Method for constructing vasculitis model in zebra fish

Abstract

The invention belongs to the technical field of biological model construction, and particularly relates to a method for constructing a vasculitis model in zebra fish. The compound is obtained by inducing a Candida albicans water soluble substance (CAWS), and the specific method is micro-injection or soaking. According to the method, whether modeling succeeds or not can be judged in the living body state of the model animal, the method is more suitable for visually observing the inflammation occurrence position, a research method for vasculitis (including Kawasaki disease) is developed, follow-up scientific research data can be richer and more accurate, and a more solid theoretical basis is laid for clinical transformation of targeted therapy of vasculitis (including Kawasaki disease).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological model construction, and particularly relates to a method for constructing a vasculitis model in zebrafish. BACKGROUND

[0002] Intraperitoneal injection of Candida albicans aqueous substance (CAWS) can induce systemic vasculitis in mice. The model is evaluated as an animal model of vasculitis and a subtype of vasculitis, Kawasaki disease (KD). However, there are the following problems in using CAWS to construct a vasculitis or KD model in mice: (1) The key indicator of whether the mouse modeling is successful is to observe whether inflammatory infiltration appears in the mouse heart tissue section by hematoxylin-eosin staining, but the heart is also the research material of KD. If the heart is used to determine whether the modeling is successful, the subsequent research material is lost; if the heart is used for research, it cannot be determined whether the mouse has really modeled successfully, and this unsolvable contradiction may lead to the mouse finally studied may not have really occurred KD, thereby causing the experimental results to deviate; (2) The extraction cycle of CAWS is long, and the yield is low. A large amount is used when modeling the mouse, and the labor cost is high; (3) The growth cycle of the mouse is slow, so the vasculitis or KD process is slow, and the related symptoms are difficult to continuously observe in the survival stage of the mouse (such as: inflammatory infiltration progress of the heart); (4) The use of CAWS to model mice cannot completely reproduce the symptoms of human KD, such as red eyes.

[0003] Therefore, a more simple and accurate vasculitis or KD modeling method is needed in the art. SUMMARY

[0004] Based on the above problems, the application provides a method for constructing a vasculitis model in zebrafish, which comprises inducing 2dpf zebrafish by using CAWS to achieve the construction of the vasculitis model.

[0005] Preferably, the CAWS-induced 2dpf zebrafish comprises two modes of microinjection and soaking;

[0006] Among them, the microinjection is to inject 10-20nL of CAWS solution with concentrations of 0.5, 4 and 8mg / ml into the yolk sac of zebrafish, and 24h of culture can obtain;

[0007] The soaking is to use the embryo culture water of the modeling zebrafish to configure CAWS solution with concentrations of 0.5, 4 and 8mg / ml, and the zebrafish is placed in it for 24h to obtain.

[0008] Compared with the prior art, the present application has the following beneficial effects: (1) the method can determine whether the modeling is successful in the live state of the model animal, (2) it is more suitable for directly observing the inflammation site, and it opens up the research method of vasculitis or KD, (3) it can also make the subsequent scientific research data more abundant and accurate, and it lays a more solid theoretical foundation for the clinical transformation of targeted treatment of vasculitis or KD. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 For the structure of zebrafish vascular endothelium under microscopic injection conditions;

[0010] Figure 2 For the structure of zebrafish vascular endothelium under immersion conditions;

[0011] Figure 3 For the aggregation of neutrophils after 2dpf zebrafish are immersed in CAWS for 24 hours;

[0012] Figure 4 For the aggregation of macrophages in the eyes and hearts of 2dpf zebrafish after 24 hours of immersion in CAWS;

[0013] Figure 5 For the transcriptome sequencing analysis results of 2dpf zebrafish after 24 hours of immersion in CAWS. DETAILED DESCRIPTION

[0014] The present application will be described in detail below in conjunction with examples, so as to facilitate the understanding of the present application by those skilled in the art. It is necessary to point out here that the examples are only used to further illustrate the present application and cannot be understood as limiting the protection scope of the present application. Non-essential improvements and adjustments to the present application made by those skilled in the art based on the above description should still fall within the protection scope of the present application. At the same time, the raw materials not described in detail are all commercially available products; the process steps or preparation methods not mentioned in detail are all known process steps or preparation methods to those skilled in the art.

[0015] Example 1: Construction of a zebrafish KD vasculitis model

[0016] Healthy and mature Tg(kdrl:mCherry) zebrafish and wild type (WT) zebrafish were selected and put into the breeding tank at a ratio of 2:1, and the next morning, healthy and high-quality embryos were selected and put into a 28°C incubator (light: 8:00-22:00, dark: 22:00-8:00 the next day). The water formula for zebrafish culture: 5 mmol / L NaCl, 0.17 mmol / L KCl, 0.4 mmol / L CaCl2, 0.16 mmol / L MgSO4, and the water was changed twice a day. When the zebrafish developed to 2dpf, zebrafish with red fluorescent vascular strips were selected for modeling. There are two methods for modeling, as follows:

[0017] (I) Microinjection

[0018] Healthy and normally developed 2dpf zebrafish with red fluorescent vascular strips were microinjected with 10-20 nL of CAWS solution at concentrations of 0, 0.5, 4, and 8 mg / ml, respectively, into the yolk sac, and then placed in an incubator for 24 h and 48 h, respectively. The zebrafish were then placed on a glass slide and photographed under a fluorescence microscope to observe the blood vessels and save the results, as shown in Figure 1 (Endothelial structure of zebrafish blood vessels under microinjection conditions): Under the condition of microinjection of CAWS at a concentration gradient of 0.5-8 mg / ml, the endothelial morphology of the blood vessels of 2dpf zebrafish embryos with red fluorescent vascular strips was disordered, suggesting that vasculitis may have occurred (white arrows indicate the site of endothelial injury; hpi: hours post injection; scale bar: 1 mm).

[0019] (II) Soaking

[0020] Healthy and normally developed 2dpf zebrafish with red fluorescent vascular strips were added to CAWS solution at concentrations of 0, 0.5, 1, 2, 4, and 8 mg / ml prepared with embryo culture water, and then placed in an incubator for 24 h and 48 h, respectively. The zebrafish were then placed on a glass slide and photographed under a fluorescence microscope to observe the blood vessels and save the results, as shown in Figure 2 (Endothelial structure of zebrafish blood vessels under soaking conditions): Under the condition of soaking with CAWS at a concentration gradient of 0.5-8 mg / ml, the endothelial morphology of the blood vessels of 2dpf zebrafish embryos with red fluorescent vascular strips was disordered, suggesting that vasculitis may have occurred (white arrows indicate the site of endothelial injury. Scale bar: 1 mm). Among them, 8 mg / ml of CAWS solution caused the death of all zebrafish after 48 h of soaking, so this part of the data is missing.

[0021] Tg(kdrl:mCherry) fish were crossed with Tg(lyz:EGFP) or Tg(mpeg1.1:GAL4, UAS:EGFP) zebrafish respectively, and embryos with both red and green fluorescence were selected. At 2dpf, 4mg / ml CAWS solution prepared with embryo culture water was added, and the zebrafish were cultured in the incubator for 24h. The blood vessels of the zebrafish were photographed under a fluorescence microscope and saved, and the results are shown in Figure 3 (CAWS immersion 2dpf zebrafish for 24h neutrophil aggregation) As shown, neutrophils were more aggregated on the blood vessels in the zebrafish, indicating the occurrence of vasculitis (yellow dotted line indicates neutrophil accumulation and aggregation on the blood vessels; scale bar is 1mm).

[0022] At the same time, as shown in Figure 4 (CAWS immersion 2dpf zebrafish for 24h macrophage aggregation in the eyes and heart) Macrophages also specifically aggregate in the heart and eyes of zebrafish, which can provide a theoretical basis for the red eye symptoms and heart damage during KD (yellow arrow indicates the heart, and blue arrow indicates the eye. Scale bar is 1mm.).

[0023] WT zebrafish embryos were added with 4mg / ml CAWS solution prepared with embryo culture water at 2dpf, and cultured in the incubator for 24h. RNA was extracted for transcriptome sequencing, and the results are shown in Figure 5 (CAWS immersion 2dpf zebrafish for 24h transcriptome sequencing analysis results) As shown, the red arrow indicates that inflammation occurs.

[0024] The blank control group above is PBS solution of the corresponding volume.

[0025] In summary, CAWS can cause vasculitis in zebrafish and can be used as an animal model for vasculitis and KD research.

[0026] The principles and implementation modes of the present application are described by applying specific examples in this paper, and the above example description is only used to help understand the method of the present application and its core idea; at the same time, for those skilled in the art, according to the idea of the present application, the specific implementation mode and application range will be changed. In summary, the content of the specification should not be understood as a limitation of the present application.

Claims

1. A method of constructing a model of vasculitis in zebrafish, characterized by: The CAWS is used to induce 2dpf zebrafish to build a blood vessel inflammation model.

2. The method of claim 1, wherein the model of vasculitis is constructed by: The CAWS is used to induce 2dpf zebrafish in two ways, namely microinjection and soaking. The microinjection is to inject 10-20nL of CAWS solution with a concentration of 0.5-8mg / ml into the yolk sac of zebrafish, and then culture for 24-48h to obtain the zebrafish; The soaking is to use the embryo culture water of the modeling zebrafish to configure the CAWS solution with a concentration of 0.5-8mg / ml, and then place the zebrafish in the solution to culture for 24-48h to obtain the zebrafish.

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