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

Construction method of zebra fish enteritis model Download PDF

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CN116369249A
CN116369249A CN202310038701.3A CN202310038701A CN116369249A CN 116369249 A CN116369249 A CN 116369249A CN 202310038701 A CN202310038701 A CN 202310038701A CN 116369249 A CN116369249 A CN 116369249A
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zebra fish
intestinal
fructose
embryo
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祝辉
廖大龙
王凝
兰朝华
肖雄俊
李林蔓
李璐
向元琳
阮茂
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Sichuan University of Science and Engineering
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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 zebra fish enteritis model
Technical Field
The invention relates to the field of animal models, and particularly discloses a method for constructing a zebra fish enteritis model.
Background
Enteritis is mainly caused by failure of intestinal barrier, which results in malnutrition, and thus, the growth performance and production efficiency of animals are reduced. When enteritis occurs, the mechanical barrier of the gut is broken, including goblet cell depletion and impaired gut tight junctions, and a large number of harmful bacteria enter the gut and multiply in the gut, producing mucosal inflammation. As the number of harmful bacteria entering the intestine increases, the chemical barrier function will be destroyed and microorganisms and endotoxins will break through the intestinal barrier into the blood circulation system.
The existing enteritis model construction method mainly comprises drug induction and gene knockout. Wherein, the gene knockout is time-consuming and labor-consuming, the method is complex, and the operation is difficult. Most methods for inducing enteritis by adopting DSS medicaments in medicament construction have the defects of high DSS concentration, high medicament dosage and unstable modeling effect. Unlike other animals (mice, rats, etc.), young zebra fish is generally treated by systemic soaking, while high concentration DSS soaking to establish enteritis of zebra fish can cause severe systemic inflammation of zebra fish, and death and consumption increase.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the problems in the background technology, the invention aims to provide a method for constructing a zebra fish enteritis model, which has the advantages of short test period, low drug dosage, simple method and better modeling effect.
In order to achieve the above effects, the invention adopts the following technical scheme:
the method for constructing the zebra fish enteritis model utilizes light to treat and induce zebra fish embryos by combining sleep deprivation with a low-concentration fructose solution.
Preferably, the zebra fish embryo is selected from 2-2.5 dpf normally developed zebra fish embryos.
Preferably, the zebra fish embryo picks up a 2 dpf normothermic zebra fish embryo.
Preferably, the specific method for sleep deprivation combined with low concentration fructose by using light comprises the following steps: transferring zebra fish embryo into embryo culture water containing low concentration fructose solution, performing sleep deprivation culture at 28-28.5deg.C under 200-300lux uninterrupted light, and culturing at intervals of 22-24 h for 3-3.5 d.
Preferably, zebra fish embryos are transferred into embryo culture water containing a low-concentration fructose solution, and subjected to sleep deprivation culture at 28.5 ℃ under uninterrupted illumination, and the culture is carried out by changing the liquid every 24 h, and 3d.
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 zebra fish is a transgenic tagged line.
The invention adopts another technical scheme that:
the method for evaluating a zebra fish enteritis model obtained by any one of the above construction methods, comprising any one or more of the following (a) - (c):
(a) Observing the aggregation condition of intestinal neutral particles of the zebra fish by using a fluorescence microscope, quantitatively analyzing the quantity of the intestinal neutral particles by using imageJ software, and detecting relevant inflammatory factors in the zebra fish embryo by qPCR;
(b) Shooting by using a fluorescence microscope, observing the generation amount of ROS in the intestinal tract of the zebra fish by combining DCFH-DA staining, and detecting the enzyme activities of CAT and SOD and the MDA content of the zebra fish embryo by using a corresponding enzyme activity kit;
(c) The pathological condition of the intestinal tissue of the zebra fish is observed by Hematoxylin Eosin (HE) staining of the intestinal tissue section of the zebra fish.
In the present invention, dpf is an abbreviation for dapest-hybridization, and means that zebra fish are fertilized for several days.
In order to facilitate visual observation of intestinal inflammation conditions of zebra fish, a transgenic marked zebra fish strain Tg (lyz: dsred) is adopted to observe aggregation conditions of intestinal neutral particles.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, sleep deprivation is carried out by adopting illumination to increase accumulation of intestinal active oxygen, and meanwhile, a low-concentration fructose solution is combined for treatment to construct the zebra fish enteritis model. The method has the advantages of short test period, good stability, simple operation, convenient observation, wide popularization, strong modeling specificity, high model success rate and stable result, and can reduce the damage of the drug to the zebra fish;
2. the dosage of the medicine is low, the molding time is shorter, and the effect is better;
3. the model is suitable for screening various medicines for preventing and treating enteritis.
Drawings
FIG. 1 is the effect of experimental, control, and comparative examples 1-2 on the accumulation of neutral particles in the intestinal tract of zebra fish in example 1; wherein A is a neutral particle aggregation phenotype diagram under the combination of a white light and a fluorescence microscope; b, quantitative graph of neutral particles in intestinal tracts of zebra fish;
FIG. 2 is the effect of experimental, control, and comparative examples 1-2 on the levels of inflammatory factors in zebra fish embryos in example 1; wherein A is the expression condition of IL-1 beta gene in zebra fish embryo; b TNF-alpha gene expression in zebra fish embryo;
FIG. 3 is the effect of the amount of ROS produced in the intestinal tract of zebra fish of example 1, control, and comparative examples 1-2; the graph shows the photographed zebra fish intestinal tract, wherein the slices are bright and represent the accumulation intensity of active oxygen in the intestinal tract, and the dotted distribution represents the enrichment condition of neutral grains in the intestinal tract;
FIG. 4 is the effect of experimental, control, and comparative examples 1-2 on oxidative stress in zebra fish embryos in example 1; wherein the upper left panel shows SOD enzyme activity (U/mgprot) in zebra fish embryos; the upper right panel shows CAT enzyme activity (U/mgprot) in zebra fish embryos; the lower left panel shows MDA content (nmol/mgpr) in zebra fish embryos;
FIG. 5 is a graph of HE staining of intestinal tissue sections of zebra fish from example 1, control, and comparative examples 1-2; wherein A is the cross section of zebra fish intestinal tract; wherein B is the longitudinal section of the intestinal tract of the zebra fish.
Detailed description of the preferred embodiments
For a better understanding of the technical solution of the present invention, the technical solution of the present invention will be further described below with reference to the accompanying drawings and examples. The manner of carrying out the invention includes, but is not limited to, the following examples, which are intended to illustrate the invention, but are not intended to limit the scope thereof. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified.
Example 1 for details of the construction of the Zebra fish enteritis model
1 Experimental method
1.1, putting the normally cultured zebra fish into a mating jar according to the number ratio of male to female of 1:1 or 1:2, and separating by a partition plate. The next morning, the barrier is removed and females begin to spawn. Embryos are collected within 30min after spawning, and the dead and unfertilized eggs, feces and other impurities are sucked out and washed 3 times with clear water. Then placed in an incubator at 28.5 ℃.
1.2, picking 2 dpf normal-development transgenic Tg (lyz: dsred) zebra fish embryos under a microscope, setting 2 groups, carrying out different treatments on the selected embryos by 20-25 embryos of each group, placing the embryos in a constant temperature incubator at 28.5 ℃ for culturing 3d, changing liquid every 22-24 h, and specifically setting 2 groups as follows:
control group: 8ml of embryo culture water, light intensity 25lux, light dark cycle conditions at 14h darkness: illuminating for 10 hours;
experimental group (light sleep deprivation+fructose group): 8ml of fructose culture water with the volume mass fraction of 2.0% is cultured under the illumination condition of 200-300lux, and illumination is continuously carried out continuously.
1.3, the treated transgenic Tg (lyz: dsred) was observed by fluorescence microscopy (Lycra M205 FA) for neutral particle aggregation in the intestinal tract of zebra fish. Meanwhile, the quantitative analysis of the quantity of intestinal neutrophils is further carried out by using imageJ software, and related inflammatory factors in zebra fish embryos, such as the transcription level of interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), are detected by qPCR.
1.4, shooting by using a fluorescence microscope, observing the generation amount of ROS in the intestinal tract of the zebra fish by combining DCFH-DA staining, and detecting the CAT and SOD enzyme activities and MDA content of the zebra fish embryo by using a corresponding enzyme activity kit.
1.5, fixing the zebra fish treated in 1.2 with 4% paraformaldehyde solution (PFA) by mass fraction for about 24 h, and dehydrating in 70% ethanol water solution for 5 times and 4-5 min/time; then respectively dehydrating in gradient ethanol water solutions with volume fractions of 80%, 90% and 100% for 1 time, and 4-5 min/time to finish dehydration;
after dehydration is completed, penetrating the dehydrated zebra fish tissue for 15-20 min by using 0.5ml of mixed solution of xylene and 0.5ml of absolute ethanol, and then penetrating the zebra fish tissue for 7-10min in 1ml of pure xylene to complete penetration treatment;
putting the zebra fish tissue subjected to penetration treatment into a mixed solution of 0.5ml of xylene and 0.5ml of paraffin, carrying out wax dipping for 25-30 min in a drying oven at about 65 ℃, then changing into 1ml of pure paraffin solution, carrying out wax dipping for 2-2.5 h in the drying oven at about 65 ℃, taking out the tissue, sealing the wax in a wax tray for at least 24 h, slicing (6 um thick) and drying;
the dried sections were stained with Hematoxylin Eosin (HE), sealed with neutral resin after staining and dried, and photographed and observed for changes in intestinal tissue in the sections using a zeiss microscope.
2. Experimental results
(a) Effects of combination of light sleep deprivation and fructose solution treatment on enrichment of neutrophils in the intestinal tract of zebra fish
As a result, as shown in fig. 1, a significant aggregation of neutral particles was observed in the zebra fish intestinal tract in the light sleep deprivation+fructose group, and the neutral particle data aggregation was more significant compared to the fructose group alone (comparative example 1) and the light sleep deprivation group alone (comparative example 2) (fig. 1a, b). In addition, further detection of the transcript levels of related inflammatory factors in zebra fish embryos by qPCR as shown in fig. 2 showed that the gene expression levels of TNF- α and IL-1β were significantly higher in the light sleep deprivation + fructose group than in the fructose group alone (comparative example 1) and the light sleep deprivation group alone (comparative example 2) (fig. 2a, b).
(b) Effects of combination of light sleep deprivation and fructose solution treatment on intestinal oxidative stress and active oxygen levels in zebra fish
As a result, as shown in FIG. 3, it was further found by fluorescent microscopy and DCFH-DA staining in combination that the amount of ROS produced in the intestinal tract of zebra fish was significantly higher in the light sleep deprivation+fructose group than in the fructose group alone (comparative example 1) and the light sleep deprivation group (comparative example 2) (FIG. 3). Also, as shown in fig. 4, CAT and SOD enzyme activities and MDA contents of zebra fish embryos were detected by the corresponding enzyme activity kit, and the results showed that CAT and SOD enzyme activities in zebra fish embryos in the light sleep deprivation + fructose group were significantly lower than those in the fructose group alone (comparative example 1) and the light sleep deprivation group (comparative example 2), and MDA contents were significantly higher than those in the fructose group alone (comparative example 1) and the light sleep deprivation group (comparative example 2) (fig. 4). It is shown that the combination treatment of light sleep deprivation and fructose can lead to more obvious oxidative stress and active oxygen accumulation in the intestinal tract of zebra fish.
(c) Effects of combination treatment of light sleep deprivation and fructose solution on intestinal tissue structure of zebra fish
As shown in fig. 5, in order to observe pathological conditions of intestinal tissues of zebra fish, the intestinal tracts were transected and longitudinally cut respectively and then HE stained, and the results showed that the fructose group alone (comparative example 1) and the light sleep deprivation group (comparative example 2) both made intestinal villi in the zebra fish intestinal tracts sparse and short. However, this phenomenon is more severe in the combination of light sleep deprivation and fructose treatment, and even presents a erosive form in the intestine (FIGS. 5A, B).
Comparative example 1
A method for constructing a zebra fish enteritis model comprises replacing 1.2 light sleep deprivation+fructose group in example 1 with 3d treated by adding fructose solution with mass fraction of 2.0%, namely separate fructose group. The method comprises the following steps:
2 dpf normally developing transgenic Tg (lyz: dsred) zebra fish embryos are selected under a microscope, transferred into 8ml fructose solution with mass fraction of 2.0% prepared by embryo culture water and placed in a constant temperature incubator at 28.5 ℃ for culture for 3d, and the individual fructose groups (fructose solution with mass fraction of 2.0% prepared by embryo culture water, light intensity of 25lux, light dark circulation condition at 14h darkness: 10h light) are changed every 22-24 h.
Comparative example 2
A method for constructing a zebra fish enteritis model comprises replacing the light sleep deprivation+fructose group of 1.2 in the embodiment 1 with a light-continuous light 3d with 200-300lux light intensity for sleep deprivation, namely a single light sleep deprivation group. The method comprises the following steps:
2 dpf normal-development transgenic Tg (lyz: dsred) zebra fish embryos are selected under a microscope, transferred into 8ml embryo culture water and placed into a constant temperature incubator under the conditions of 200-300lux illumination at the temperature of 28.5 ℃ for culture, and the liquid is changed every 22-24 h, and illumination is continuously carried out for 3d.
Comparative example 3
A method for constructing a zebra fish enteritis model comprises replacing the light sleep deprivation+fructose group of 1.2 in the embodiment 1 with only 3.5d of fructose solution with the mass fraction of 3.0%. The method comprises the following steps:
2 dpf normal-development transgenic Tg (lyz: dsred) zebra fish embryos are selected under a microscope, transferred into 8ml fructose solution with mass fraction of 3.0% prepared by embryo culture water, and placed into a constant temperature incubator with temperature of 28.5 ℃ and 25lux illumination condition for culturing 3.5d, liquid is changed every 22-24 h, and the light and dark circulation condition is that the liquid is dark for 14 hours: and (5) illuminating for 10 hours.
Comparative example 4
A method for constructing a zebra fish enteritis model comprises replacing the light sleep deprivation+fructose group of 1.2 in the embodiment 1 with continuous light irradiation with 200-300lux light intensity for 3.5d for sleep deprivation. The method comprises the following steps:
2 dpf normal-development transgenic Tg (lyz: dsred) zebra fish embryos are selected under a microscope, transferred into 8ml embryo culture water and placed into a constant temperature incubator under the conditions of 200-300lux illumination at the temperature of 28.5 ℃ for culture, and the liquid is changed every 22-24 h, and illumination is continuously carried out for 3.5 days.
Example 2
A method for constructing a zebra fish enteritis model comprises the step of reducing the fructose concentration in a 1.2 light sleep deprivation and fructose group in the embodiment 1 to 1.0 mass percent of fructose culture water for 3d. The method comprises the following steps:
2 dpf normal-development transgenic Tg (lyz: dsred) zebra fish embryos are selected under a microscope, transferred into 8ml fructose solution with mass fraction of 1.0% prepared by embryo culture water, and placed into a constant temperature incubator with temperature of 28.5 ℃ and 200-300lux illumination condition for culture, and liquid is changed every 22-24 h, and continuous illumination is carried out for 3 days. The rest of the procedure was the same as in example 1.
Example 3
A method for constructing a zebra fish enteritis model comprises the step of improving the fructose concentration in a 1.2 light sleep deprivation and fructose group in the embodiment 1 to 3.0% of fructose culture water by mass fraction for 3d. The method comprises the following steps:
2 dpf normal-development transgenic Tg (lyz: dsred) zebra fish embryos are selected under a microscope, transferred into 8ml fructose solution with mass fraction of 3.0% prepared by embryo culture water, and placed into a constant temperature incubator with temperature of 28.5 ℃ and 200-300lux illumination condition for culture, and liquid is changed every 22-24 h, and continuous illumination is carried out for 3 days. The rest of the procedure was the same as in example 1.
TABLE 1 mutagenesis rates and zebra fish embryo mortality rates for enteritis models of groups
Figure SMS_1
。
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (10)

1. The method for constructing the zebra fish enteritis model is characterized in that the method for combining sleep deprivation with low-concentration fructose solution is used for treating and inducing zebra fish embryos by utilizing light.
2. The method of claim 1, wherein the zebra fish embryo is selected from the group consisting of 2-2.5 dpf normothermic zebra fish embryos.
3. The method of claim 2, wherein the zebra fish embryo is selected from 2 dpf normothermic zebra fish embryos.
4. The construction method according to claim 1, wherein the specific method for sleep deprivation combined with low concentration fructose by using light is as follows: transferring zebra fish embryo into embryo culture water containing low concentration fructose solution, performing sleep deprivation culture at 28-28.5deg.C under 200-300lux uninterrupted light, and culturing at intervals of 22-24 h for 3-3.5 d.
5. The method according to claim 4, wherein the zebra fish embryo is transferred into embryo culture water containing low concentration fructose solution, and subjected to sleep deprivation culture at 28-28.5 ℃ under uninterrupted illumination, and the culture medium is changed every 24 h, and the culture medium is cultured for 3d.
6. A method of construction as claimed in claim 1 or 3, wherein the low concentration fructose solution has a mass fraction of 1.0% to 3.0%.
7. The method according to claim 6, wherein the low concentration fructose solution has a mass fraction of 2.0%.
8. The method of claim 1, wherein the zebra fish is a transgenic tagged line.
9. The construction method according to claim 8, wherein the transgenic marker line is Tg (lyz: dsred).
10. The method for evaluating a zebra fish enteritis model obtained by the method of any one of claims 1-9, comprising any one or more of the following (a) - (c):
(a) Observing the aggregation condition of intestinal neutral particles of the zebra fish by using a fluorescence microscope, quantitatively analyzing the quantity of the intestinal neutral particles by using imageJ software, and detecting relevant inflammatory factors in the zebra fish embryo by qPCR;
(b) Shooting by using a fluorescence microscope, observing the generation amount of ROS in the intestinal tract of the zebra fish by combining DCFH-DA staining, and detecting the enzyme activities of CAT and SOD and the MDA content of the zebra fish embryo by using a corresponding enzyme activity kit;
(c) The pathological condition of the intestinal tissue of the zebra fish is observed by Hematoxylin Eosin (HE) staining of the intestinal tissue section of the zebra fish.
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Publication number Priority date Publication date Assignee Title
CN115786246A (en) * 2022-12-16 2023-03-14 四川轻化工大学 Method for constructing zebra fish immune unresponsive heart failure model

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