CN112226405A

CN112226405A - Method for constructing zebra fish iron death model

Info

Publication number: CN112226405A
Application number: CN202011098946.8A
Authority: CN
Inventors: 王明勇; 徐又佳; 曹子厚
Original assignee: Suzhou Murui Biotechnology Co ltd
Current assignee: Suzhou Murui Biotechnology Co ltd
Priority date: 2020-10-14
Filing date: 2020-10-14
Publication date: 2021-01-15

Abstract

The scheme relates to a method for constructing a zebra fish iron death model, which comprises the steps of breeding zebra fish, treating the zebra fish by utilizing Erastin solution or ferric ammonium citrate solution to construct the iron death model, verifying the iron death model by using trypan blue, and successfully constructing the zebra fish iron death model after cell death occurs. The invention firstly uses the zebra fish to define and research the phenomenon of iron death, and the invention discovers that the iron death inducer can obviously increase the generation of ROS in the zebra fish body, induces the number of cell death to be obviously increased, and changes the expression of genes related to iron death. The method has the advantages of convenience, rapidness, high success rate, easiness in repetition and the like, can realize the qualitative and quantitative determination of the iron death at the individual level, enables the zebra fish to become a convenient model to research the iron death phenomenon, and models of cancers, blood vessels, osteoporosis, metabolism and other human diseases related to the iron death, and can also provide a model for high-throughput screening of medicines related to the iron death.

Description

Method for constructing zebra fish iron death model

Technical Field

The invention belongs to the field of biotechnology model construction, and particularly relates to a construction method of a zebra fish iron death model.

Background

Cell death is one of the major links in mammalian development, maintenance of homeostasis, and disease development. Iron death (Ferroptosis) is a mode of cell death and has been studied more at the cellular level, and at the mouse level. Iron death is iron-dependent apoptosis under the control of Glutathione Peroxidase 4(Glutathione Peroxidase 4, GPX 4). The nomenclature committee for cell death was formally named in 2014, and iron death had the following 4-point characteristics: cell death and increased iron level have a causal relationship, and iron needs to participate in the iron death process; lipid peroxide increase and NADPH decrease; mitochondria have special morphological changes; can be reversed by lipophilic antioxidants and iron chelators. Iron death is associated with a variety of pathological cell deaths, for example, some degenerative pathological changes are cell death due to a reduced ability to repair lipid peroxides; iron death may have a tumor-inhibiting function that can remove cells that are unavailable for key trophic factors or are infected and damaged by environmental changes.

Zebra fish is a tropical carp family fish, and is widely applied to new medicine screening at present. The zebra fish has clear genetic background, the similarity with human genes is up to 87%, and the organ pathological process is very close to that of human. Meanwhile, the zebra fish embryo development is completely transparent, the pathological process can be clearly observed by adding proper pigment inhibition treatment, and the zebra fish is small in size, short in experiment period, low in maintenance cost and very suitable for high-throughput experiments. However, zebrafish, as the third model organism in biomedical research, have no applicable techniques or methods for iron death.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the zebra fish iron death model construction method which is low in cost, quick in effect, stable in model performance and wide in applicability.

In order to achieve the purpose, the invention provides the following technical scheme:

a construction method of a zebra fish iron death model comprises the following steps:

1) breeding zebra fish: placing sexually mature zebra fish into a mating fish tank according to the ratio of male to female being 1:2, inserting a partition plate, and separating the male and female zebra fish; turning on the lamp after night for changing water, taking out the partition plate, and collecting the fish eggs after 30-60 minutes; placing the collected roes in a culture dish containing E3, selecting fertilized roes, dividing the fertilized roes into disks according to the density of 100 roes per disk, adding an embryo culture solution, and incubating at a constant temperature of 28 ℃ for 5 hours;

2) construction of iron death model: selecting the zebra fish which are normally incubated in the step 1), distributing the zebra fish in a 6-hole plate according to the density of 10-20 strips/hole, adding an E3 culture solution, and incubating at the constant temperature of 28 ℃ for 24 h; removing the E3 culture solution, randomly dividing the fertilized eggs in the 6-well plate into a DMSO negative control group, an Erastin treatment group and an ammonium ferric citrate (FAC) treatment group, respectively adding DMSO, Erastin solution and FAC solution into the groups, and carrying out constant-temperature culture for 48 h;

3) verification of iron death model: and (3) carrying out vital staining on the zebra fish of the DMSO negative control group, the Erastin treatment group and the FAC treatment group in the step 2) by using trypan blue, and detecting a cell death phenomenon to obtain the zebra fish iron death model.

Further, the concentration of the Erastin solution is 20 mu mol/L, and the concentration of the FAC solution is 200 mu g/ml.

Further, in the step 2), the conditions for the constant temperature culture are 28 ℃ and the light-shielding treatment.

The invention has the beneficial effects that: zebrafish have not been reported in the field of iron death as a biomedical model, nor has there been a method for defining iron death in zebrafish. The invention uses zebrafish for the first time to define and study the phenomenon of iron death, treats zebrafish juvenile fish with an iron death inducer, and then defines dead cells by trypan blue staining. The invention discovers that the iron death inducer can obviously increase the ROS generation in the zebra fish body, induce the cell death number to be obviously increased and change the expression of iron death related genes. The method has the advantages of convenience, rapidness, high success rate, easiness in repetition and the like, can realize the qualitative and quantitative determination of the iron death at the individual level, enables the zebra fish to become a convenient model to research the iron death phenomenon, and models of cancers, blood vessels, osteoporosis, metabolism and other human diseases related to the iron death, and can also provide a model for high-throughput screening of medicines related to the iron death.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

Fig. 1 is a schematic representation of ROS generation after vital staining of zebrafish in the DMSO negative control group, Erastin-treated group, and FAC-treated group in this case.

Fig. 2 is a schematic diagram of the iron death results of zebrafish vital staining in the DMSO negative control group, Erastin treatment group, and FAC treatment group in the present application.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1: zebra fish reproduction

Placing sexually mature zebra fish into a mating fish tank according to the ratio of male to female being 1:2, inserting a partition plate, and separating the male and female zebra fish; turning on the lamp after night for changing water, taking out the partition plate, and collecting the fish eggs after 30-60 minutes; the collected roe was placed in a petri dish containing E3, unfertilized roe was removed, fertilized roe was divided into dishes at a density of 100 eggs/dish, embryo culture solution was added, and incubation was performed at a constant temperature of 28 ℃ for 5 hours.

Example 2: construction of iron death model

Selecting the zebra fish which are normally incubated in the embodiment 1, distributing the zebra fish in a 6-well plate according to the density of 10-20 strips/well, adding an E3 culture solution, and incubating at the constant temperature of 28 ℃ for 24 h; removing the E3 culture solution, randomly dividing the fertilized eggs in the 6-well plate into a DMSO negative control group, an Erastin treatment group and a FAC treatment group, respectively adding DMSO, Erastin solution and FAC solution into the fertilized eggs, culturing the fertilized eggs at 28 ℃ in the dark for 48h, and replacing the corresponding solution every 24 h; the concentration of Erastin solution was 20. mu. mol/L and the concentration of FAC solution was 200. mu.g/ml.

Example 3: validation of iron death model

And (3) carrying out vital staining on the zebra fish of the DMSO negative control group, the Erastin treatment group and the FAC treatment group in the example 2 by using trypan blue, and obtaining the zebra fish iron death model if a cell death phenomenon is detected.

From fig. 1, it can be seen that the FAC group and Erastin-treated group are brighter than the DMSO negative control group, and the brighter is the more ROS, which indicates that the ROS in the zebrafish is significantly increased, and the number of induced cell death is increased, and from fig. 2, it can also be seen that the black spots on the surface of the zebrafish (the black spots indicate the number of iron death) are significantly increased, which indicates that the invention successfully constructs the zebrafish iron death model.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

1. A construction method of a zebra fish iron death model is characterized by comprising the following steps:

1) breeding zebra fish: placing sexually mature zebra fish into a mating fish tank according to the ratio of male to female being 1:2, inserting a partition plate, and separating the male and female zebra fish; turning on the lamp after night for changing water, taking out the partition plate, and collecting the fish eggs after 30-60 minutes; placing the collected roes in a culture dish containing E3, selecting fertilized roes, performing disc division according to the density of 100 roes per disc, adding an embryo culture solution, and incubating at a constant temperature of 28 ℃ for 5 hours;

2) construction of iron death model: selecting the zebra fish which are normally incubated in the step 1), distributing the zebra fish in a 6-hole plate according to the density of 10-20 strips/hole, adding an E3 culture solution, and incubating at the constant temperature of 28 ℃ for 24 h; removing the E3 culture solution, randomly dividing the fertilized eggs in the 6-pore plate into a DMSO negative control group, an Erastin treatment group and a ferric ammonium citrate treatment group, respectively adding a DMSO solution, an Erastin solution and a ferric ammonium citrate solution, and culturing at constant temperature for 48 h;

3) verification of iron death model: and (3) carrying out vital staining on the zebra fish in the DMSO negative control group, the Erastin treatment group and the ferric ammonium citrate treatment group by using trypan blue, and detecting a cell death phenomenon to obtain the zebra fish iron death model.

2. The method for constructing the iron death model of zebrafish according to claim 1, wherein the concentration of the Erastin solution is 20 μmol/L, and the concentration of the ammonium ferric citrate solution is 200 μ g/ml.

3. The method for constructing the zebrafish iron death model according to claim 1, wherein in the step 2), the conditions for isothermal culture are 28 ℃ and light-shielding treatment.