CN116369249A - Construction method of zebra fish enteritis model - Google Patents

Abstract

The invention belongs to the field of animal models, and particularly discloses a method for constructing a zebra fish enteritis model. The light causes insufficient sleep, and can relate to the field of animal models, so that active oxygen is continuously accumulated in the intestinal tract, oxidative damage is caused to the intestinal tract, the barrier function of the intestinal tract is destroyed, and finally apoptosis of intestinal cells is caused. Meanwhile, after fructose enters the intestinal tract, the fructose is decomposed by fructokinase to cause endoplasmic reticulum stress and intestinal inflammation, so that the barrier of the intestinal tract is damaged, and the death rate of zebra fish is increased due to high concentration of fructose. Therefore, the invention adopts light to carry out sleep deprivation to increase the accumulation of the intestinal active oxygen, and simultaneously combines with low-concentration fructose solution to construct the zebra fish enteritis model. The method has the advantages of simple manufacturing process, low cost, wide popularization, strong modeling specificity, high model success rate and stable result, and can reduce the damage of the medicine to the zebra fish.

Description

Construction method of zebrafish enteritis model

technical field

The invention relates to the field of animal models, and specifically discloses a method for constructing a zebrafish enteritis model.

Background technique

Enteritis is mainly due to the failure of the intestinal barrier, leading to malnutrition, which reduces the growth performance and production efficiency of animals. When enteritis occurs, the intestinal mechanical barrier is destroyed, including the reduction of goblet cells and the damage of intestinal tight junction proteins, and a large number of harmful bacteria enter the intestinal tract and multiply in the intestinal tract, resulting in mucosal inflammation. As the number of harmful bacteria entering the intestinal tract continues to increase, the chemical barrier function will be destroyed, and microorganisms and endotoxins will break through the intestinal barrier and enter the blood circulation system.

The existing enteritis model construction methods mainly include drug induction and gene knockout. Among them, gene knockout is time-consuming, complicated and difficult to operate. However, most of the methods for drug construction using DSS drugs to induce enteritis have high DSS concentrations, high drug dosages, and unstable modeling effects. Zebrafish is different from other model animals (mice, rats, etc.), juvenile zebrafish are generally treated with drugs by whole-body immersion, and high-concentration DSS immersion to establish zebrafish enteritis will lead to severe systemic inflammation in zebrafish , easily lead to death and increase consumption.

In view of this, the present invention is proposed.

Contents of the invention

In order to solve the problems in the background technology, the object of the present invention is to provide a method for constructing a zebrafish enteritis model with short test period, low drug dosage, simple method and better modeling effect.

In order to achieve the above-mentioned effect, the technical scheme adopted in the present invention is:

The construction method of the zebrafish enteritis model is to induce zebrafish embryos by using light to perform sleep deprivation combined with low-concentration fructose solution.

Preferably, the zebrafish embryos selected are 2-2.5 dpf zebrafish embryos with normal development.

Preferably, the zebrafish embryos are selected from zebrafish embryos with normal development at 2 dpf.

Preferably, the specific method of using light to perform sleep deprivation combined with low-concentration fructose is: move zebrafish embryos into embryo culture water containing a low-concentration fructose solution, and implement uninterrupted light at an intensity of 200-300 lux at 28-28.5°C Perform sleep deprivation culture, change the medium every 22-24 h, and culture for 3-3.5 days.

Preferably, the zebrafish embryos are transferred into embryo culture water containing a low-concentration fructose solution, subjected to sleep deprivation culture at 28.5°C under uninterrupted light conditions, and the medium is changed every 24 h for 3 days.

Preferably, the mass fraction of the low-concentration fructose solution is 1.0%-3.0%.

Preferably, the mass fraction of the low-concentration fructose solution is 2.0%.

Preferably, the zebrafish is a transgenic marker strain.

Another technical scheme that the present invention adopts is:

The evaluation method of the zebrafish enteritis model obtained by any of the construction methods described above includes any one or more of the following (a)-(c):

(a) The aggregation of intestinal neutrophils in zebrafish was observed by fluorescence microscope, and the number of intestinal neutrophils was quantitatively analyzed by ImageJ software, and the relevant inflammatory factors in zebrafish embryos were detected by qPCR;

(b) The amount of ROS production in the zebrafish intestine was observed by fluorescence microscopy and combined with DCFH-DA staining, and the CAT and SOD enzyme activities and MDA content of zebrafish embryos were detected by corresponding enzyme activity kits;

(c) Hematoxylin and eosin (HE) staining of zebrafish intestinal tissue sections to observe the pathological conditions of zebrafish intestinal tissue.

It should be noted that, in the present invention, dpf is the abbreviation of daypost-fertilization, and refers to a few days after fertilization of zebrafish.

In order to observe the intestinal inflammation of zebrafish intuitively, the transgenic marked zebrafish line Tg (lyz: Dsred) was used to observe the accumulation of neutrophils in the intestine.

Compared with prior art, the beneficial effect of the present invention is:

1. In the present invention, sleep deprivation by light is used to increase the accumulation of reactive oxygen species in the intestine, and at the same time, a low-concentration fructose solution is combined with treatment to construct a zebrafish enteritis model. This method has short test period, good stability, simple operation, easy observation, can be widely promoted, has strong modeling specificity, high model success rate, stable results, and can reduce the damage of drugs to zebrafish;

2. Low drug dose, shorter molding time and better effect;

3. The model is suitable for drug screening for various prevention and treatment of enteritis diseases.

Description of drawings

Figure 1 is the effect of the experimental group, control group, and comparative examples 1-2 on the aggregation of neutrophils in the intestinal tract of zebrafish in Example 1; wherein A is the phenotype diagram of neutrophil aggregation under the combination of white light and fluorescence microscopy; B zebrafish Quantitative map of neutrophils in the gut;

Fig. 2 is the influence of experimental group, control group, comparative example 1-2 on zebrafish embryo inflammatory factor level in embodiment 1; Wherein A is IL-1β gene expression situation in zebrafish embryo; B TNF-α in zebrafish embryo Gene expression status;

Figure 3 is the effect of the experimental group, control group, and comparative example 1-2 zebrafish intestinal ROS production in Example 1; the figure is the photographed zebrafish intestinal tract, and the bright patches represent the accumulation intensity of active oxygen in the intestinal tract , the dotted distribution represents the enrichment of intestinal neutrophils;

Fig. 4 is the influence of experimental group, control group, comparative example 1-2 on the oxidative stress of zebrafish embryo in embodiment 1; Wherein upper left subgraph is SOD enzyme activity (U/mgprot) in zebrafish embryo; The picture shows the activity of CAT enzyme in zebrafish embryos (U/mgprot); the sub-picture on the lower left shows the content of MDA in zebrafish embryos (nmol/mgpr);

Fig. 5 is the HE staining diagram of the zebrafish intestinal tissue section in the experimental group, the control group, and comparative examples 1-2 in Example 1; wherein A is the cross section of the zebrafish intestinal tract; wherein B is the longitudinal section of the zebrafish intestinal tract .

Specific implementation method

In order to better understand the technical solution of the present invention, the technical solution of the present invention will be further described below in conjunction with the drawings and embodiments. The way to realize the present invention includes but not limited to the following examples, the following examples are used to illustrate the present invention, but not to limit the protection scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.

Example 1 is used to describe the construction method of the zebrafish enteritis model in detail

1 Experimental method

1.1. Put the normally cultured zebrafish into the mating tank at a ratio of 1:1 or 1:2 for males and females, and separate them with partitions. The next morning, the barrier is removed and the females begin laying eggs. Embryos were collected within 30 minutes after spawning, and dead and unfertilized eggs, feces and other debris were sucked out and rinsed with clean water 3 times. Then place in a 28.5°C incubator.

1.2. Select 2 dpf transgenic Tg (lyz: Dsred) zebrafish embryos with normal development under the microscope, set up 2 groups, 20-25 embryos in each group, carry out different treatments on the selected embryos and place them in a constant temperature incubator at 28.5°C Cultivate for 3 days, change the medium every 22-24 hours, and the specific settings of the two groups are as follows:

Control group: 8ml of embryo culture water, light intensity 25lux, light-dark cycle conditions: 14h dark: 10h light;

Experimental group (light sleep deprivation + fructose group): 8ml of fructose culture water with a volume mass fraction of 2.0%, cultured under 200-300lux light conditions, and continued light without interruption.

1.3. Use a fluorescence microscope (Leica M205FA, Germany) to observe the aggregation of neutrophils in the intestinal tract of transgenic Tg (lyz: Dsred) zebrafish after treatment. At the same time, ImageJ software was further used to quantitatively analyze the number of intestinal neutrophils, and qPCR was used to detect the transcription of related inflammatory factors in zebrafish embryos, such as interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α). level.

1.4. Use fluorescent microscope to shoot and combine with DCFH-DA staining to observe the amount of ROS production in the zebrafish intestine, and use the corresponding enzyme activity kit to detect the CAT and SOD enzyme activities and MDA content of zebrafish embryos.

1.5. Fix the zebrafish treated in 1.2 with 4% paraformaldehyde solution (PFA) for about 24 h, and then dehydrate in 70% ethanol aqueous solution for 5 times, 4-5 min/ and then dehydration in gradient ethanol aqueous solution with a volume fraction of 80%, 90%, and 100% respectively, 4-5 min/time, to complete the dehydration;

After the dehydration is completed, use a mixed solution of 0.5ml xylene and 0.5ml absolute ethanol to penetrate the dehydrated zebrafish tissue for 15-20 minutes, and then penetrate the zebrafish tissue in 1ml of pure xylene for 7-10 minutes to complete the penetration. Permeable treatment;

Put the zebrafish tissue that has been penetrated into a mixed solution of 0.5ml xylene and 0.5ml paraffin and soak it in an oven at about 65°C for 25-30 minutes, then change to 1ml pure paraffin solution and soak it in an oven at about 65°C Wax for 2-2.5 h, take out the tissue and put it in the wax dish for at least 24 h, slice it (6um thick) and dry it;

The dried sections were stained with hematoxylin and eosin (HE), sealed with neutral resin after staining and dried, then photographed and observed the changes of the intestinal tissues in the sections with a Zeiss microscope.

2. Experimental results

(a) Effects of combination treatment of light sleep deprivation and fructose solution on neutrophil enrichment in zebrafish gut

The results are shown in Figure 1. Significant neutrophil aggregation was observed in the zebrafish gut in the light sleep deprivation + fructose group, and it was compared with the fructose alone group (comparative example 1) and the light sleep deprivation group alone ( Compared with Comparative Example 2), the clustering of neutrophil data was more significant (Fig. 1A,B). In addition, as shown in Figure 2, further detection of the transcript levels of relevant inflammatory factors in zebrafish embryos by qPCR showed that the gene expression levels of TNF-α and IL-1β in the light sleep deprivation + fructose group were significantly higher than those in the fructose alone group (for Ratio 1) and the light sleep deprivation group alone (Comparative Ratio 2) (Fig. 2A,B).

(b) Effects of light sleep deprivation and fructose solution combined treatment on oxidative stress and reactive oxygen species levels in zebrafish gut

The results are shown in Figure 3. Further use of fluorescence microscopy and DCFH-DA staining combined to find that the ROS production in the zebrafish gut in the light sleep deprivation + fructose group was significantly higher than that in the fructose alone group (Comparative Example 1) and the light sleep deprivation group (Comparative Example 2) (Fig. 3). Moreover, as shown in Figure 4, the CAT and SOD enzyme activities and MDA content of zebrafish embryos were detected by corresponding enzyme activity kits, and the results showed that the CAT and SOD enzyme activities in zebrafish embryos in the light sleep deprivation + fructose group were significantly lower Compared with the fructose alone group (comparative example 1) and the light sleep deprivation group (comparative example 2), the MDA content was significantly higher than the single fructose group (comparative example 1) and the light sleep deprivation group (comparative example 2) (Figure 4). It was shown that light sleep deprivation + fructose combination treatment would lead to more obvious oxidative stress response and reactive oxygen species accumulation in the zebrafish gut.

(c) Effects of combination treatment of light sleep deprivation and fructose solution on the intestinal structure of zebrafish

The results are shown in Figure 5. In order to observe the histopathological conditions of the intestinal tract of zebrafish, cross-section and longitudinal section and subsequent HE staining were performed on the intestinal tract respectively. Comparative example 2) can make the intestinal villi in the intestine of zebrafish thinner and shorter. However, this phenomenon was more severe in the light sleep deprivation + fructose combination treatment group, and the intestinal tract even showed a kind of erosion (Fig. 5A, B).

Comparative example 1

A method for constructing a zebrafish enteritis model, in which the light sleep deprivation + fructose group in 1.2 of Example 1 is replaced by only adding 2.0% fructose solution for 3 days, that is, the fructose alone group. Specifically:

Transgenic Tg (lyz:Dsred) zebrafish embryos with normal development at 2 dpf were selected under a microscope, transferred into 8 ml of fructose solution with a mass fraction of 2.0% in embryo culture water and placed in a constant temperature incubator at 28.5°C for 3 days. The medium was changed every 22-24 h, and the fructose group alone (embryo culture water was prepared with 2.0% fructose solution, the light intensity was 25 lux, and the light-dark cycle condition was 14h dark: 10h light).

Comparative example 2

A method for constructing a zebrafish enteritis model, replacing the light sleep deprivation + fructose group in 1.2 of Example 1 with only 200-300 lux light intensity and continuous light for 3 days for sleep deprivation, that is, the light sleep deprivation group alone. Specifically:

Under the microscope, 2 dpf transgenic Tg (lyz: Dsred) zebrafish embryos with normal development were selected, transferred to 8ml of embryo culture water, and placed in a constant temperature incubator with a temperature of 28.5°C and 200-300lux light conditions for cultivation. Every 22-24 h, change the liquid, and continue to illuminate for 3 days without interruption.

Comparative example 3

A method for constructing a zebrafish enteritis model, replacing the light sleep deprivation + fructose group of 1.2 in Example 1 with only 3.0% fructose solution for 3.5 days. Specifically:

Select 2 dpf transgenic Tg (lyz:Dsred) zebrafish embryos with normal development under a microscope, transfer them into 8ml fructose solution with a mass fraction of 3.0% prepared with embryo culture water, and place them in constant temperature culture at 28.5°C and 25lux light conditions Cultivate in the box for 3.5 days, change the medium every 22-24 hours, and the light-dark cycle condition is 14h dark: 10h light.

Comparative example 4

A method for constructing a zebrafish enteritis model, in which the light sleep deprivation + fructose group in 1.2 of Example 1 is replaced with only 200-300 lux light intensity and continuous light for 3.5 days for sleep deprivation. Specifically:

Under the microscope, 2 dpf transgenic Tg (lyz: Dsred) zebrafish embryos with normal development were selected, transferred to 8ml of embryo culture water and placed in a constant temperature incubator with a temperature of 28.5°C and 200-300lux light conditions for cultivation. Every 22- The medium was changed every 24 hours, and the light continued for 3.5 days without interruption.

Example 2

A method for constructing a zebrafish enteritis model, in which the fructose concentration in the light sleep deprivation+fructose group of 1.2 in Example 1 is reduced to 1.0% fructose culture water for 3 days. Specifically:

Select 2 dpf transgenic Tg (lyz:Dsred) zebrafish embryos with normal development under a microscope, transfer them into 8 ml of fructose solution with a mass fraction of 1.0% prepared in embryo culture water, and place them in a 28.5°C, 200-300lux light conditions. The culture was carried out in a constant temperature incubator, the medium was changed every 22-24 h, and the light was continuously illuminated for 3 days. All the other operations are the same as in Example 1.

Example 3

A method for constructing a zebrafish enteritis model. The fructose concentration in the light sleep deprivation+fructose group of 1.2 in Example 1 is increased to 3.0% fructose culture water for 3 days. Specifically:

Select 2 dpf transgenic Tg (lyz:Dsred) zebrafish embryos with normal development under a microscope, transfer them into 8ml of fructose solution with a mass fraction of 3.0% prepared in embryo culture water, and place them in a 28.5°C, 200-300lux light conditions. The culture was carried out in a constant temperature incubator, the medium was changed every 22-24 h, and the light was continuously illuminated for 3 days. All the other operations are the same as in Example 1.

Table 1 The mutagenesis rate and zebrafish embryonic mortality of enteritis model in each group

。

.

It should be noted that at last: above each embodiment is only in order to illustrate technical scheme of the present invention, and is not intended to limit; Although the present invention has been described in detail with reference to foregoing each embodiment, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. range.

Claims (10)

1. The construction method of the zebrafish enteritis model is characterized in that, utilizing light to carry out sleep deprivation combined with the method of low-concentration fructose solution to process and induce zebrafish embryos. 2. The construction method according to claim 1, wherein the zebrafish embryos are selected from zebrafish embryos with normal development of 2-2.5 dpf. 3. The construction method according to claim 2, wherein the zebrafish embryos are selected from zebrafish embryos with normal development at 2 dpf. 4. The construction method according to claim 1, characterized in that, the specific method of utilizing light to carry out sleep deprivation in combination with low-concentration fructose is: moving zebrafish embryos into embryo culture water containing low-concentration fructose solution, at 28- Under 28.5°C, the intensity of 200-300lux uninterrupted light was implemented for sleep deprivation culture, and the medium was changed every 22-24 h, and the culture was 3-3.5d. 5. The construction method according to claim 4, wherein the zebrafish embryos are moved into embryo culture water containing a low-concentration fructose solution, and sleep deprivation is carried out at 28-28.5°C under uninterrupted light conditions, and every 24 Change the medium for 3 h and culture for 3 d. 6. The construction method according to claim 1 or 3, wherein the mass fraction of the low-concentration fructose solution is 1.0%-3.0%. 7. The construction method according to claim 6, wherein the mass fraction of the low-concentration fructose solution is 2.0%. 8. The construction method according to claim 1, wherein the zebrafish is a transgenic marker strain. 9. The construction method according to claim 8, wherein the transgenic marker line is Tg (lyz: Dsred). 10. The evaluation method of the zebrafish enteritis model obtained by the construction method according to any one of claims 1-9, characterized in that, comprising any one or more of the following (a)-(c): (a) The aggregation of intestinal neutrophils in zebrafish was observed by fluorescence microscope, and the number of intestinal neutrophils was quantitatively analyzed by ImageJ software, and the relevant inflammatory factors in zebrafish embryos were detected by qPCR; (b) The amount of ROS production in the zebrafish intestine was observed by fluorescence microscopy and combined with DCFH-DA staining, and the CAT and SOD enzyme activities and MDA content of zebrafish embryos were detected by corresponding enzyme activity kits; (c) Hematoxylin and eosin (HE) staining of zebrafish intestinal tissue sections to observe the pathological conditions of zebrafish intestinal tissue.

Images