Zebra fish embryo model for 5-FU side effect research and construction method and evaluation method thereof

Abstract

The invention belongs to the field of animal models, and particularly relates to a zebrafish embryo model for 5-FU side effect research and a construction method and an evaluation method thereof. The construction method comprises the following steps: selecting zebrafish embryos for later use; and putting the zebrafish embryo into a culture hole, and then adding 5-FU for modeling treatment, thereby finishing the construction of the zebrafish embryo model for 5-FU side effect research. The 5-FU induced intestinal mucositis model is constructed by adopting a 3-5-day zebrafish embryo and analyzing information such as the survival rate, the travel trajectory, the intestinal morphology and ROS fluorescent living imaging of zebrafish, and the 5-FU induced intestinal mucositis model has the advantages of being simple in observation index, easy to observe, simple to operate, capable of saving operation time, convenient, rapid, high in efficiency and the like. The method has the characteristics of simple operation, high cost, cheaper mouse model, qualitative and quantitative properties and the like, and is suitable for high-throughput screening of 5-FU induced intestinal mucositis model drugs.

Description

Technical Field

[0001] The present invention belongs to the field of animal models, and particularly relates to a zebrafish embryo model for 5-FU side effect research, and a construction method and an evaluation method thereof. Background Art

[0002] The incidence and mortality of malignant tumors are both on the rise. Chemotherapy plays an extremely important role in the treatment of malignant tumors. It exerts an anti-tumor effect by inhibiting cell growth and / or cell division to kill rapidly growing cells. At the same time, it induces various side effects such as myelosuppression, gastrointestinal toxicity, neurotoxicity, and myocardial toxicity. Intestinal epithelium is easily damaged by chemotherapeutic drugs due to its high proliferation potential, resulting in DNA damage and cell death, with the most obvious damage to intestinal crypt stem cells. This intestinal toxicity is also called chemotherapy-induced intestinal mucositis (CIM). 5-FU is a basic treatment regimen for malignant tumors such as colorectal cancer. Studies have shown that about 50-80% of patients using 5-FU will eventually develop mucositis and stop chemotherapy, leading to an imbalance between absorption and secretion in the small intestine, mainly manifested as abdominal pain, diarrhea, etc., which is one of the main factors leading to chemotherapy termination and poor prognosis of patients. However, the mechanism of side effects such as chemotherapy-induced intestinal mucositis is not clear, and clinically available drugs are very limited.

[0003] At present, the research on the side effects of chemotherapeutic drugs mainly focuses on in vivo and in vitro cell experiments, but there are the following problems: the cost of mammals required for in vivo experiments is high, the time for constructing models is long, and the ethical review of animal use is difficult. The in vitro cell experiment system is single, and the environment cannot simulate the in vivo metabolic environment, etc., which all hinder the mechanism research and drug development of chemotherapeutic drug side effects.

[0004] In recent years, with the rapid development of traditional Chinese medicine chemoinformatics, the research on multi-components and multi-targets of traditional Chinese medicine has been gradually deepened. Most of the components screened by researchers using computer virtual screening cannot reflect the true degree of clinical efficacy. The reason is that there is a gap between in vitro models and in vivo animal models, which cannot balance the high throughput and accuracy of drug screening and cannot establish a docking relationship between components and efficacy.

[0005] 87% of the genes of zebrafish are similar to those of humans. Zebrafish have the advantages of fast development, easy reproduction, convenient behavioral and anatomical observation, low cost, short experimental cycle, and being qualitative and quantitative. Zebrafish have a digestive system and a metabolic system similar to those of mammals. In recent years, zebrafish have been widely used in the research of the mechanisms of diseases in systems such as cardiovascular, endocrine, nerve, and gastrointestinal, and in the evaluation of drug toxicity. Zebrafish have potential application value in the research of side effects such as chemotherapy-induced enteritis, and can provide a high-throughput drug screening model organism and a direct and visual evaluation tool for the research of drug treatment of chemotherapy-induced enteritis and other diseases. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a method for constructing a zebrafish embryo model for the study of 5-FU side effects. The construction method includes the following steps:

[0007] (1) Select zebrafish embryos for standby;

[0008] (2) Put the zebrafish embryos into culture wells, and then add 5-FU for modeling treatment, that is, complete the construction of the zebrafish embryo model for the study of 5-FU side effects.

[0009] Preferably, in step (1), the zebrafish embryos are wild zebrafish embryos 3 to 5 days after fertilization.

[0010] Preferably, in step (2), the zebrafish embryos are placed in culture wells for culture at a density of 8 to 10 embryos per well.

[0011] Preferably, during the culture process, E3 medium is added. The E3 medium includes 34.8 g / L of NaCl, 1.6 g / L of KCl, 5.8 g / L of CaCl2·2H2O, and 9.78 g / L of MgCl2·6H2O.

[0012] Preferably, in step (2), the concentration of 5-FU added is 200 to 1400 μM.

[0013] Preferably, in step (2), the time of modeling treatment is 120 h.

[0014] Based on the same technical concept, the present invention further provides a zebrafish embryo model for the study of 5-FU side effects obtained by the above construction method.

[0015] Based on the same technical concept, the present invention further provides an evaluation method for the above construction method. The evaluation method is as follows: Use the obtained zebrafish embryo model as the model group and set a normal control group, and detect the following indicators of the model group and the normal control group:

[0016] (1) Survival index;

[0017] (2) Parade trajectory index;

[0018] (3) Morphological evaluation;

[0019] (4) Oxidative stress index;

[0020] Using statistical methods to perform differential analysis on the above index data of the model group and the normal control group, and setting qualified values for the above indexes. If there are significant differences between the index data or scores of the model group and the normal control group, and the model group reaches the qualified value, it is considered that the model construction is successful.

[0021] The beneficial effects of the present invention are as follows:

[0022] The present invention uses 3 - 5 - day zebrafish embryos to construct a 5 - FU - induced intestinal mucositis model. By analyzing information such as zebrafish survival rate, parade trajectory, intestinal morphology, ROS fluorescence in vivo imaging, etc., a 5 - FU - induced intestinal mucositis model is constructed. This model has the characteristics of simple observation indexes, easy to observe, simple operation, saving operation time, convenient and fast, relatively low cost compared with mouse models, and can be qualitatively and quantitatively analyzed, and is suitable for high - throughput screening of drugs for 5 - FU - induced intestinal mucositis models. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is a result diagram of zebrafish parade trajectory analysis.

[0025] Figure 2 It is the zebrafish intestinal morphology (HE staining).

[0026] Figure 3 It is a ROS in vivo imaging diagram of zebrafish embryos. Detailed Embodiments

[0027] To make the purpose, technical solutions and advantages of the present invention clearer, the following will describe the technical solutions of the present invention in detail. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0028] Example 1

[0029] In this example, a zebrafish embryo model for studying the side effects of 5-FU was first constructed. The construction method includes the following steps:

[0030] (1) Select wild zebrafish embryos 3 to 5 days after fertilization and set aside.

[0031] (2) Place the wild zebrafish embryos into a 12-well plate at a density of 10 embryos per well, with each group repeated 3 times. Then, put the 5-FU prepared with E3 medium into the culture wells, and the concentration of 5-FU is 0 μM to 1600 μM; among them, the E3 medium includes 34.8 g / L of NaCl, 1.6 g / L of KCl, 5.8 g / L of CaCl2·2H2O, and 9.78 g / L of MgCl2·6H2O.

[0032] (3) Replace the medium every day, observe the embryo mortality rate, and a zebrafish embryo model can be obtained after administering the drug for 120 hours.

[0033] In the survival rate index, after step (3) is completed, observe the death situation of the embryos in each group under a stereomicroscope. It is found that when the concentration is 1200 μM and above, the mortality rate of zebrafish embryos exceeds 50%, and the details are shown in Table 1.

[0034] Table 1 Survival rate of zebrafish embryos after intervention with different concentrations of 5-FU

[0035]

[0036]

[0037] Among them, the survival rate = (total number of zebrafish embryos - number of dead zebrafish embryos) / total number of zebrafish embryos * 100%.

[0038] According to the results in Table 1, 5-FU with a concentration greater than or equal to 1200 μM is selected as the modeling concentration.

[0039] Example 2

[0040] In this example, a zebrafish embryo model for studying the side effects of 5-FU was first constructed. The construction method includes the following steps:

[0041] (1) Select wild zebrafish embryos 3 to 5 days after fertilization and set aside.

[0042] (2) Transfer the wild zebrafish embryos into a 48-well plate containing fresh E3 buffer at a density of 10 embryos per well. The E3 medium includes 34.8 g / L of NaCl, 1.6 g / L of KCl, 5.8 g / L of CaCl2·2H2O, and 9.78 g / L of MgCl2·6H2O;

[0043] (3) The 48-well plate was placed in the DanioVision behavior recording system, which was equipped with relevant motion tracking and analysis software (EthoVision XT, Noldus, Netherlands). The parameters of light intensity, circulating water system (the observation tank was maintained at 28 °C), and light cycle (light 5 min / dark 5 min / light 5 min / dark 5 min) were set. At the same time, video acquisition and analysis of the zebrafish movement trajectories were performed.

[0044] Result description: As Figure 1 shown, in this example, it was found that after 120 h of 5-FU intervention, the swimming trajectories of zebrafish showed weakened movement. Compared with the Control group, the maximum acceleration, minimum acceleration, movement distance, movement speed, and cumulative movement duration of zebrafish intervened with 1200 μM 5-FU all decreased (p < 0.01).

[0045] Example 3

[0046] In this example, a zebrafish embryo model for studying the side effects of 5-FU was first constructed. The construction method includes the following steps:

[0047] (1) Wild zebrafish embryos 3 - 5 days after fertilization were selected and reserved.

[0048] (2) The wild zebrafish embryos were placed in a 12-well plate at a density of 10 embryos per well, with each group repeated 3 times. Then, 5-FU prepared with E3 medium was added to the culture wells, and the concentration of 5-FU was 0 μM - 1600 μM; among them, the E3 medium included 34.8 g / L of NaCl, 1.6 g / L of KCl, 5.8 g / L of CaCl2·2H2O, and 9.78 g / L of MgCl2·6H2O.

[0049] (3) The culture medium was changed every day, and the embryo mortality was observed. A zebrafish embryo model could be obtained after 120 hours of drug administration.

[0050] Further, when step (3) is completed, tricaine methanesulfonate is added to the zebrafish embryo model system until the concentration of tricaine methanesulfonate in the system is 0.002 - 0.004 wt% to anesthetize the embryonic zebrafish. Then it is fixed with 4 wt% paraformaldehyde (PFA) for 2 - 4 h at room temperature, and then the zebrafish embryos are rinsed in running water for about 2 h. Subsequently, they are dehydrated with ethanol of different concentrations (dehydrated with 50% ethanol for more than 2 h, 70% ethanol for more than 2 h, 80% ethanol for 2 h, 90% ethanol for 1.5 h, 95% ethanol for 1.5 h, absolute ethanol for 40 min). The dehydrated zebrafish embryos are treated with ethanol:xylene with a volume ratio of 1:1 for 1 h, then treated with xylene for 10 min, then soaked in xylene:paraffin with a volume ratio of 1:1 for 1 h, and then soaked in paraffin for 40 min to 1 h. The zebrafish embryos are taken out of the paraffin, placed in an embedding cassette, poured with paraffin, and embedded. Then they are sectioned on a microtome, and the section thickness is set to 4 - 8 μm. The cut thin slices are gently placed in a 38°C water bath. After being fully unfolded, they are transferred to a glass slide coated with glycerin jelly and dried in an incubator at 60°C for about 12 h. The glass slide is taken out and dewaxed with xylene for 10 min, dewaxed with absolute ethanol for 1 min, treated with 95% ethanol for dewaxing for 1 min, 90% ethanol for dewaxing for 1 min, 80% ethanol for dewaxing for 1 min, 70% ethanol for dewaxing for 1 min, and then hematoxylin-eosin staining is carried out. The specific staining steps are as follows: stained with hematoxylin solution for 10 min, then differentiated and washed with distilled water and hydrochloric acid ethanol for a moment respectively, continuously rinsed with running water for more than 10 min, rinsed with 70% ethanol for 1 min, 80% ethanol for 1 min, 95% ethanol for 1 min, then stained in eosin solution for 1 min, enter the dehydration stage, dehydrated with 95% ethanol for 1 min, 100% absolute ethanol for 1 min, xylene for 5 min. Finally, neutral gum is dropped on the glass slide, and then the cover glass is gently placed on the glass slide to prevent air bubbles. For the sections of 10 zebrafish embryos, 5 sections are randomly selected from each tail for microscopic observation of the intestinal cross-section, and the corresponding results are recorded.

[0051] In addition, the pathological score of zebrafish intestinal injury is shown in Table 2 (in this example, the pathological score is set to be mild injury when it is greater than or equal to 1 and less than 2; moderate injury when it is greater than or equal to 2 and less than 3; severe intestinal injury when it is greater than or equal to 3), and the scores of some concentrations are shown in Table 3.

[0052] Table 2

[0053] Item Disappearance of intestinal folds Cell shedding Inflammatory cell infiltration Score +1 +1 +1

[0054] Table 3

[0055] Concentration Score 0 μM 0 600 μM 0 800 μM 1 1000 μM 2 1200 μM 3 1400 μM 3

[0056] It can be seen from Figure 2 that in zebrafish embryos intervened at a concentration of 1200 μM and above, intestinal folds disappeared, cells shed, and inflammatory cell infiltration occurred. A pathological score of greater than or equal to 3 was the standard for zebrafish intestinal mucositis.

[0057] Example 4

[0058] In this example, a zebrafish embryo model for 5-FU side effect research was first constructed. The construction method included the following steps:

[0059] (1) Select wild zebrafish embryos 3 - 5 days after fertilization and set aside;

[0060] (2) Place the wild zebrafish embryos in a 12-well plate at a density of 10 embryos per well, with 3 replicates for each group. Then put 5-FU prepared with E3 medium into the culture wells. The concentration of 5-FU was 1200 μM. Among them, the E3 medium included 34.8 g / L of NaCl, 1.6 g / L of KCl, 5.8 g / L of CaCl2·2H2O, and 9.78 g / L of MgCl2·6H2O;

[0061] (3) Replace the medium every day, observe the embryo mortality rate, and a zebrafish embryo model can be obtained after administering the drug for 120 hours.

[0062] Furthermore, when step (3) was completed, staining was performed for 45 minutes at a rate of 1 μL DCFH-DA (concentration 10 mM) per milliliter of culture solution, washed three times with the medium, and tricaine methanesulfonate was added to the zebrafish embryo model system until the concentration of tricaine methanesulfonate in the system was 0.002 - 0.004% to anesthetize the embryonic zebrafish. Subsequently, observation of bright field and ROS fluorescence expression was carried out under a two-photon confocal microscope, and the results were as Figure 3 shown. It can be seen from Figure 3 that in zebrafish embryos intervened with 5-FU at a concentration of 1200 μM, an increase in ROS expression occurred (p < 0.05), and oxidative stress damage was suffered.

[0063] As mentioned above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. A method for constructing a zebrafish embryo model for 5-FU side effect research, characterized in that, The construction method includes the following steps: (1) Select zebrafish embryos for standby; (2) Place the zebrafish embryos into culture wells, and then add 5-FU for modeling treatment, thus completing the construction of the zebrafish embryo model for the study of 5-FU side effects.

2. The construction method of the zebrafish embryo model for 5-FU side effect research according to claim 1, characterized in that, In step (1), the zebrafish embryos are wild zebrafish embryos 3 to 5 days after fertilization.

3. The construction method of the zebrafish embryo model for 5-FU side effect research according to claim 1, wherein, In step (2), the zebrafish embryos are placed into culture wells at a density of 8 to 10 embryos per well for culture.

4. The method for constructing a zebrafish embryo model for 5-FU side effect research according to claim 3, characterized in that During the culture process, E3 medium is added. The E3 medium includes 34.8 g / L of NaCl, 1.6 g / L of KCl, 5.8 g / L of CaCl2·2H2O, and 9.78 g / L of MgCl2·6H2O.

5. The construction method of the zebrafish embryo model for 5-FU side effect research according to claim 1, characterized in that In step (2), the concentration of 5-FU added is 200 to 1400 μM.

6. The construction method of the zebrafish embryo model for 5-FU side effect research according to claim 1, characterized in that, In step (2), the time of modeling treatment is 120 h.

7. The zebrafish embryo model for the study of 5-FU side effects obtained by the construction method according to any one of claims 1 to 6.

8. A method for evaluating the construction method according to any one of claims 1 to 6, characterized in that, The evaluation method is as follows: Use the obtained zebrafish embryo model as the model group and set up a normal control group, and detect the following indicators of the model group and the normal control group: (1) Survival index; (2) Swimming trajectory index; (3) Morphological evaluation; (4) Oxidative stress index; Use statistical methods to perform differential analysis on the above index data of the model group and the normal control group, and set a qualified value for the above indicators. If the index data or score of the model group has a significant difference compared with the normal control group, and the model group reaches the qualified value, it is considered that the model construction is successful.

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