CN115786246B - Method for constructing a zebrafish immune-unresponsive heart failure model - Google Patents

Abstract

The invention discloses a method for constructing a zebra fish immune non-responsive heart failure model, which utilizes butyl p-hydroxybenzoate to induce zebra fish embryos so as to obtain the zebra fish immune non-responsive heart failure model. Specifically, selecting a zebra fish embryo with normal development of 5-6hpf, transferring the zebra fish embryo into a zebra fish embryo culture solution added with 1.2-2.0mg/L of butyl p-hydroxybenzoate, culturing at 28-28.5 ℃, changing the solution every 22-24h, and culturing to 3-3.5dpf to obtain the zebra fish immune non-responsive heart failure model. The model test period constructed by the invention is short, and the method is simple to operate.

Description

Method for constructing a zebrafish immune-unresponsive heart failure model

Technical Field

The invention relates to the field of animal models, and in particular to a method for inducing a zebrafish immune unresponsive heart failure model by utilizing butyl parahydroxybenzoate.

Background Art

Heart failure is a clinical syndrome of heart failure caused by various heart diseases. It is also the final outcome of most cardiovascular diseases. Its main symptoms include dyspnea, fatigue, and pulmonary edema. When heart failure occurs, the heart cannot pump enough blood to meet the body's metabolic needs. In severe cases, it can threaten people's lives. Heart failure syndrome can be caused by lesions at any level of the heart, including myocardium, cardiovascular system, pericardium, heart valves, and heart abnormalities.

Compared with traditional animal models (mice, rats, rabbits, etc.), zebrafish have the advantages of short growth cycle, high reproductive capacity, transparent body during embryonic period, easy to observe, and high-throughput screening. In addition, the genome sequence of zebrafish is 87% homologous to that of humans, and the molecular and cellular mechanisms of development are very similar to those of mammals, which can effectively simulate the pathological characteristics of human diseases. The heart of zebrafish consists of one ventricle and one atrium, similar to the heart of a 3-week-old human pup. Its heart morphology, physiology, molecular and pathological characteristics are highly similar to those of humans, and the heart is the first organ to develop and function in zebrafish, so zebrafish is an ideal animal model for studying heart disease.

At present, the main methods for establishing heart failure models include: pressure overload method, volume load method, myocardial ischemia/myocardial infarction method and drug method. However, these methods are basically based on animal models such as mice, rats, and rabbits, and they have the main disadvantages of long experimental cycle, complex methods, and difficult operation. For example, literature 1 (Qiu Boyong, Wei Yihong, Yuan Suyun, et al. Study on the protective effect and mechanism of Shenhe powder on the heart of rats with pressure overload heart failure [J]. Chinese Journal of Traditional Chinese Medicine, 2022 (004): 040.) uses the pressure overload method to establish a rat heart failure model. For example, in literature 2 (Wang Siyuan, Chen Weidan, Chen Xinxin. Study on the heart failure model induced by doxorubicin in young rats [J]. Journal of Guangzhou Medical College, 2019, 047 (001): 10-12, 16.), the doxorubicin drug method is used to construct a rat heart failure model.

Summary of the invention

The technical problem to be solved by the present invention is to provide a method for constructing a zebrafish immune unresponsive heart failure model with a short cycle and simple method.

The technical scheme of the present invention is: a method for constructing a zebrafish immune unresponsiveness heart failure model, in which butyl parahydroxybenzoate is used to induce zebrafish embryos to obtain the zebrafish immune unresponsiveness heart failure model.

Furthermore, 5-6 hpf normally developed zebrafish embryos were selected and transferred into zebrafish embryo culture medium supplemented with 1.2-2.0 mg/L butylparaben, cultured at 28-28.5°C, with the medium changed every 22-24 hours, and cultured to 3-3.5 dpf to obtain a zebrafish immune unresponsive heart failure model.

Furthermore, the added amount of butyl parahydroxybenzoate is 1.8 mg/L.

Furthermore, zebrafish embryos with normal development at 5 hpf were selected.

Furthermore, the culture temperature was 28.5°C.

Furthermore, the zebrafish immune unresponsive heart failure model was obtained by culturing to 3 dpf.

Furthermore, the medium was changed every 24 h.

Furthermore, in order to facilitate the intuitive observation of the physiological condition of zebrafish, the zebrafish adopts transgenic marked zebrafish strains, such as Tg:(myl7:GFP), Tg(myl7:GFP)×Tg(gata1:Dsred) hybrid zebrafish, Tg(myl7:GFP)×Tg:(mpeg1:mcherry), Tg(myl7:GFP)×Tg(lyz:Dsred) hybrid zebrafish, etc.

In the present invention, hpf is the abbreviation of hour post-fertilization, which refers to a few hours after fertilization of zebrafish. Dpf is the abbreviation of day post-fertilization, which refers to a few days after fertilization of zebrafish.

The present invention also discloses a method for evaluating a zebrafish immune unresponsive heart failure model, comprising one or more of the following (a)-(c):

(a) The cardiac development of the transgenic Tg:(myl7:GFP) zebrafish model was observed using a fluorescence microscope, and video recording was further used to obtain the end-systolic and end-diastolic images of the heart using tracker software.

(b) The number of red blood cells, blood volume and blood flow velocity in the heart of the Tg(myl7:GFP)×Tg(gata1:Dsred) hybrid zebrafish model were observed by fluorescence confocal and stereo fluorescence microscopy combined with o-dianisidine staining; and the transcription levels of endocardial blood flow response genes and atrial natriuretic peptide and brain natriuretic peptide were detected by qPCR.

(c) Fluorescence confocal imaging was used to observe the number of macrophages and neutrophils in the myocardial tissue of the Tg(myl7:GFP)×Tg:(mpeg1:mcherry) and Tg(myl7:GFP)×Tg(lyz:Dsred) hybrid zebrafish models; and a stereofluorescence microscope was used to photograph the number of macrophages and neutrophils in the whole body of the Tg(mfap4:GFP) and Tg(lyz:Dsred) transgenic zebrafish models; and qPCR was used to detect the transcription levels of related inflammatory factors in the heart region.

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

1. The test cycle is short and the method is simple to operate.

2. Dynamic changes in the heart area can be observed in real time.

3. High-throughput screening can be performed to rescue drugs that cause or aggravate heart failure caused by macrophage defects or immune unresponsiveness.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effects of different concentrations of butyl paraben solution on zebrafish heart development in the implementation case; wherein A is a heart phenotype diagram under white light and fluorescence microscope; B is a diagram of zebrafish heart rate changes; C is a diagram of zebrafish mortality changes; D is a diagram of zebrafish cardiomyocyte area; and E is a diagram of zebrafish bulbus arteriosus-sinus venosus distance or atrioventricular canal distance.

Figure 2 shows the effects of different concentrations of butyl paraben on the contractile ability of zebrafish embryonic heart in the implementation case; wherein A is a schematic diagram of the zebrafish heart and the end-systolic and end-diastolic diagrams; B is the ventricular area (size); C is the shortening fraction; D is the pumping fraction; and E is the stroke volume.

Figure 3 shows the effects of exposure to different concentrations of butyl paraben on the heart blood supply capacity and blood flow rate of zebrafish in the implementation case; wherein A is a representative graph of the number of heart blood cells; B is a representative graph of heart blood volume; C is a statistical graph of heart blood cell intensity; D is a statistical diagram of tail vein blood flow rate; E is a statistical graph of tail vein blood flow rate; F is the transcription level of the endocardial blood flow response gene kfl2a; E is the transcription level of the recognized indicator of heart failure.

Figure 4 shows the effects of exposure to different concentrations of butylparaben on macrophages and neutrophils in zebrafish myocardial tissue in the implementation case; wherein A is a representative graph of the number of macrophages in myocardial tissue; B is a representative graph of the number of neutrophils in myocardial tissue; C is a statistical graph of the number of macrophages in myocardial tissue; D is a statistical graph of the number of neutrophils in myocardial tissue; E is a representative graph of the number of macrophages in the whole body of zebrafish; F is a representative graph of the number of neutrophils in the whole body of zebrafish; G is a statistical graph of the number of macrophages in the whole body of zebrafish; H is a statistical graph of the number of neutrophils in the whole body of zebrafish; I is the transcription level of inflammatory factors in the heart region.

DETAILED DESCRIPTION

The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

1. Experimental Methods

1. Place the normally cultured zebrafish in a mating tank in a ratio of 1:1 or 1:2 between male and female, and separate them with a partition. The next morning, remove the barrier and the female begins to lay eggs. Collect the embryos within 30 minutes after spawning, and suck out dead and unfertilized eggs, feces and other debris, and rinse with clean water 3 times. Then place in a 28.5℃ incubator.

2. Select 5-6 hpf normally developed zebrafish embryos under a microscope, and transfer them into butyl p-hydroxybenzoate solutions of different concentration gradients (0.6 mg/L, 1.2 mg/L, 1.8 mg/L, etc.) prepared with embryo culture water. At the same time, set up a control group (incubated with the same volume of embryo culture water) and place them in an incubator at 28-28.5°C, changing the solution every 22-24 hours.

3. The heart development of transgenic Tg:(myl7:GFP) zebrafish after treatment was observed using a fluorescence microscope (Leica M205 FA, Germany). Video recording was also used to obtain images of the end of systole and end of diastole using tracker software. The ventricular area (size), shortening fraction, pumping fraction, and stroke volume were obtained using the short axis length a and the long axis length b, respectively (as shown in Figure 2).

Ventricular area = π × ab/2;

Ventricular volume = a2 × b × 0.523;

Fractional shortening = (end-diastolic area - end-systolic area)/end-diastolic area;

Pump fraction = (end-diastolic volume - end-systolic volume)/end-diastolic volume;

Stroke volume = end-diastolic volume - end-systolic volume.

4. Using Tg(myl7:GFP)×Tg(gata1:Dsred) hybrid zebrafish, the number of red blood cells, blood volume and blood flow velocity in the zebrafish heart were observed by fluorescence confocal and stereo fluorescence microscopy combined with o-dianisidine staining; and qPCR was used to detect the transcription levels of endocardial blood flow response genes and atrial natriuretic peptide and brain natriuretic peptide.

5. Observe the number of macrophages and neutrophils in the myocardial tissue of Tg(myl7:GFP)×Tg:(mpeg1:mcherry) and Tg(myl7:GFP)×Tg(lyz:Dsred) hybrid zebrafish by fluorescence confocal imaging; and use a stereofluorescence microscope to photograph the number of macrophages and neutrophils in the transgenic zebrafish Tg(mfap4:GFP) and Tg(lyz:Dsred); and use qPCR to detect the transcription levels of related inflammatory factors in the heart area.

2. Experimental Results

(a) Effects of different concentrations of butyl paraben solution on the contractile ability of zebrafish heart.

As shown in Figure 1, the initial phenotypes of pericardial edema, cardiac linearization, and decreased heart rate were observed after treatment with butyl paraben (A and B in Figure 1). And with the increase in the concentration and treatment time of butyl paraben, the survival rate and heart rate of zebrafish decreased (B and C in Figure 1), and the state of pericardial edema and cardiac linearization became more serious (A, D, E in Figure 1). In addition, as shown in Figure 2, further video recording was performed by tracker software to obtain the end-systolic and end-diastolic images of the heart (A in Figure 2). Through statistics, it was found that butyl paraben solution caused the zebrafish ventricular area (size) (B in Figure 2), shortening fraction (C in Figure 2), pumping fraction (D in Figure 2), and stroke volume (E in Figure 2) to decrease significantly with increasing concentration. Among them, shortening fraction and pumping fraction are key indicators of cardiac contractility, and insufficient cardiac contractility meets one of the key characteristics of heart failure.

(b) Effects of different concentrations of butyl paraben solution on the zebrafish heart's blood supply capacity and blood flow velocity.

The results are shown in Figure 3. Further fluorescence confocal and o-dianisidine staining revealed that a decrease in cardiac blood cells and blood volume led to a decrease in cardiac blood supply (reduced cardiac blood flow) (A, B, C in Figure 3). It was also found that it led to blood circulation disorders (reduced blood flow rate) (D, E in Figure 3). The reduction in cardiac blood flow was further verified by endocardial blood flow response gene detection (F in Figure 3). That is, the key characteristics of heart failure were met phenotypically. And the key molecular indicators leading to heart failure were verified by increased transcription levels of atrial natriuretic peptide (nppa) and brain natriuretic peptide (nppb) (G in Figure 3).

(c) Butylparaben solution causes macrophage deficiency and immune cell response defects in the zebrafish heart.

The results are shown in Figure 4. By recording macrophages and neutrophils in the zebrafish heart region through fluorescence confocal microscopy (A and B in Figure 4), it was found that after treatment with butylparaben, myocardial tissue and heart region did not recruit more macrophages and neutrophils (C and D in Figure 4), and the number of heart-resident macrophages was lost (B in Figure 4); and the morphology and size of macrophages were abnormally changed (arrow A in Figure 4). By photographing zebrafish macrophages and neutrophils throughout the body through stereo fluorescence microscopy, it was found that butylparaben did not significantly affect the overall macrophage and neutrophil status (E, G, F, H in Figure 4); but by QPCR, the transcription level of inflammatory factors in the isolated heart was detected (I in Figure 4), and it was found that there was a significant inflammatory response in the heart region; the results showed that butylparaben inhibited the recruitment of macrophages and neutrophils in myocardial tissue and heart region and led to defects in heart-resident macrophages.

Claims (9)

1. The method for constructing the zebra fish immune non-responsive heart failure model is characterized by selecting 5-6 hpf normal-development zebra fish embryos, transferring the zebra fish embryos into zebra fish embryo culture solution added with 1.2-1.8mg/L butyl p-hydroxybenzoate, culturing at 28-28.5 ℃, changing the solution every 22-24 h, and culturing until the temperature reaches 3-3.5 dpf to obtain the zebra fish immune non-responsive heart failure model.

2. The construction method according to claim 1, wherein the addition amount of butyl parahydroxybenzoate is 1.8mg/L.

3. The method of claim 1, wherein 5 hpf normal-developing zebra fish embryos are selected.

4. The method according to claim 1, wherein the culturing temperature is 28.5 ℃.

5. The method of claim 1, wherein culturing to 3dpf results in a zebra fish immune non-responsive heart failure model.

6. The method of claim 1, wherein the liquid is changed every 24 hours.

7. The method of claim 1, wherein the zebra fish is a transgenic tagged line.

8. The construction method according to claim 7, wherein the transgenic marker line is one or more of Tg (myl: GFP), tg (myl: GFP) x Tg (gata 1: dsred), tg (myl: GFP) x Tg (mpeg 1: mcherry) or Tg (myl: GFP) x Tg (lyz: dsred).

9. A method of assessing a model of zebra fish immune non-responsive heart failure as claimed in any one of claims 1-8, comprising one or more of the following (a) - (c):

(a) Observing the heart development condition of the transgenic Tg (myl 7:GFP) zebra fish model by using a fluorescence microscope, and obtaining images of the end systole and the end diastole of the heart by using video recording and tracker software;

(b) Observing the quantity, blood volume and blood flow velocity of red blood cells at the heart of the hybridized zebra fish model by fluorescence confocal, integral fluorescence microscopy shooting and combining o-dianisidine staining to observe Tg (myl 7:GFP) x Tg (gata 1: dsred); detecting endocardial blood flow reaction genes and the transcription level of the natriuretic peptide and the brain natriuretic peptide by qPCR;

(c) The numbers of macrophages and neutral particles in myocardial tissue of the hybridized zebra fish model were observed by fluorescence confocal imaging for Tg (myl: GFP). Times.Tg (mpeg 1: mcherry), tg (myl: GFP). Times.Tg (lyz: dsred); shooting the quantity conditions of whole macrophages and neutral grains of the transgenic zebra fish model with a split fluorescent microscope according to Tg (mfap 4:GFP) and Tg (lyz: dsred); and detecting transcription level of the inflammatory factors related to the heart region by qPCR.