A zebrafish model, its establishment method and application

Abstract

This invention relates to a zebrafish model, its establishment method, and its applications, relating to the biomedical field. The establishment method includes the following steps: injecting zebrafish with polyinosinic-polycytidylic acid (poly I:C), resuscitating, and culturing. This method can obtain a stable zebrafish model that can simulate an inflammatory state similar to that in humans following viral infection. This model can be used to assess whether antiviral drugs inhibit viral activity and the differences in efficacy among different antiviral drugs, thereby enabling preliminary screening of antiviral drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a zebrafish model, its establishment method, and its application. Background Technology

[0002] Viral infectious diseases are characterized by high infectivity, rapid spread, and severe consequences. Besides vaccines, antiviral drugs play a crucial role in treating viral infections. Due to the characteristics of viruses, screening experiments for antiviral drugs must be conducted in high-level biosafety laboratories (Biosafety Level 3 or 4 laboratories). Therefore, large-scale screening of antiviral drugs remains inconvenient, making the development of safe and reliable antiviral drug screening methods essential. One approach is to establish animal models to evaluate drug efficacy and screen antiviral drugs. However, commonly used mouse models are costly to build and struggle to simultaneously represent a similar inflammatory state to humans following viral infection while also being easily observable. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a method for establishing a zebrafish model. This method can obtain a stable zebrafish model that can simulate an inflammatory state similar to that of humans after viral infection. This model can be used to evaluate whether antiviral drugs have an inhibitory effect on the virus and the differences in efficacy among different antiviral drugs, thereby enabling preliminary screening of antiviral drugs.

[0004] To achieve the above objectives, the present invention provides a method for establishing a zebrafish model, comprising the following steps: injecting zebrafish with polyinosinic acid, reviving, and culturing.

[0005] During their research, the inventors discovered that while mice are commonly used animal models, their long modeling cycle, typically requiring pre-drug administration for more than 7 days, is not only costly but also unsuitable for large-scale drug screening, making them unsuitable for high-throughput screening. Therefore, mouse models cannot be stably and effectively used for antiviral drug screening. Zebrafish (Danio rerio), a small tropical freshwater fish native to the Indian subcontinent, is an emerging model organism with a genome highly homologous to the human genome and similar inflammatory cells and receptors. It can effectively simulate the inflammatory state following viral infection in humans, allowing the established zebrafish model to better reflect the efficacy of antiviral drugs. This enables the assessment of whether antiviral drugs inhibit viral activity and the differences in efficacy among different antiviral drugs. Furthermore, zebrafish have advantages such as short spawning cycles, high egg production, transparent embryos, and in vitro fertilization and development. Therefore, using zebrafish as the modeling basis results in a shorter experimental cycle, lower costs, and suitability for high-throughput screening, allowing for simultaneous comparison of efficacy differences among multiple drugs.

[0006] Given that zebrafish were chosen as the modeling basis, the inventors used polyinosinic-polycytidylic acid (PolyIC) injection to establish a zebrafish model. PolyIC, as a synthetic double-stranded viral RNA mimic, is a common viral replication intermediate that can be recognized and activated by Toll-like receptor 3 (TLR3). It participates in regulating the body's immune function, apoptosis, and other pathological processes, inducing the body to produce antiviral immune responses and inflammatory responses. Therefore, injecting polyIC can enable the zebrafish model to better simulate the inflammatory state of humans after viral infection.

[0007] In one embodiment, the injection is a microinjection, and the injection site is the yolk sac of the zebrafish.

[0008] In one embodiment, the concentration of polyinosinic acid is 0.8-1.2 mg / mL, and the amount of polyinosinic acid used is 1.5-2.5 nL.

[0009] Because zebrafish, especially juveniles three days after fertilization, are relatively fragile, it is necessary to strictly control the concentration and dosage of polyinosinic acid (PIA). Otherwise, zebrafish may die or die from yolk sac rupture.

[0010] In one embodiment, the resuscitation is achieved using embryo culture medium, the resuscitation temperature is 27-29°C, and the culture time is 10-14 hours.

[0011] In one embodiment, the method further includes anesthetizing the zebrafish before injecting them with polyinosinic acid; the anesthetic agent for anesthesia includes tricaine.

[0012] The present invention also provides a zebrafish model obtained by the aforementioned method.

[0013] The zebrafish model described above is relatively stable and can simulate an inflammatory state similar to that of humans after viral infection. It can be used to assess whether antiviral drugs inhibit the virus and the differences in efficacy among different antiviral drugs, thereby enabling preliminary screening of antiviral drugs.

[0014] The present invention also provides a method for evaluating antiviral drugs, comprising: after the zebrafish model described in claim 6 is resuscitated, soaking it in the drug to be tested, culturing it, performing tissue staining detection, neutrophil detection, inflammatory factor detection and / or survival rate analysis, and evaluating the efficacy of the drug to be tested based on the detection results.

[0015] In one embodiment, the culture time in the evaluation method of the antiviral drug is 10-14 hours.

[0016] In one embodiment, the detection results include increased neutrophil migration and aggregation, low neutrophil count, low expression of inflammatory factors, and / or high survival rate, which indicate high efficacy of the drug being tested.

[0017] In one embodiment, the inflammatory factors include TNF-α and / or IL-6.

[0018] The present invention also provides the application of the zebrafish model in the evaluation of antiviral drugs.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention discloses a method for establishing a zebrafish model, which can obtain a stable zebrafish model that can simulate an inflammatory state similar to that of humans after viral infection. This model can be used to evaluate whether antiviral drugs have an inhibitory effect on the virus and the differences in efficacy among different antiviral drugs, thereby enabling preliminary screening of antiviral drugs. Attached Figure Description

[0021] Figure 1 This is a fluorescence image of the yolk sac of zebrafish after microinjection of PolyIC in Example 2;

[0022] Figure 2 This is a statistical diagram of neutrophils in zebrafish after microinjection of PolyIC in Example 2;

[0023] Figure 3 This is a pathological tissue image of zebrafish after microinjection of PolyIC in Example 2;

[0024] Figure 4 The inflammatory factors (of which) were microinjected into zebrafish in Example 2 after PolyIC injection Figure 4 A represents IL-6. Figure 4 B represents the mRNA expression map of TNF-α.

[0025] Figure 5 The image shows the survival rate of zebrafish microinjected with PolyIC within 72 hours in Example 2. 1 represents PBS, 2 represents Model, 3 represents LQYP-400 μg / mL, 4 represents LQYP-600 μg / mL, 5 represents LQYP-800 μg / mL, 6 represents LQKL-400 μg / mL, 7 represents LQKL-600 μg / mL, 8 represents LQKL-800 μg / mL, and 9 represents DEX-5 μg / mL. Detailed Implementation

[0026] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] definition:

[0029] 3dpf: DPF is an abbreviation for days after fertilization. 3dpf refers to 3 days after fertilization.

[0030] Embryo culture medium: refers to the E2 culture medium commonly used for zebrafish in this technical field, which was prepared by the inventors themselves. The specific components are: potassium chloride (0.25mM), sodium chloride (7.5mM), magnesium sulfate (0.5mM), potassium dihydrogen phosphate (75uM), sodium dihydrogen phosphate (0.25mM), calcium chloride (0.5mM), sodium bicarbonate (0.35mM), and methylene blue (1%). The above components were weighed and prepared into a culture medium with pure water according to their final working concentration.

[0031] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all experimental methods are conventional experimental methods in this field.

[0032] Example 1

[0033] A zebrafish model and its construction method.

[0034] I. Preparation for microinjection experiment.

[0035] Weigh out agarose powder, pour it into pure water and stir. Heat the mixture in a microwave oven, pour it into a petri dish while hot, and allow it to cool and solidify to prepare a 3% agar plate. Then, use a needle puller to heat and pull open a capillary glass tube for injection to obtain a glass needle. When injecting, trim the thin end to the size of two squares under a 6x microscope, and inject the solution into it for microinjection.

[0036] That evening, select healthy adult zebrafish, with a male-to-female ratio of 1:1, and place them in a zebrafish spawning tank, separating the males and females with a baffle. The baffle is removed when the light begins the next day. After the zebrafish mate, collect the eggs and place them in culture water containing 0.02% methylene blue, then incubate them in a constant temperature incubator at 28.5℃.

[0037] II. Modeling zebrafish using PolyIC microinjection.

[0038] Healthy 3dpf zebrafish juveniles were selected, anesthetized with 0.03% tricaine, placed on an agar plate, and 2 nL of PolyIC (1 mg / mL) solution was injected into the yolk sac of the zebrafish using microinjection. Immediately after injection, the embryos were revived with embryo culture medium at 28.5℃ and then cultured for 12 hours to obtain the zebrafish model.

[0039] Example 2

[0040] An evaluation method for antiviral drugs.

[0041] I. Establish a zebrafish model and observe neutrophils.

[0042] 1. Preparation for microinjection experiment.

[0043] Weigh out agarose powder, pour it into pure water and stir. Heat the mixture in a microwave oven, pour it into a petri dish while hot, and allow it to cool and solidify to prepare a 3% agar plate. Then, use a needle puller to heat and pull open a capillary glass tube for injection to obtain a glass needle. When injecting, trim the thin end to the size of two squares under a 6x microscope, and inject the solution into it for microinjection.

[0044] That evening, select healthy adult zebrafish, with a male-to-female ratio of 1:1, and place them in a zebrafish spawning tank, separating the males and females with a baffle. The baffle is removed the next day when the light begins. After the zebrafish mate, collect the eggs and place them in an embryo culture medium containing 0.02% methylene blue, then incubate them in a constant temperature incubator at 28.5℃.

[0045] 2. Microinjection of PolyIC into zebrafish and observation of neutrophils using fluorescence.

[0046] Healthy 3dpf zebrafish juveniles were selected, and 30 were randomly selected as the normal control group (without PolyIC injection). The remaining zebrafish juveniles were anesthetized with 0.03% tricaine, placed on an agar plate, and 2 nL of PolyIC (1 mg / mL) solution was injected into the yolk sac of the zebrafish using a microinjection method. Immediately after injection, the zebrafish were revived with embryo culture medium at 28.5℃ and then randomly assigned to the model control group, dexamethasone group, traditional Forsythia suspensa decoction group, and Forsythia suspensa formula granule group, with 30 zebrafish in each group.

[0047] In addition to the normal control group (Control) and the model control group (Model), each group was administered 2 mL of dexamethasone sodium phosphate solution, Forsythia suspensa slices, or Forsythia suspensa granule solution. The treatment groups were given 5 μg / mL of dexamethasone (DEX), and the Forsythia suspensa slices (LQYP, purchased from Kangmei Pharmaceutical Co., Ltd., Shanxi Province, batch number 221103881) and Forsythia suspensa granule group (LQKL, purchased from Guangdong Yifang Pharmaceutical Co., Ltd., batch number A2020113) were randomly divided into three groups with doses of 400 μg / mL (low dose), 600 μg / mL (medium dose), and 800 μg / mL (high dose). After administration (specifically, zebrafish juveniles were immersed in the drug-containing embryo culture medium for each group), and cultured for 12 hours, the juveniles were anesthetized and placed on agar plates. The distribution and number of neutrophils in the yolk sac were observed using a stereomicroscope, and the number of neutrophils in the yolk sac was counted by photographing.

[0048] II. Establish a zebrafish model and analyze zebrafish pathological tissues.

[0049] Healthy 3dpf zebrafish juveniles were selected and injected with PolyIC using the microinjection method described in step one of this embodiment. After administration, the fish were cultured in an incubator at 28.5°C for 12 hours and then collected. They were then placed in 4% paraformaldehyde and fixed at room temperature for 24 hours before dehydration, embedding, sectioning, and HE staining.

[0050] Dehydration steps: 80% ethanol (2h), 90% ethanol (2h), 95% ethanol (overnight), anhydrous ethanol I (0.5h), anhydrous ethanol II (0.5h), anhydrous ethanol III (1h), xylene I (0.5h), xylene II (0.5h), xylene III (0.5h), paraffin I (0.5h), paraffin II (0.5h), paraffin III (1h).

[0051] Embedding procedure: The juvenile fish were transferred to an embedding box and quickly placed on an ice plate to cool and fix. After the paraffin was completely solidified, the sections were sliced ​​with a thickness of 4μm and then baked for 1 hour.

[0052] HE staining procedure: The slides were sequentially immersed in xylene 1 (3 min), xylene 2 (3 min), xylene 3 (3 min), anhydrous ethanol 1 (2 min), anhydrous ethanol 2 (2 min), 95% ethanol (1 min), 90% ethanol (1 min), 80% ethanol (1 min), hematoxylin staining (10 min), 1% hydrochloric acid alcohol (10 s), rinsing with running water for "blue return" (10 min), eosin staining (3 min). Then, 80% ethanol (10 s), 90% ethanol (10 s), 95% ethanol (2 min), anhydrous ethanol 3 (3 min), anhydrous ethanol 4 (3 min), anhydrous ethanol 5 (3 min), xylene 4 (3 min), xylene 5 (3 min), xylene 6 (3 min), and finally mounted with neutral resin. The slides were observed and photographed under an optical microscope.

[0053] III. Establish a zebrafish model and detect the expression of inflammatory factor mRNA by qPCR.

[0054] Healthy 3dpf zebrafish juveniles were selected and injected with PolyIC using the microinjection method described in step one of this embodiment. After administration, the fish were incubated at 28.5℃ for 12 hours and then collected. The zebrafish were washed twice with PBS, and total RNA was extracted from them. 1 mL of Trizol solution was added to the zebrafish, and tissue was aspirated using a sterile syringe. Chloroform was added, and the mixture was shaken and centrifuged at 13000 rpm for 15 min. The supernatant was collected, and an equal volume of isopropanol was added. After mixing, the mixture was centrifuged at 13000 rpm for 15 min. The supernatant was discarded, leaving a white precipitate. 75% ethanol was added to this precipitate, and the mixture was vortexed and centrifuged at 13000 rpm for 15 min. The supernatant was discarded, and the RNA precipitate was obtained. After evaporation, ddH2O was added, and the RNA concentration was determined after vortexing.

[0055] RNA was reverse transcribed to obtain cDNA. The reverse transcription conditions were: Step 1, 37℃, 15 min; Step 2, 85℃, 5 s; Step 3, 4℃, then terminated. The obtained cDNA was subjected to Real-time PCR under the following conditions: Step 1, pre-denaturation, 95℃, 30 s, 1 cycle; Step 2, PCR reaction, 95℃, 5 s, then 60℃, 30 s, 40 cycles; Step 3, melting curve, 95℃, 5 s, then 65℃, 60 s, then 95℃, 1 s, 1 cycle, then incubated at 4℃. 2 -ΔΔCt The expression levels of zebrafish inflammatory factors TNF-α and IL-6 were obtained by relative quantitative analysis of the expression levels of the internal reference actin (Bactin) using the primer sequences shown in the table below.

[0056] Table 1 Primer sequences for zebrafish embryo experiments

[0057]

[0058] IV. Establish a zebrafish model and analyze the 72-hour survival rate.

[0059] Healthy 3dpf zebrafish juveniles were selected and injected with PolyIC using the microinjection method described in step one of this embodiment. After administration, the fish were cultured at 28.5℃ for 12 hours. Their survival status was observed and the number of deaths was recorded over 72 hours. The medicated embryo culture medium was replaced every 24 hours. Statistical analysis was performed using survival proportions.

[0060] V. Results Analysis.

[0061] 1. Results of fluorescence observation of neutrophils.

[0062] Fluorescence image of yolk sac after microinjection of PolyIC in zebrafish is shown below. Figure 1 As shown, the neutrophil count results are as follows: Figure 2 As shown in the figure, after microinjection of PolyIC into the yolk sac of zebrafish, the number of neutrophils in the yolk sac of the model group increased significantly. Compared with the model control group, the number of neutrophils in the low, medium and high dose groups of Forsythia suspensa traditional decoction pieces and Forsythia suspensa formula granules was significantly reduced. Among them, the number of neutrophils in the medium dose group of Forsythia suspensa traditional decoction pieces and Forsythia suspensa formula granules was similar to that in the dexamethasone group, while the number of neutrophils in the high dose group of Forsythia suspensa traditional decoction pieces and Forsythia suspensa formula granules was lower than that in the dexamethasone group.

[0063] 2. Pathological tissue analysis results of zebrafish.

[0064] The results of HE staining of zebrafish microinjection PolyIC pathological tissue are as follows: Figure 3 As shown in the figure, compared with the normal control group, neutrophil migration and aggregation were significantly increased in zebrafish juveniles after microinjection of polyIC in other groups. Both formulations of Forsythia suspensa showed varying degrees of inhibition of neutrophil aggregation into the yolk sac; at the same dose, the Forsythia suspensa granule formulation was slightly more effective than the Forsythia suspensa slices formulation.

[0065] 3. Results of qPCR detection of inflammatory factor mRNA expression.

[0066] The expression diagram of inflammatory factor mRNA after microinjection of PolyIC in zebrafish is shown below. Figure 4 As shown, where, Figure 4 A represents the results of IL-6 expression. Figure 4 B represents the expression results of TNF-α. After microinjection of PolyIC into the yolk sac of zebrafish, inflammatory factors in the yolk sac of the model group were significantly increased, while the levels of inflammatory factor mRNA in the low, medium, and high dose groups of Forsythia suspensa traditional decoction pieces and Forsythia suspensa formula granules were significantly reduced.

[0067] 4. 72-hour survival rate analysis results.

[0068] The survival rate of zebrafish within 72 hours after microinjection of PolyIC is shown in the figure. Figure 5 As shown, after microinjection of PolyIC into the yolk sac of zebrafish, the survival rate of zebrafish in the model group decreased significantly, while the survival rates of the low, medium and high dose groups treated with traditional Forsythia suspensa slices and Forsythia suspensa granules were significantly improved.

[0069] 5. Differences in efficacy among different antiviral drugs.

[0070] By comparing the Forsythia suspensa granule formulation group and the Forsythia suspensa decoction group, it was found that the Forsythia suspensa granule formulation group, at medium and high doses, was superior to the Forsythia suspensa decoction group at medium and high doses in inhibiting neutrophil aggregation and reducing inflammatory factors. Therefore, this zebrafish model can be used to compare the efficacy differences of different antiviral drugs, thereby enabling preliminary screening of antiviral drugs.

[0071] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0072] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for establishing a zebrafish model, characterized in that, Includes the following steps: Zebrafish were injected with polyinosinic-cytosine, revived, and cultured. The zebrafish are 3dpf zebrafish juveniles; the injection is a microinjection, and the injection site is the yolk sac of the zebrafish; the concentration of polyinosinic-polycytidylic acid is 0.8-1.2 mg / mL, and the amount of polyinosinic-polycytidylic acid used is 1.5-2.5 nL; the resuscitation is achieved using E2 culture medium, the resuscitation temperature is 27-29℃, and the culture time is 10-14 h.

2. The method for establishing according to claim 1, characterized in that, The method further includes anesthetizing the zebrafish before injecting them with polyinosinic-polycytidylic acid; the anesthetic agent used for anesthesia includes tricaine.

3. A method for evaluating antiviral drugs, characterized in that, include: After the zebrafish model is revived using the method described in any one of claims 1-2, it is immersed in the drug to be tested, cultured, and subjected to tissue staining detection, neutrophil detection, inflammatory factor detection and / or survival rate analysis. The efficacy of the drug to be tested is evaluated based on the test results.

4. The evaluation method according to claim 3, characterized in that, The detection results, including increased neutrophil migration and aggregation, low neutrophil count, low expression of inflammatory factors, and / or high survival rate, indicate high efficacy of the drug being tested.

5. The evaluation method according to claim 4, characterized in that, The inflammatory factors include TNF-α and / or IL-6.

6. The application of the zebrafish model obtained by the method described in any one of claims 1-2 in the evaluation of antiviral drugs.

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