CN112841089A - Preparation method of zebra fish thrombus model - Google Patents
Preparation method of zebra fish thrombus model Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention relates to a preparation method of a zebra fish thrombus model, which comprises the following steps: 1) selecting 30 normal-developing 2dpf zebra fishes, transferring into 10-70 mu mol/L carrageenan solution prepared from embryo culture water, culturing in an incubator for 24h, removing culture water, and dyeing in 6 micro-porous plates with ortho-dianisidine dyeing solution in a dark place for 15 min; 2) removing the staining solution, and rapidly washing with DMSO for 3 times; 3) transferring zebra fish into a new 6-microporous plate, and adding DMSO (dimethyl sulfoxide); 4) placing the zebra fish on a glass slide, and taking a picture of the zebra fish under a microscope and storing the zebra fish; 5) with NIS-ElementsDTMImage processing software carries out quantitative analysis on the staining intensity of heart red blood cells and calculates the incidence rate of thrombus; the invention has the beneficial effects that: the preparation method of the zebra fish thrombus model has the advantages of simple and convenient construction method, cheap reagent and stable molding rate.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a preparation method of a zebra fish thrombus model.
Background
With the aging of population, the incidence of cardiovascular diseases is continuously increased, the number of deaths caused by cardiovascular diseases is far higher than that of other diseases such as tumor, and the death rate of the cardiovascular diseases is the first of the death rates of various diseases, wherein the thrombotic cardiovascular diseases account for a great proportion.
The zebra fish is a common tropical fish, a pair of zebra fish can lay eggs about 200 at a time, the eggs are fertilized in vitro, the in vitro development is realized, the embryo development is fast, the embryo body is transparent, the blood vessel development condition can be observed visually under a microscope, the homology between the blood vessel of the zebra fish and the nervous system and human and mammals on a molecular signal path reaches more than 85 percent, and the zebra fish is an excellent model for researching angiogenesis, blood vessel inhibition, thrombus diseases and the like.
At present, arachidonic acid, phenylhydrazine, adrenaline hydrochloride and laser are used for inducing and generating a zebra fish thrombus model, and the methods have the defects of low molding rate, strong reagent toxicity, expensive reagent or expensive instruments, so a new method for constructing the zebra fish thrombus model is needed to be found.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a novel method for constructing a zebra fish thrombus model, which has the advantages of simple and convenient construction method, cheap reagent and stable molding rate. Carrageenan is a sulfated polysaccharide, which can cause thrombosis through local vascular inflammation and endothelial cell damage, and has the mechanism that inflammation damages vascular endothelial cells, leads inflammatory cells to chemotaxis and infiltration under the action of IL-1 beta, activates an extrinsic coagulation system to cause thrombosis, and inhibits a fibrinolysis system after thrombosis. The invention utilizes carrageenan to induce the thrombosis of zebra fish.
The preparation process of the invention is as follows:
1. selecting about 30 normal-developing 2dpf zebra fishes, transferring into 10-70 μmol/L carrageenan solution prepared from embryo culture water, culturing in an incubator for 24h, removing culture water, and dyeing in 6 micro-porous plates with o-dianisidine staining solution for 15min in dark place;
2. removing the staining solution, and rapidly washing with DMSO for 3 times;
3. transferring zebra fish into a new 6-microporous plate, and adding DMSO (dimethyl sulfoxide);
4. placing the zebra fish on a glass slide, and taking a picture of the zebra fish under a microscope and storing the zebra fish;
5. using NIS-Elements DTMImage processing software carries out quantitative analysis on the staining intensity of heart red blood cells and calculates the incidence rate of thrombus;
the formula of the water for culturing the zebra fish comprises the following components: 5mmol/L NaCl, 0.17mmol/L KCl, 0.4mmol/L CaCl2，0.16mmol/LMgSO4；
The formula of the o-dianisidine staining solution comprises the following components: contains 0.6mg/mL o-dianisidine, 0.01mol/L sodium acetate, and 0.65% H2O2，0.16mmol/LMgSO4。
Preferably, the method for preparing the animal model of the invention comprises the following steps:
selecting healthy and mature zebra fish, putting the zebra fish into a fish tank according to the proportion of male and female 2:1, obtaining embryos in the morning of the next day, selecting healthy and high-quality embryos, putting the zebra fish into an incubator at 28 ℃, changing water twice every day, selecting 30 normally-developed zebra fish when the zebra fish develops to 2dpf, adding a carrageenan solution with the concentration of 40 mu mol/l prepared by using embryo culture water, putting the zebra fish into the incubator for continuous culture for 24h, sucking the culture water, sucking the isovolumetric o-dianisidine staining solution by using a liquid transfer gun, and staining the zebra fish in a 6-lucifugal microplate for 15 min; removing the staining solution, and rapidly washing with DMSO for 3 times; placing the zebra fish on a glass slide, and taking a picture of the zebra fish under a microscope and storing the zebra fish; using NIS-Elements DTMThe image processing software carries out quantitative analysis on the staining intensity of the heart red blood cells and calculates the incidence rate of thrombus.
Designing 3 experimental groups in addition, wherein a blank control group is embryo culture water, a model control group is added with a carrageenan solution with the concentration of 40 mu mol/L prepared by the embryo culture water, a positive drug group is added with clopidogrel with the concentration of 5mg/L prepared by the embryo culture water when zebrafish grows to 42hpf for pretreatment for 6h, the carrageenan solution is added until the final concentration is 40 mu mol/L, the culture is continued for 24h, the culture water is discarded, and an equal volume of o-dianisidine staining solution is absorbed by a pipette gun and is dyed in 6 microwell plates for 15min in a dark place; removing the staining solution, and rapidly washing with DMSO for 3 times; placing the zebra fish on a glass slide, and taking a picture of the zebra fish under a microscope and storing the zebra fish; using NIS-Elements DTMThe image processing software carries out quantitative analysis on the staining intensity of the heart red blood cells and calculates the thrombus inhibition rate.
The calculation method of the thrombus inhibition rate comprises the following steps:
another object of the present invention is to provide a method for using the zebrafish thrombosis animal model:
1. preparing a zebra fish thrombus animal model according to the above contents;
2. adding the drug to be tested, and continuing culturing for a corresponding time;
3. calculating the thrombus inhibition rate and evaluating the thrombolytic effect of the medicament.
The terms appearing in the specification are explained and illustrated:
dpf Days post fertilization development of Days past fertilization Zebra fish embryos
Hpf number of hours of development after fertilization of Hourpast fertilization Zebra embryo
DMSO Dimethyl Sulphoxide
The invention has the beneficial effects that:
the preparation method of the zebra fish thrombus model has the advantages of simple and convenient construction method, cheap reagent and stable molding rate.
Drawings
FIG. 1: tail thrombus, A is 10 mu mol/L carrageenan; b, 40 mu mol/L carrageenan; c: 70 mu mol/L carrageenan; d, 5mg/L clopidogrel and 40 mu mol/L carrageenan
FIG. 2: rate of thrombosis
FIG. 3: staining pattern of heart red blood cells, A: 10. mu. mol/L carrageenan; b, 40 mu mol/L carrageenan; c: 70 mu mol/L carrageenan; d, 5mg/L clopidogrel and 40 mu mol/L carrageenan
FIG. 4: thrombus inhibition rate
Detailed Description
The present invention is further illustrated in detail by the following examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1: preparation of animal models of the invention
Experimental materials: carrageenan type I, purchased from SIGMA corporation, was made up in 10-70. mu. mol/L with embryo culture water; DMSO was purchased from Beijing Solaibao technologies, Inc.; o-dianisidine was purchased from SIGMA; NaCl, KCl, CaCl2、MgSO4Sodium acetate, H2O2Purchased from Guangzhou chemical, 6-well plates from Shanghai Nest Biotech; clopidogrel was purchased from Shanghai crystal pure practice Co., Ltd and was prepared into 7.5g/L of a mother liquor with 100% DMSO.
An experimental instrument: the Zeiss type microscope Stereo Discovery V8 was purchased from Zhongji science and technology Co., Ltd, Beijing ruike; the constant temperature incubator BJPX-250-II was purchased from Jinnanlaibao medical instruments Co.
Zebra fish breeding environment: zebra fish for experiment is AB system, and is bred in Guangzhou institute of biological medicine and health by the Westefield culture method, the water temperature is 28 ℃, the conductivity is 450-.
The experimental method comprises the following steps: selecting healthy mature AB-series zebra fish, putting the healthy mature AB-series zebra fish into a fish tank according to the proportion of male and female 2:1, obtaining embryos in the morning of the next day, selecting healthy high-quality embryos, putting the healthy high-quality embryos into a 28 ℃ incubator, changing water twice a day, selecting 30 normally-developed zebra fish from a blank control group, a model control group and a positive drug group when the zebra fish develops to 2dpf, wherein the blank control group is embryo culture water, the model control group is added with 10 mu mol/L, 20 mu mol/L, 30 mu mol/L, 40 mu mol/L, 50 mu mol/L, 60 mu mol/L and 70 mu mol/L carrageenan solution prepared by the embryo culture water, culturing for 24h, the positive drug group is added with 5mg/L of clopidogrel prepared by the embryo culture water when the zebra fish develops to 42hpf, pre-treating for 6h, and is added with 40 mu mol/L of carrageenan solution, continuing to culture for 24h, sucking and removing culture water, and sucking the isovolumetric o-dianisidine staining solution into a 6-micropore plate by using a pipette gun to dye for 15min in a dark place; removing the staining solution, and rapidly washing with DMSO for 3 times; taking the zebra fish, placing the zebra fish on a glass slide, and taking a picture of the zebra fish under a microscope and storing the zebra fish; by NIS-element DTMThe image processing software carries out quantitative analysis on the staining intensity of the heart red blood cells and calculates the thrombus inhibition rate.
As can be seen from FIGS. 1 and 2, carrageenan can form thrombus in zebra fish, the dosage of 40 mu mol/L is proper, the clopidogrel pretreatment can prevent the thrombus formation, and the thrombus inhibition rate is 63.04% as can be seen from FIGS. 3 and 4.
Example 2 comparison of this model with the phenylhydrazine induction model
Experimental materials: carrageenan type I, purchased from SIGMA corporation, was made up with embryo culture water to 40. mu. mol/L; DMSO was purchased from Beijing Solaibao technologies, Inc.; o-dianisidine was purchased from SIGMA; NaCl, KCl, CaCl2、MgSO4Sodium acetate, H2O2Purchased from Guangzhou chemical, 6-well plates from Shanghai Nest Biotech; phenylhydrazine was purchased from Shanghai boat-Fuv Biotech Ltd and prepared into 1.5mmol/L mother liquor with 100% ethanol.
An experimental instrument: the Zeiss type microscope Stereo Discovery V8 was purchased from Zhongji science and technology Co., Ltd, Beijing ruike; the constant temperature incubator BJPX-250-II was purchased from Jinnanlaibao medical instruments Co.
Zebra fish breeding environment: zebra fish for experiment is AB system, and is bred in Guangzhou institute of biological medicine and health by the Westefield culture method, the water temperature is 28 ℃, the conductivity is 450-.
The experimental method comprises the following steps: selecting healthy mature AB-series zebra fishes, putting the healthy mature AB-series zebra fishes into a fish tank according to the proportion of male and female parts of 2:1, obtaining embryos in the morning of the next day, selecting healthy high-quality embryos, putting the healthy high-quality embryos into an incubator at 28 ℃, changing water twice a day, selecting 30 normally-developed zebra fishes from a blank control group, a carrageenan model group and a phenylhydrazine model group when the zebra fishes develop to 2dpf, wherein the blank control group is embryo culture water, the carrageenan model group is 40 mu mol/L, and the phenylhydrazine model group is 1.5 mu mol/L, continuously culturing for 24h, sucking the culture water, sucking the ortho-isovolume anthranilamide staining solution into a 6 micropore plate by using a liquid transfer gun, and staining for 15min in a dark place; removing the staining solution, and rapidly washing with DMSO for 3 times; placing Zebra fish on glass slide, and placing on microscopeTaking a picture of the zebra fish and storing the zebra fish; using NIS-Elements DTMThe image processing software carries out quantitative analysis on the staining intensity of the heart red blood cells and calculates the thrombosis rate.
TABLE 1 Carrageenan groups vs. phenylhydrazine groups
Claims (3)
1. A preparation method of a zebra fish thrombus model is characterized by comprising the following steps:
1) selecting 30 normally developed 2dpf zebra fishes, transferring into 10-70 mu mol/L carrageenan solution prepared by embryo culture water, placing in an incubator for culturing for 24h, removing the culture water, and dyeing in 6 micro-porous plates for 15min in a dark place by using an o-dianisidine dyeing solution;
2) removing the staining solution, and rapidly washing with DMSO for 3 times;
3) transferring zebra fish into a new 6-microporous plate, and adding DMSO (dimethyl sulfoxide);
4) placing the zebra fish on a glass slide, and taking a picture of the zebra fish under a microscope and storing the zebra fish;
5) using NIS-Elements DTMImage processing software carries out quantitative analysis on the staining intensity of heart red blood cells and calculates the incidence rate of thrombus;
the formula of the water for culturing the zebra fish comprises the following components: 5mmol/L NaCl, 0.17mmol/L KCl, 0.4mmol/L CaCl2，0.16mmol/L MgSO4；
The formula of the o-dianisidine staining solution comprises the following components: contains 0.6mg/mL o-dianisidine, 0.01mol/L sodium acetate, and 0.65% H2O2，0.16mmol/L MgSO4。
2. A preparation method of a zebra fish thrombus model is characterized by comprising the following steps:
1) selecting 30 normal-developing 2dpf zebra fishes, transferring into a carrageenan solution of 40 mu mol/L prepared by embryo culture water, culturing in an incubator for 24h, removing the culture water, and dyeing in a 6-micropore plate for 15min in a dark place by using an o-dianisidine dyeing solution;
2) removing the staining solution, and rapidly washing with DMSO for 3 times;
3) transferring zebra fish into a new 6-microporous plate, and adding DMSO (dimethyl sulfoxide);
4) placing the zebra fish on a glass slide, and taking a picture of the zebra fish under a microscope and storing the zebra fish;
5) using NIS-Elements DTMImage processing software carries out quantitative analysis on the staining intensity of heart red blood cells and calculates the incidence rate of thrombus;
the formula of the water for culturing the zebra fish comprises the following components: 5mmol/L NaCl, 0.17mmol/L KCl, 0.4mmol/L CaCl2，0.16mmol/L MgSO4；
The formula of the o-dianisidine staining solution comprises the following components: contains 0.6mg/mL o-dianisidine, 0.01mol/L sodium acetate, and 0.65% H2O2，0.16mmol/L MgSO4。
3. The method of using the zebrafish thrombus model of claim 2, comprising the steps of:
1) preparing a zebrafish thrombus animal model according to the method of claim 2;
2) adding the drug to be tested, and continuing culturing for a corresponding time;
3) calculating the thrombus inhibition rate and evaluating the thrombolytic effect of the medicament.
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