents

Abstract

The invention provides a construction method of an animal model of acute pancreatitis, which induces animals to form acute pancreatitis by using high-fat feed and alcohol together. Obesity and alcohol caused by high-fat feed can synergistically cause pathological damage to the pancreas of a mouse, local inflammation reaction of the pancreas is caused, and lung and general inflammation reaction can be caused. The animal model is closer to human morbidity due to large amount of drinking in the basic obesity state, has obvious pancreatic injury and is accompanied with the expression of multi-organ dysfunction syndrome, and accords with the clinical characteristic that the obese acute pancreatitis patient is easy to become severe. The animal model has the advantages of simple modeling method, good repeatability and strong clinical relevance, can be used for researching pathogenesis of obesity and alcohol-related acute pancreatitis, and provides an important basis for screening of treatment targets and research of medical transformation.

Description

Construction method and application of an acute pancreatitis animal model

technical field

The invention belongs to the field of biotechnology, and in particular relates to a construction method and application of an acute pancreatitis animal model.

Background technique

Acute pancreatitis (AP) is an inflammatory injury such as pancreatic edema, hemorrhage, and necrosis caused by the self-digestion of pancreatic tissue due to various etiologies. With the change of people's lifestyle, the incidence of AP is increasing year by year. As a common disease in gastroenterology, AP is mainly manifested as acute and persistent middle and upper abdominal pain, accompanied by nausea, vomiting, etc., blood amylase or lipase increase more than 3 times the upper limit of normal, accompanied by typical images In severe cases, inflammation spreads to the whole body, often combined with systemic or local complications, and the prognosis is poor.

It is extremely important to study the pathogenesis and drug treatment of AP, but there are still ethical disputes in clinical research. Animal models can well simulate the pathogenesis of AP and provide a good research tool for studying its pathogenesis, drug treatment, and preventive care. .

The current AP animal models mainly use physical, chemical, biological and complex pathogenic factors to act on animals, causing local damage to the animal's pancreas with or without damage to other organs in the body. Features. For example, the most commonly used method is intraperitoneal injection of cerulein (chemical drugs), which is simple and easy to repeat, but has no clinical relevance; secondly, retrograde injection of bile salts (physics combined with chemical drugs) into the pancreaticobiliary duct is also common. This model There is a certain clinical correlation with biliary AP, but the operation is complicated and difficult to repeat; the other AP models have basically no clinical correlation, so the current research is seldom used, and it is difficult to provide a comparison for the actual clinical treatment of AP. good reference.

Over the years, a large number of studies on AP have shown that its pathogenic causes include biliary tract disease, alcohol, hyperlipidemia, etc. Obesity and fatty liver have also been classified as risk factors for the prognosis of AP. However, the research on the specific association mechanism between the above-mentioned various factors and AP is still unclear, so it is difficult to effectively guide the establishment of animal models with high clinical relevance and the development of new drugs for the treatment of pancreatitis.

Therefore, there is an urgent need to develop an AP animal model with strong clinical relevance and significant AP characteristics based on AP risk factors.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an acute pancreatitis animal model and its construction method and use.

The invention provides a method for constructing an acute pancreatitis animal model, which uses high-fat feed and alcohol to induce acute pancreatitis in animals.

Further, the above method comprises the steps:

(1) High-fat diet: the animals are continuously fed with high-fat feed for 10 to 15 weeks; the high-fat feed is feed with a fat energy supply ratio higher than 45%.

(2) Alcohol induction: An ethanol solution was administered to the animals.

Further, the high-fat feed in step (1) is a feed with a fat function ratio of 60%.

Further, the feeding time of step (1) is 12 weeks.

Further, the ethanol solution in step (2) is a 35-45% ethanol solution prepared with water or physiological saline, preferably a 37.5% ethanol solution prepared with physiological saline.

Further, the method for administering the ethanol solution in step (2) is: gavage or injection; the injection is intraperitoneal injection or intravenous injection.

Further, the method for administering the ethanol solution in step (2) is intraperitoneal injection, the number of intraperitoneal injections is 1 to 3 times, and each time interval is 1 hour; the injection dose for each time is 5 to 15 μL/g;

Preferably, the number of intraperitoneal injections is 2 times, with an interval of 1 hour between each time; the dose of each injection is 10 μL/g.

Furthermore, 1 hour after the second injection in step (2), intraperitoneal injection of physiological saline is performed; the injection dose of the physiological saline is 5-15 μL/g, preferably 10 μL/g.

Further, the above-mentioned animal is any one of mice, rats, dogs, and pigs, preferably mice.

The present invention also provides the application of the above-mentioned acute pancreatitis animal model in the study of acute pancreatitis disease, and the purpose of the study is to diagnose or treat non-diseases.

Furthermore, the above-mentioned acute pancreatitis disease research is a research on the treatment mechanism of acute pancreatitis, or a research on screening drugs for treating acute pancreatitis disease.

The experimental results show that the animal model of the present invention has the following beneficial effects: based on the high prevalence of obesity, many complications and the epidemiological burden of alcoholism, the pathogenesis of AP is not completely clear and the clinical status of AP without specific therapeutic drugs, Combining obesity and alcohol as common risk factors for AP, an innovative obesity-alcoholic AP model was established. This model simulates the onset of heavy drinking in the basal obesity state, which is closer to the human onset. Pancreatic injury is obvious and accompanied by multiple organ dysfunction syndrome, which is in line with the clinical characteristics of obese AP patients who are prone to severe aggravation. The animal model modeling method of the invention is simple, has high success rate, good repeatability and strong clinical correlation, and can be used to conduct research on the pathogenesis of obesity and alcohol-related acute pancreatitis through the model, and provides the basis for the screening of therapeutic targets and medical transformation research. important foundation.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 is a schematic diagram of the induction of an animal model of obese alcoholic acute pancreatitis. HFD, high-fat diet; EtOH, alcohol.

Figure 2 shows changes in body weight and fat of mice. Changes in (A) body weight and (B) abdominal fat in mice fed a high-fat diet for 12 weeks. ***P<0.001 compared to the control group. CD, control diet; HFD, high-fat diet.

Figure 3 shows the changes of serum amylase and lipase levels in mice. After inducing the obese alcoholic AP model, the levels of (A) pancreatic amylase and (B) lipase in serum were detected. Data are presented as mean ± standard error. Compared with the blank control group, **P<0.01, ***P<0.001; compared with the alcohol control group, #P<0.05. CD, blank control; HFD, high-fat control; EtOH, alcohol control; HFD+EtOH, the obese alcoholic acute pancreatitis mouse model modeled in Example 1.

Figure 4 shows the pathological changes of mouse pancreas. (A) Histopathological sections of pancreas were stained with HE and double-blind to score (B) edema, (C) inflammatory infiltration, (D) necrosis and (E) total score. Data are presented as mean ± standard error. Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the alcohol control group, ###P<0.001. CD, blank control; HFD, high-fat control; EtOH, alcohol control; HFD+EtOH, the obese alcoholic acute pancreatitis mouse model modeled in Example 1.

Figure 5 shows the changes of systemic inflammatory response in mice. (A) MPO level in pancreatic tissue, (B) MPO level in lung tissue and (C) serum IL-6 level. Data are presented as mean ± standard error. Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the alcohol control group, #P<0.05, ##P<0.01, ###P<0.001. CD, blank control; HFD, high-fat control; EtOH, alcohol control; HFD+EtOH, obese alcoholic acute pancreatitis mouse model modeled in Example 1; MPO, myeloperoxidase; IL-6, interleukin -6.

Figure 6 shows the changes of liver and kidney function in mice. (A) serum CREA level, (B) serum UREA level, (C) serum ALT level and (D) serum AST level. Data are presented as mean ± standard error. Compared with the blank control group, **P<0.01, ***P<0.001; compared with the alcohol control group, #P<0.05, ##P<0.01. CD, blank control; HFD, high-fat control; EtOH, alcohol control; HFD+EtOH, a mouse model of obese alcoholic acute pancreatitis modeled in Example 1; CREA, creatinine; UREA, urea; ALT, alanine aminotransferase; AST , Aspartate aminotransferase.

Figure 7 shows the changes of serum lactate dehydrogenase and calcium ion levels in mice. (A) Serum lactate dehydrogenase level and (B) serum calcium ion level. Data are presented as mean ± standard error. Compared with the blank control group, ^** P<0.01, ^*** P<0.001; compared with the alcohol control group, ^#P <0.05, ^### P<0.001. CD, blank control; HFD, high-fat control; EtOH, alcohol control; HFD+EtOH, obese alcoholic acute pancreatitis mouse model modeled in Example 1; LDH, lactate dehydrogenase; Ca ²⁺ , calcium ion.

Figure 8 shows the changes of circulating histones in mouse serum. (A) Western blot detection of histone expression in serum, (B) Gray value analysis of Western blot results and statistics. Data are presented as mean ± standard error. ^*** P<0.001 compared with the blank control group; ^### P<0.001 compared with the alcohol control group. CD, blank control; HFD, high-fat control; EtOH, alcohol control; HFD+EtOH, the obese alcoholic acute pancreatitis mouse model modeled in Example 1.

Detailed ways

1. Experimental animals

SPF grade male C57BL/6J mice (4-5 weeks) were purchased from Beijing Huafukang Biotechnology Co., Ltd., license: SCXK (Beijing) 2020-0004. They were kept in separate cages in the animal room of the Experimental Animal Center, West China Hospital, Sichuan University, with 5 animals per cage, constant temperature (25±2°C) and lighting control (12h day/night cycle), free food and water, and adaptive feeding after 1 week. Start the experiment. This experiment was reviewed and approved by the Ethics Committee of the Experimental Animal Center of West China Hospital, Sichuan University (ethics number 2021016A). All animal experiments and related operations were performed in accordance with school and national standards.

Example 1. Construction of acute pancreatitis mouse model

The mice were fed a high-fat diet continuously for 12 weeks, and feed was added weekly to ensure their quality. The high-fat feed used in this example is 60% high-fat feed (HFD, fat energy supply ratio of 60%, H10060, Huafukang, Beijing) in the obesity model series diets (DIO series Diets); The energy supply ratio is shown in Table 1. Alcohol induction was performed after 12 weeks, and all mice were fasted 12 hours before modeling, but could not be allowed to water. The 37.5% ethanol solution was prepared with physiological saline, which was prepared and used now, and the mice were injected with 37.5% ethanol solution (10 μL/g) by intraperitoneal injection for 2 consecutive injections with an interval of 1 h. Rehydration was performed 1 h after the last alcohol injection, that is, intraperitoneal injection of normal saline (10 μL/g). The induction process is shown in Figure 1.

Table 1 Feed composition and energy supply ratio

The beneficial effects of the present invention are demonstrated below through experimental examples.

Experimental Example 1. Obesity and alcohol synergistically induce pancreatic pathological changes and multiple organ damage in mice

1. Animal grouping and treatment (8 animals per group)

(1) Obese alcoholic acute pancreatitis group (ie, Example 1): fed with a high-fat diet for 12 weeks, and given 37.5% ethanol solution for 2 consecutive injections with an interval of 1 h. The specific operation is carried out according to Example 1.

(2) Blank control group: normal diet (using control feed (CD, fat energy supply ratio of 10%, H10010, Huafukang, Beijing), the specific composition and energy supply ratio of the feed are shown in Table 1) for 12 weeks, given and The same amount of normal saline of the ethanol solution used in Example 1 was continuously injected twice with an interval of 1 h, and the rest of the operations were the same as those in Example 1.

(3) High-fat control group: fed with high-fat diet for 12 weeks, and injected with the same amount of normal saline as the ethanol solution used in Example 1 for 2 consecutive injections with an interval of 1 h, and other operations were the same as those in Example 1.

(4) Alcohol control group: normal diet for 12 weeks, 37.5% ethanol solution in the same amount as the ethanol solution used in Example 1 was injected for 2 consecutive times, with an interval of 1 h, and other operations were the same as those in Example 1.

2. Material sampling method

After modeling, the samples were collected 12 hours after the first alcohol injection. After induction of anesthesia with isoflurane gas, blood collection from the heart is performed. The specific operations are as follows: put the mouse into an anesthesia induction box and anesthetize the mouse. After the mouse is anesthetized, a mask is used to maintain it. The chest cavity is quickly opened, the heart is exposed and cut open, and the blood is quickly drawn with a 1 mL syringe. , after resting at room temperature for 30 min, centrifuged at 1500g for 15 min at room temperature, aspirated the upper serum, frozen in -80 ℃ refrigerator, used to measure serum biochemistry, interleukin-6 (IL-6) and circulating histone levels. After blood collection, the right lower lung was taken out, rinsed in normal saline, dried with filter paper, quickly frozen in liquid nitrogen, and then stored in a -80°C refrigerator for the determination of myeloperoxidase (MPO). After that, the abdominal cavity was quickly opened, and the pancreatic tissue was divided into three parts. The whole part of the pancreatic body was put into the embedding box, immersed in 10% neutral formalin solution for more than 48 hours, dehydrated and embedded in paraffin as hematoxylin Serum-eosin (hematoxylin-eosin, HE) staining; the other two parts of the pancreatic tissue were taken out, snap-frozen in liquid nitrogen, and then stored in a -80°C refrigerator for the determination of MPO and other experiments.

3. Experimental results

(1) High-fat diet-induced obesity

Obesity is manifested as excessive weight gain and excess body fat accumulation. As shown in Figure 2A, there was no significant difference in body weight between the high-fat diet and control diet groups before starting feeding (20.9 vs 21.2 g, P>0.05). After feeding the two groups with different diets for 12 weeks, the body weight increased compared with that before feeding. The body weight of the high-fat diet group increased to 32.9g, which was significantly higher than that of the control diet group (26.5g, P<0.001). On dissection (Fig. 2B), mice in the high-fat diet group exhibited significant fat accumulation, especially visceral fat.

The above results indicate that high-fat diet can induce obesity in mice, and provide a basic state of obesity for further induction of obese alcoholic acute pancreatitis model.

(2) Changes of serum amylase, lipase and pancreatic pathology

Elevated levels of circulating amylase and lipase are important signs of AP, and clinical guidelines take amylase and lipase elevated to more than three times the upper limit of normal as one of the diagnostic criteria. The levels of pancreatic amylase and pancreatic lipase in serum were detected by an automatic biochemical analyzer. The results are shown in Figure 3A-B. There was no significant difference in the levels of amylase and lipase between the high-fat control group and the blank control group, indicating that only high-fat induced, the successful construction of the AP model could not be achieved.

After alcohol injection, the levels of amylase and lipase in each group were increased, which were statistically significant compared with the blank control group (P<0.01). Among them, amylase increased to 21768U/L and lipase level increased to 1595U/L in the obese alcoholic acute pancreatitis group, which were significantly higher than those in the alcohol control group (P<0.05), indicating that high-fat diet and alcohol-induced production A significant synergistic effect was achieved, and an animal model with significant AP characteristics was successfully constructed.

Pathological diagnosis is often regarded as the gold standard of disease. To further observe the pathological changes of mouse pancreatic tissue, the pancreatic tissue was stained with HE staining on paraffin-embedded sections and observed under a microscope. The results are shown in Figure 4A. It can be seen that the pancreatic acinar cells in the blank control group and the high-fat control group are intact and regularly arranged, without interstitial edema, inflammatory cell infiltration and cell necrosis, indicating that the AP model cannot be achieved only by high-fat induction. successful build.

After alcohol injection, the alcohol control group showed a slight increase in pancreatic tissue space, and scattered acinar cells were separated by edema; while in the obese alcoholic AP group induced by high fat and alcohol, the pancreatic tissue space was significantly increased, acinar cells were separated by edema, and ducts and ducts were separated. Inflammatory cell infiltration can be seen between lobules, and acinar cell necrosis can be seen locally. Two pathologists used a double-blind method to stain the pathological sections of mouse pancreatic tissue with HE, and scored from three aspects: edema, inflammatory infiltration and necrosis. The results are shown in Figure 4B-E, there was no significant difference in the edema, inflammatory infiltration, necrosis and total score between the high-fat control group and the blank control group, and the edema, necrosis and total score of the alcohol control group were slightly higher than those of the blank control group ( P<0.05), there was no significant difference in inflammatory infiltration compared with the blank control group, indicating that the establishment of an AP model with significant pathological characteristics cannot be achieved only by alcohol induction.

The edema, inflammatory infiltration, necrosis and total scores of the obese alcoholic acute pancreatitis group induced by high-fat diet and alcohol were 1.98, 1.10, 1.27 and 4.35, respectively, which were significantly higher than those in the blank control group (P<0.001), and more Furthermore, the pancreatic pathological score indexes in the obese alcoholic AP group were significantly higher than those in the alcoholic control group (P<0.001). The above results show that obesity and alcohol synergistically can cause increased serum amylase and lipase levels and pancreatic pathological damage in mice, marking the occurrence of acute pancreatitis. Through the synergistic induction method of high-fat diet and alcohol of the present invention, it is possible to effectively construct obvious features. AP model.

(3) Changes in systemic inflammatory response

When AP occurs, local inflammation occurs in the pancreas, and the inflammatory reaction can also develop to the whole body, resulting in the involvement of distant organs, and the lungs are the most vulnerable distal organs. Changes in MPO level and activity represent the function and activity state of neutrophils, so MPO can be used as an inflammatory marker. A large number of studies have shown that MPO activity in pancreas and lung tissue is significantly increased when AP occurs, suggesting that there is a large number of neutrophil infiltration in pancreas and lung tissue. As a member of cytokines, IL-6 is involved in numerous inflammatory diseases. When AP occurs, circulating IL-6 levels are closely related to the severity of AP. Therefore, we detected the MPO activity in pancreatic tissue to reflect the local inflammatory response of the pancreas, and the MPO activity and circulating IL-6 level in the lung tissue to reflect the systemic inflammatory response.

The results are shown in Figure 5A-B, there was no significant difference in the levels of MPO in the pancreas and lung tissues of the high-fat control group and the blank control group, indicating that only high-fat induction was used, and the inflammatory response was not significant. After alcohol injection, the levels of MPO in the pancreas and lung tissue of each group were increased, which were statistically significant compared with the blank control group (P<0.05), indicating that the use of alcohol alone can promote the occurrence of inflammatory reaction to a certain extent. But the effect is not good. The MPO level of the obese alcoholic acute pancreatitis group induced by both high fat and alcohol was 100%, which was significantly higher than that of the alcohol control group (P<0.05).

The serum IL-6 level of mice was detected by a commercial kit (Figure 5C), and there was no significant difference between the high-fat control group and the blank control group, and there was no significant difference between the alcohol control group and the blank group. It shows that only using alcohol induction or only using high-fat induction can not lead to obvious inflammatory response. After high-fat diet-induced alcohol injection, the serum IL-6 level in the obese alcoholic AP group increased from 16.97 pg/mL in the blank control group to 139.71 pg/mL (P < 0.01). Significantly higher than the alcohol control group (P<0.01).

The above results indicate that obesity and alcohol synergistically can increase the level of local pancreas, lung MPO and serum IL-6 level, which is the manifestation of local and systemic inflammatory response in acute pancreatitis. Through the synergistic induction method of high-fat diet and alcohol of the present invention, an AP model with obvious characteristics can be effectively constructed.

(4) Changes in multiple organ damage

AP can further develop into severe AP, accompanied by organ failure of one or more organs, and the incidence of renal damage is second only to pulmonary dysfunction. We detected creatinine (CREA) and urea (UREA) in mouse serum to reflect renal damage.

The results are shown in Figure 6A and B, there is no significant difference between the high-fat control group, the alcohol control group and the blank control group, indicating that the high-fat induction or alcohol induction alone has no obvious effect on renal function damage. After high-fat diet-induced alcohol injection, the serum CREA and UREA levels in the obese alcoholic AP group were significantly higher than those in the blank control group (P<0.01), and were significantly higher than those in the alcohol control group (P<0.01). <0.05), indicating that the co-induction of high-fat and alcohol significantly promoted renal damage.

Alcohol can cause liver damage, which is also common in patients with severe AP. We detected serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to reflect liver damage.

The results are shown in Figure 6C and D, there was no significant difference between the high-fat control group and the blank control group, and the serum ALT and AST levels were significantly increased after alcohol injection (P<0.001). The increase in the control group was more significant (P<0.01), indicating that the co-induction of high fat and alcohol significantly promoted liver function damage.

Severe AP is often accompanied by changes in a series of biomarkers. Lactate dehydrogenase (LDH) is an indicator of cell death.

The LDH level in serum was detected. The results are shown in Figure 7A. There was no significant difference between the high-fat control group and the blank control group. After alcohol injection, the serum LDH level increased (P<0.001). The increase in alcohol control group was more significant (P<0.001). Decreased serum calcium is a symptom of severe AP and suggests a poor prognosis. As shown in Figure 7B, there was no significant difference between the high-fat control group and the blank control group. After alcohol injection, the blood calcium level of the alcohol control group had no significant change, while the blood calcium level of the obese alcoholic AP group was higher than that of the blank control group and the alcohol control group. Significantly decreased (P<0.05). It shows that the co-induction of high fat and alcohol significantly promotes the development of AP aggravation.

Histones are highly conserved basic structural proteins in eukaryotic cell nuclei. Upon tissue damage and cell death, histones are released outside the cell and enter the circulation, namely circulating histones. Circulating histones were significantly elevated in severe AP.

The levels of circulating histones were detected by immunoblotting, and quantitative analysis was carried out using standard substances. The results are shown in Figure 8A and B. The serum of the blank control group and the high-fat control group had almost no histones. After alcohol injection, the serum of the alcohol control group had almost no histone. Compared with the blank control group, the histone level was slightly increased, but the difference was not statistically significant, while the serum histone level in the obese alcoholic AP group induced by both high fat and alcohol was significantly higher than that of the blank control group and the alcohol control group (P<0.001). , further confirmed that the co-induction of high-fat diet and alcohol can effectively induce the occurrence of AP.

The above results show that obesity and alcohol synergistically can cause abnormal liver and kidney function in mice, and the manifestations of acute pancreatitis, such as increased serum lactate dehydrogenase, decreased blood calcium, and increased circulating histones, are associated with multiple organ function damage and aggravation of acute pancreatitis. The clinical characteristics of obese AP patients that are prone to severe aggravation have strong clinical correlation.

Overall, the 8 mice of the obese alcoholic acute pancreatitis group of the present invention modeled according to the method of Example 1 were all successfully modeled, and an animal model with significant AP characteristics was obtained, indicating that the method of the present invention has a high modeling success rate, Good repeatability.

To sum up, the present invention provides a method for constructing an animal model of obesity-alcoholic acute pancreatitis. Obesity and alcohol can synergistically cause pathological damage to the pancreas in mice, cause local inflammatory response in the pancreas, and can lead to inflammatory responses in the lungs and the whole body. The model completely adopts obesity and alcohol, two independent risk factors of AP, to synergistically induce AP. It has strong clinical correlation, high modeling success rate and good reproducibility. It is suitable for the study of the pathogenesis of acute pancreatitis and the screening of therapeutic drugs. provides an important foundation.

Claims (8)

1. A method for constructing an animal model of acute pancreatitis is characterized in that high-fat feed and alcohol are used for inducing animals to form the acute pancreatitis, and the method comprises the following steps:

(1) high fat diet: continuously feeding animals with high-fat feed for 10-15 weeks; the high-fat feed is a feed with the energy supply ratio of fat higher than 45%;

(2) alcohol induction: administering an ethanol solution to the animal; the ethanol solution is 35-45% ethanol solution prepared by water or normal saline; the method for administering the ethanol solution is intraperitoneal injection, wherein the number of intraperitoneal injection is 1-3, and each time interval is 1 hour; the injection dosage of each time is 5-15 mu L/g; injecting normal saline into the abdominal cavity 1 hour after the last injection of the ethanol solution into the abdominal cavity; the injection dosage of the normal saline is 5-15 mu L/g;

the animal is a mouse.

2. The method of claim 1, wherein the high fat diet of step (1) is a diet having a fat energy ratio of 60%.

3. The method of claim 1, wherein the period of feeding in step (1) is 12 weeks.

4. The method of claim 1, wherein the ethanol solution of step (2) is a 37.5% ethanol solution in normal saline.

5. The method of claim 1, wherein the number of intraperitoneal injections in step (2) is 2, each time at 1 hour intervals; the injection dose is 10 μ L/g.

6. The method of claim 1, wherein the normal saline is injected in a dose of 10 μ L/g.

7. Use of an animal model of acute pancreatitis prepared by the method of any one of claims 1-6 in the study of acute pancreatitis, for the purpose of diagnosis or treatment of non-diseases.

8. The use of claim 7, wherein the acute pancreatitis disease study is a study screening for drugs to treat acute pancreatitis disease.

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