Construction method and application of zebra fish acute pancreatitis model

Abstract

The invention relates to the field of pancreatitis models, and provides a construction method and application of a zebra fish acute pancreatitis model. The construction method of the acute pancreatitis model of the zebrafish comprises the following steps: performing intravenous injection on 1-4 dpf zebrafish to give rain frog element, culturing for 3-5 days, and then testing the pancreatic apoptotic cell fluorescence intensity and / or ROS fluorescence intensity of the zebrafish. According to the method, the zebra fish acute pancreatitis model is successfully constructed by optimizing influence factors such as model making concentration and evaluation index selection, the detection accuracy is high, the model making period is short, and the detection cost is reduced. The invention also provides application of the acute pancreatitis model of zebra fish obtained by the construction method in evaluation of acute pancreatitis treatment efficacy of drugs, health food or food.

Description

Technical Field

[0001] The invention relates to the field of pancreatitis models, and in particular to a method for constructing a zebrafish acute pancreatitis model and an application thereof. Background Art

[0002] Acute pancreatitis is a pancreatic autodigestive disease caused by abnormal activation of pancreatic enzymes, and may cause dysfunction of other organs. Acute pancreatitis usually occurs in adults, with an annual incidence of 5 / 100,000 to 30 / 100,000, and the incidence is increasing year by year. Commonly used animal models of acute pancreatitis are rats, mice and rabbits, such as Xi Junyan, Yu Zhenfan, Yang Ying, et al. Effects of tea polyphenols on caerulein-induced acute pancreatitis in mice and mechanism analysis [J]. Journal of Xiangnan University (Medical Edition), 2018, 20(04): 28-31. However, the results of these animal models are not intuitive and intestinal experiments often have high requirements for animals. If necessary, endpoint studies will be conducted on animals, causing their death, resulting in unnecessary waste. Therefore, it is very necessary to select a suitable animal model for exploring the disease mechanism of acute pancreatitis, evaluating new treatments, conducting drug screening and toxicity testing, exploring disease risk factors, and developing and validating biomarkers. Summary of the invention

[0003] The present invention provides a method for constructing a zebrafish acute pancreatitis model and application thereof, optimizes influencing factors such as modeling concentration and evaluation index selection, successfully constructs a zebrafish acute pancreatitis model, and has high detection accuracy, short modeling cycle, and reduced detection cost.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: A method for constructing a zebrafish acute pancreatitis model comprises the following steps: administering caerulein by intravenous injection to 1-4 dpf zebrafish, and testing the fluorescence intensity of pancreatic apoptotic cells and / or ROS fluorescence intensity of the zebrafish after culturing for 3-5 days.

[0005] Preferably, the zebrafish used to analyze the fluorescence intensity of pancreatic apoptotic cells is a wild-type AB strain zebrafish.

[0006] Preferably, the zebrafish used for analyzing the ROS fluorescence intensity is an Albino strain zebrafish with a melanin allele mutation.

[0007] Preferably, the zebrafish are reared at a temperature of 27-29°C, and the light cycle needs to be controlled to 14 hours of light and 10 hours of darkness from fertilization until 1-4 dpf.

[0008] Preferably, the injection amount of caerulein per zebrafish is 100-200 ng.

[0009] As a further preference, the injection amount of caerulein per zebrafish is 200 ng.

[0010] Preferably, caerulein is prepared into an injection solution of 80-100 mg / mL using DMSO.

[0011] Preferably, the culture temperature is 27-29°C; more preferably, it is 28°C.

[0012] Preferably, the test method for the fluorescence intensity of apoptotic cells is: taking out the cultured zebrafish, staining the apoptotic cells with acridine orange, photographing the zebrafish under a fluorescence microscope, analyzing and collecting data with image processing software, and analyzing the fluorescence intensity of apoptotic cells in the zebrafish pancreas. The statistical analysis results of this indicator are used to evaluate whether the acute pancreatitis model is successfully induced.

[0013] As a preferred method for testing the ROS fluorescence intensity, the cultured zebrafish are taken out and the ROS fluorescence intensity is measured by using Cell ROX TM Green Reagent was used for ROS cell staining, zebrafish were placed under a fluorescence microscope and photographed, image processing software was used to analyze and collect data, and the fluorescence intensity of zebrafish pancreatic apoptotic cells was analyzed. The statistical analysis results of this indicator were used to evaluate whether the acute pancreatitis model was successfully induced.

[0014] The present invention also provides application of the zebrafish acute pancreatitis model obtained by the construction method in evaluating the acute pancreatitis therapeutic efficacy of drugs, health foods or foods.

[0015] Therefore, the beneficial effects of the present invention are as follows: by exploring the influencing factors such as modeling concentration and selection of evaluation indicators, a zebrafish acute pancreatitis model was successfully constructed, the detection accuracy was high, the detection method was scientific and reasonable, and the modeling cycle was short, which greatly reduced the detection cost, providing practical feasibility for popularizing and promoting the detection method to the general public, enterprises, and governments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a typical graph of the fluorescence intensity of apoptotic cells in zebrafish after treatment with caerulein in Example 1.

[0017] Figure 2 This is a typical graph of the ROS fluorescence intensity of zebrafish after caerulein treatment in Example 2. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below through specific embodiments.

[0019] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the embodiments, unless otherwise specified, are all conventional methods in the art. Unless otherwise specified, parts are all weight parts, temperatures are expressed in ° C or at ambient temperature, and pressures are atmospheric pressure or near atmospheric pressure. There are many variations and combinations of reaction conditions (such as component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction ranges) and conditions that can be used to optimize the purity and yield of the product obtained by the method, and only reasonable routine experiments will be required to optimize such method conditions.

[0020] Example The main symptom of acute pancreatitis is the sudden onset of persistent upper abdominal pain, which may be accompanied by nausea, vomiting, abdominal distension, fever and other symptoms. Severe patients may experience hypotension or shock, and have multiple organ dysfunction, resulting in a high mortality rate. Most patients with acute pancreatitis experience a mild course that often resolves on its own. However, severe acute pancreatitis accompanied by organ dysfunction can be life-threatening, with a mortality rate of up to 30%. Acute pancreatitis is a more common type of acute abdomen, and approximately one-fifth to one-quarter of patients will develop severe acute pancreatitis. In this case, patients will develop a systemic inflammatory response syndrome, which often results in a poor prognosis and a higher risk of death.

[0021] Caerulein is a cholinergic stimulant that is mainly used to induce an animal model of acute pancreatitis in a laboratory environment. Caerulein overstimulates the pancreas to secrete digestive enzymes by simulating the physiological stimulation during food digestion, leading to damage and inflammatory response of pancreatic cells, and then inducing acute pancreatitis. The pathogenesis of acute pancreatitis involves mitochondrial dysfunction in pancreatic acinar cells, which leads to excessive production of reactive oxygen species (ROS), decreased mitochondrial membrane potential, reduced ATP production, oxidative damage to mitochondrial DNA and its release, which in turn activates a cascade reaction related to cell death and inflammatory signals, ultimately leading to pancreatic necrosis and systemic inflammation.

[0022] Zebrafish has become an ideal model for many research fields due to its advantages such as low breeding cost, strong reproductive capacity, high genetic similarity with humans, simple operation, and short experimental cycle. The pancreas of zebrafish and mammals is highly similar in structure and function. The zebrafish pancreas consists of two parts, endocrine and exocrine, which are similar to the pancreatic structure of mammals. At the molecular level, the expression and function of many genes and signaling pathways during pancreatic development are also conserved in zebrafish and mammals, indicating that zebrafish can be used as a powerful model for studying human pancreatic diseases and regeneration mechanisms. The advantages of using zebrafish fry for modeling in the present invention include: 1. Zebrafish fry experiments are fast and efficient, and the efficiency is much higher than that of adult fish experiments; 2. The experimental results of zebrafish are intuitive and can be accurately located, and with the help of pictures, it is clear whether there is a therapeutic effect.

[0023] The present invention provides a method for constructing a zebrafish acute pancreatitis model to simulate the pathological process of human acute pancreatitis in order to better study the disease mechanism, evaluate new treatment methods, conduct drug screening and toxicity testing, explore disease risk factors, and develop and verify biomarkers. This model is crucial for understanding the complex biological process of acute pancreatitis, helps promote the development of prevention and treatment strategies, and provides a scientific basis for clinical treatment. The specific modeling method is as follows: A method for constructing a zebrafish acute pancreatitis model, comprising the following steps: (1) Zebrafish breeding Zebrafish were kept in aquaculture water at 28°C (water quality: 200 mg instant sea salt was added to 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO 3 ), bred and provided by the fish farming center of Hangzhou Huante Biotechnology Co., Ltd., with the experimental animal use license number: SYXK(Zhejiang)2022-0004, and the breeding and management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0024] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 1-4 days post-fertilization (1-4 dpf) were used to establish the acute pancreatitis model. From fertilization, zebrafish need to control the light cycle to 14:10 (14 hours of light and 10 hours of darkness).

[0025] The melanin allele mutant Albino strain zebrafish was bred by natural pair mating. Zebrafish aged 1-4 days (1-4 dpf) were used to establish the acute pancreatitis model. From fertilization, the zebrafish needed to control the light cycle to 14:10 (14 hours of light and 10 hours of darkness).

[0026] (2) Preparation of inducer Caerulein, white powder, batch number O18IS229227, Shanghai Yuanye Biotechnology Co., Ltd., stored frozen. Prepared into 80-100 mg / mL caerulein injection with DMSO, stored at -20℃ for later use.

[0027] (3) Intravenous injection of zebrafish The 1-4 dpf zebrafish raised in step (1) are intravenously injected with the caerulein injection solution prepared in step (2), and the caerulein injection amount per zebrafish is 100-200 ng, and more preferably 200 ng.

[0028] (4) Cultivating zebrafish The injected zebrafish are cultured at 27-29° C. (more preferably 28° C.) for 3-5 days.

[0029] (5) Testing Test the fluorescence intensity of apoptotic cells: Take out the cultured wild-type AB strain zebrafish, use acridine orange to stain apoptotic cells, randomly select a few zebrafish from each experimental group and take pictures under a fluorescence microscope, use NIS-Elements D3.20 advanced image processing software to analyze and collect data, analyze the fluorescence intensity of apoptotic cells in the zebrafish pancreas, and use the statistical analysis results of this indicator to evaluate whether the acute pancreatitis model is successfully induced. The statistical processing results are expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0030] Test ROS fluorescence intensity: Take out the cultured Albino zebrafish with melanin allele mutation and use Cell ROX TM Green Reagent was used for ROS cell staining. Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data. The zebrafish ROS fluorescence intensity was analyzed, and the statistical analysis results of this indicator were used to evaluate whether the acute pancreatitis model was successfully induced. The statistical processing results were expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0031] The present invention also provides an application of the zebrafish acute pancreatitis model obtained by the construction method, which is used to evaluate the acute pancreatitis therapeutic efficacy of drugs, health foods or foods.

[0032] Specifications of instruments, consumables and reagents: Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); electric focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); microinjection instrument (IM300, Narishige, Japan); needle puller (PC-10, Narishige, Japan).

[0033] Methylcellulose (Batch No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Acridine orange (Batch No. C12894919, Shanghai McLean Biochemical Technology Co., Ltd., China); Dimethyl sulfoxide (DMSO, Batch No. BCCD8942, Sigma, Switzerland); Cell ROX TM Green Reagent (lot number 2581648, Invitrogen, USA).

[0034] Example 1 A method for constructing a zebrafish acute pancreatitis model, the steps are as follows: (1) Zebrafish breeding Wild-type AB zebrafish were raised in 28°C fish water (water quality: 200 mg instant sea salt was added to 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO 3 ), bred and provided by the fish farming center of Hangzhou Huante Biotechnology Co., Ltd., with the experimental animal use license number: SYXK(Zhejiang)2022-0004, and the breeding and management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0035] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 3 days post fertilization (3dpf) were used to establish the acute pancreatitis model. From fertilization, zebrafish need to control the light cycle to 14:10 (14h light, 10h dark).

[0036] (2) Preparation of inducer Caerulein, white powder, batch number O18IS229227, Shanghai Yuanye Biotechnology Co., Ltd., stored frozen. Prepared into 100 mg / mL caerulein injection with DMSO, stored at -20℃ for later use.

[0037] (3) Intravenous injection and culture of zebrafish 3dpf wild-type AB strain zebrafish were randomly selected in 6-well plates, with 30 fish in each well (experimental group). They were given intravenous injection of caerulein (doses shown in Table 1), and a normal control group was set up at the same time, with a volume of 3mL per well. After culturing at 28℃ for 3 days, the maximum detection dose (MTD) of caerulein in the samples for normal zebrafish was determined.

[0038] Table 1. Results of the maximum detection dose of caerulein (n=30) As can be seen from Table 1, under the experimental conditions, the maximum detection dose of caerulein to zebrafish is 200 ng / tail.

[0039] (4) Testing the fluorescence intensity of apoptotic cells The zebrafish cultured in step (3) were tested for the fluorescence intensity of apoptotic cells. Specifically, acridine orange was used to stain apoptotic cells. Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The data were analyzed and collected using NIS-ElementsD 3.20 advanced image processing software. The fluorescence intensity of apoptotic cells in the zebrafish pancreas was analyzed, and the statistical analysis results of this indicator were used to evaluate whether the acute pancreatitis model was successfully induced. The statistical processing results were expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant. The results are shown in Tables 2 and Figure 1 As shown, Figure 1 a is an example, b is a normal control group, and c is a 200 ng / tail caerulein group. Figure 1 It can be seen that under the experimental conditions, caerulein can induce acute pancreatitis in zebrafish, which is specifically manifested by increasing the fluorescence intensity of apoptotic cells.

[0040] Table 2. Experimental results of fluorescence intensity of apoptotic cells (n=10) Compared with the normal control group, **p<0.01 Comparative Example 1 The difference from Example 1 is that the injection method is intraperitoneal injection. The details are as follows: A method for constructing a zebrafish acute pancreatitis model, the steps are as follows: (1) Zebrafish breeding Wild-type AB zebrafish were raised in 28°C fish water (water quality: 200 mg instant sea salt was added to 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO 3), bred and provided by the fish farming center of Hangzhou Huante Biotechnology Co., Ltd., with the experimental animal use license number: SYXK(Zhejiang)2022-0004, and the breeding and management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0041] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 3 days post fertilization (3dpf) were used to establish the acute pancreatitis model. From fertilization, zebrafish need to control the light cycle to 14:10 (14h light, 10h dark).

[0042] (2) Preparation of inducer Caerulein, white powder, batch number O18IS229227, Shanghai Yuanye Biotechnology Co., Ltd., stored frozen. Prepared into 100 mg / mL caerulein injection with DMSO, stored at -20℃ for later use.

[0043] (3) Intraperitoneal injection and culture of zebrafish 3dpf wild-type AB zebrafish were randomly selected and placed in a 6-well plate, with 30 fish in each well (experimental group). Caerulein (doses shown in Table 2) was intraperitoneally injected, and a normal control group was set up at the same time, with a volume of 3 mL in each well. The plates were cultured at 28°C for 3 days.

[0044] (4) Testing the fluorescence intensity of apoptotic cells The zebrafish cultured in step (3) were tested for the fluorescence intensity of apoptotic cells. Specifically, acridine orange was used to stain apoptotic cells, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope, and NIS-ElementsD 3.20 advanced image processing software was used to analyze and collect data, and the fluorescence intensity of apoptotic cells in the zebrafish pancreas was analyzed. The statistical analysis results of this indicator were used to evaluate whether the acute pancreatitis model was successfully induced. The statistical processing results were expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0045] The results are shown in Table 2. As can be seen from Table 2, under the experimental conditions, the significance of caerulein (intraperitoneal injection) in inducing acute pancreatitis in zebrafish is not as good as caerulein (intravenous injection). In addition, the intravenous injection used in the present invention has the following advantages over intraperitoneal injection: 1. Rapidly reach high concentration: intravenous injection can quickly deliver the drug directly into the blood circulation, thereby quickly reaching the required drug concentration. This is particularly important in the study of acute pancreatitis, because it is necessary to quickly establish and maintain effective drug levels to observe its effects on the pancreas. 2. Reduce operational complexity: Although intraperitoneal injection is simple to operate and widely used in the establishment of acute pancreatitis models, multiple intraperitoneal injections may increase the complexity and time cost of the experiment. Intravenous injection usually only needs one or several times to complete, simplifying the experimental process. 3. Avoid traumatic effects: intraperitoneal injection may cause certain trauma to the animal, especially when it is difficult to find a suitable position, this trauma may affect the experimental results. Intravenous injection avoids this trauma and helps to keep the physiological state of the animal stable. In summary, compared with intraperitoneal injection, intravenous injection is fast, simple, and avoids trauma in the cerulein-induced acute pancreatitis model, making it a better choice.

[0046] Example 2 A method for constructing a zebrafish acute pancreatitis model, the steps are as follows: (1) Zebrafish breeding The melanin allele mutant Albino strain zebrafish were raised in 28°C fish farming water (water quality: 200 mg instant sea salt was added to 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO 3 ), bred and provided by the fish farming center of Hangzhou Huante Biotechnology Co., Ltd., with the experimental animal use license number: SYXK(Zhejiang)2022-0004, and the breeding and management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0047] The melanin allele mutant Albino strain zebrafish was bred by natural pair mating. Zebrafish aged 3 days post fertilization (3dpf) were used to establish the acute pancreatitis model. From fertilization, the zebrafish needed to control the light cycle to 14:10 (14h light, 10h dark).

[0048] (2) Preparation of inducer Caerulein, white powder, batch number O18IS229227, Shanghai Yuanye Biotechnology Co., Ltd., stored frozen. Prepared into 100 mg / mL caerulein injection with DMSO, stored at -20℃ for later use.

[0049] (3) Intravenous injection and culture of zebrafish 3dpf melanin allele mutant Albino zebrafish were randomly selected and placed in a 6-well plate, with 30 fish in each well (experimental group). 200ng / tail of caerulein were intravenously injected, and a normal control group was set up at the same time, with a volume of 3mL per well. The culture was carried out at 28℃ for 3 days.

[0050] (4) Test ROS fluorescence intensity The ROS fluorescence intensity of zebrafish cultured in step (3) was tested. Specifically, Cell ROX TM Green Reagent was used for ROS cell staining. Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data. The zebrafish ROS fluorescence intensity was analyzed, and the statistical analysis results of this indicator were used to evaluate whether the acute pancreatitis model was successfully induced. The statistical processing results were expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant. The results are shown in Table 3 and Figure 2 As shown, Figure 2 a is an example, b is a normal control group, and c is a 200 ng / tail caerulein group. Figure 2 It can be seen that under the experimental conditions, caerulein can induce acute pancreatitis in zebrafish, which is specifically manifested by an increase in ROS fluorescence intensity.

[0051] Table 3. ROS fluorescence intensity experimental results (n=10) Group Dose (ng / tail) ROS fluorescence intensity (pixels, mean ± SE) Normal control group - 42869±2164 caerulein 200 57915±3448** Compared with the normal control group, **p<0.01 The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for constructing a zebrafish acute pancreatitis model, characterized in that: The following steps are involved: 1-4 dpf zebrafish were intravenously injected with caerulein, and the fluorescence intensity of apoptotic cells and / or ROS in the pancreas of the zebrafish was tested after 3-5 days of culture.

2. The method for constructing a zebrafish acute pancreatitis model according to claim 1, characterized in that: Wild-type AB strain zebrafish were used to analyze the fluorescence intensity of pancreatic apoptotic cells.

3. The method for constructing a zebrafish acute pancreatitis model according to claim 1, characterized in that: The zebrafish used to analyze the ROS fluorescence intensity were Albino strain zebrafish with melanin allele mutation.

4. A method for constructing a zebrafish acute pancreatitis model according to claim 1, 2 or 3, characterized in that: The zebrafish are kept at a temperature of 27-29°C and the light cycle needs to be controlled to 14 h of light and 10 h of darkness from fertilization until 1-4 dpf.

5. The method for constructing a zebrafish acute pancreatitis model according to claim 1, characterized in that: The injection amount of caerulein per zebrafish was 100-200 ng.

6. The method for constructing a zebrafish acute pancreatitis model according to claim 5, characterized in that: The injection amount of caerulein per zebrafish was 200 ng.

7. A method for constructing a zebrafish acute pancreatitis model according to claim 1, 5 or 6, characterized in that: The culture temperature is 27-29 ℃.

8. The method for constructing a zebrafish acute pancreatitis model according to claim 1, characterized in that: The test method for the fluorescence intensity of apoptotic cells is as follows: taking out the cultured zebrafish, staining the apoptotic cells, placing the zebrafish under a fluorescence microscope and photographing the zebrafish, using image processing software to analyze and collect data, and analyzing the fluorescence intensity of apoptotic cells in the zebrafish pancreas.

9. The method for constructing a zebrafish acute pancreatitis model according to claim 1, characterized in that: The test method for ROS fluorescence intensity is: take out the cultured zebrafish, perform ROS cell staining, place the zebrafish under a fluorescence microscope and take pictures, use image processing software to analyze and collect data, and analyze the fluorescence intensity of zebrafish pancreatic apoptotic cells.

10. Use of the model obtained by the construction method according to any one of claims 1 to 9 in evaluating the therapeutic efficacy of drugs, health foods or foods for acute pancreatitis.

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