CN112271000A - Construction and detection method of zebra fish heart failure model - Google Patents
Construction and detection method of zebra fish heart failure model Download PDFInfo
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Abstract
The invention discloses a construction and detection method of a zebra fish heart failure model, wherein cmlc2 is characterized in that a GFP transgenic zebra fish couples a green fluorescent protein reporter gene to the downstream of a cmlc2 promoter, so that the zebra fish heart with green fluorescence can be observed under a fluorescence microscope; the nppb is characterized in that luciferase reporter genes are coupled at the downstream of an nppb promoter in lucerise transgenic zebra fish, the expression level of the nppb is identified through luceriase values, and the NPpb is treated with AA or cmlc2 is treated with GFP to cause the expression level of heart failure marker genes nppb to be increased, interfere the heart development of the zebra fish and reduce the survival rate of the zebra fish, so that the zebra fish heart failure model is constructed. The constructed model induces the heart failure of the zebra fish 72 hours after the zebra fish is fertilized, and simultaneously performs the expression analysis of cardiac development and heart failure marker genes on the animal level and the tissue level of the zebra fish, so that the construction and the analysis of the heart failure model can be quickly completed; high stability and good repeatability, and can be applied to high-throughput drug screening and analysis.
Description
Technical Field
The invention relates to the field of medicines, in particular to a construction and detection method of a zebra fish heart failure model.
Background
The zebra fish is an important vertebrate model organism, and the zebra fish embryo is transparent and has high development speed. The zebrafish have high similarity with human genes and have cardiovascular systems, nervous systems, metabolic systems and the like similar to those of mammals. Compared with the traditional model organism, the zebra fish has unique advantages in cardiovascular diseases, and the zebra fish is fertilized in vitro, develops in vitro and has high cardiac development speed. Zebrafish embryos started beating at 24 hours (24hpf) after fertilization, cardiac cyclization was completed 48 hours (48hpf) after fertilization, forming an S-shaped heart, and differentiation of the heart chambers was completed 72 hours (72hpf) after fertilization. The heart morphological and functional abnormality can be detected in zebra fish living embryo. The wild zebra fish embryo is exposed to small molecular compounds, has high correlation with human toxicity reaction, and plays an important role in drug toxicity research.
Aristolochic Acid (AA), also known as total aristolochic acid and phagocytic acid, is a class of nitrophenanthrene organic acids found in natural products, and has a wide range of pharmacological actions, such as enhancing cellular immunity, resisting tumors, resisting bacteria, resisting platelet aggregation, and the like. The Chinese herbal medicine containing aristolochic acid can be dozens of kinds, such as caulis Aristolochiae Manshuriensis, caulis Aristolochiae, radix Aristolochiae Fangchi, radix Aristolochiae, herba asari, etc. Studies report that aristolochic acid has definite renal toxicity and may be an important factor for inducing liver cancer. Thus, many countries have strict regulations for the use of aristolochic acid containing drugs. According to the application, aristolochic acid is used for treating nppb: luc and cmlc2: GFP zebra fish embryos, so that the expression level of a heart failure marker gene nppb is increased, the heart development of zebra fish is interfered, the survival rate of zebra fish is reduced, and the zebra fish heart failure model is established. The established model is efficient, reliable, simple and convenient, the establishment and analysis of the heart failure model can be completed 3 days after the zebra fish is fertilized, the method is suitable for high-throughput drug screening, and the method has important significance for searching related drugs for myocardial protection or cardiotoxicity.
Disclosure of Invention
The invention aims to provide a construction and detection method of a zebra fish heart failure model, which comprises the steps of treating two transgenic zebra fish embryos of nppb: luc or cmlc2: GFP by using AA to increase the expression level of a heart failure marker gene nppb, interfere the heart development of zebra fish and reduce the survival rate of the zebra fish, so as to construct the zebra fish heart failure model, the constructed model induces the zebra fish heart failure 72 hours after the zebra fish is fertilized, and simultaneously, the heart development and the expression analysis of the heart failure marker gene are carried out on the animal level and the tissue level of the zebra fish, so that the construction and the analysis of the heart failure model can be rapidly completed; high stability and good repeatability, and can be applied to high-throughput drug screening and analysis.
The purpose of the invention can be realized by the following technical scheme:
a zebra fish heart failure model is constructed, namely cmlc2, GFP transgenic zebra fish couples a green fluorescent protein reporter gene to the downstream of a cmlc2 promoter, so that the zebra fish heart with green fluorescence can be observed under a fluorescence microscope; the method comprises the following steps of coupling luciferase reporter genes at the downstream of an nppb promoter by a luciferase transgenic zebra fish, identifying the expression level of the nppb through the luciferase value, and treating zebra fish embryos with two transgenes, namely, luc or cmlc2 and GFP, by using AA to enable the expression level of heart failure marker genes nppb to be increased, interfere the heart development of the zebra fish and reduce the survival rate of the zebra fish, so that a zebra fish heart failure model is constructed, wherein the construction method comprises the following steps:
s1, determining the developmental stage of the zebra fish;
s2, drug treatment: setting the microporous plate into two experimental groups which are respectively a drug treatment group and a control group;
s3, adopting a zebra fish cardiomyopathy model to analyze heart failure;
s4, analyzing the cardiac development of the zebra fish;
and S5, analyzing the survival rate of the zebra fish.
Further, in the step S1, nppb: luc or cmlc2: GFP strain transgenic zebra fish parents are respectively mated and hatched, and zebra fish which develop 24hpf are selected and respectively transplanted into corresponding microplates and are marked for distinguishing.
Further, the drug treatment group is prepared by dissolving AA powder in DMSO solution to prepare 10mM AA storage concentration, adding 1 μ L10 mM AA into a zebra fish micropore plate containing 1mL fish water, and performing drug treatment on zebra fish, wherein the treatment concentration of AA is 10 μ M.
Further, the control group was zebrafish embryos treated with DMSO at the same concentration as the drug-treated group.
Further, the same volume of water for fish culture was added to both the drug-treated group and the control group, and the incubation was performed at the same temperature.
Further, the heart failure analysis of the step S3 is to add luciferase reagent into each well of the microplate after drug treatment of nppb: luc line zebra fish to 72hpf, incubate in the dark, measure the value of luciferase, and observe the expression level of the heart failure marker gene nppb.
Further, the step S4 of analyzing cardiac development is to treat drug-treated cmlc2 GFP strain zebrafish to 72hpf, anaesthetize the zebrafish, and observe the cardiac morphology of the zebrafish under a fluorescence microscope.
Further, the step S5 of analyzing the survival rate is to treat drug-treated cmlc2 GFP strain zebrafish to 96hpf, observe the heartbeat of zebrafish under a microscope, and count the survival rate of zebrafish.
A detection method for construction of a zebra fish heart failure model comprises the following steps:
t1, drug treatment cmlc2, separating zebrafish heart after GFP strain zebrafish to 72 hpf;
t2, extracting RNA, carrying out reverse transcription to obtain cDNA, carrying out fluorescent quantitative PCR, and detecting the mRNA expression levels of the heart failure marker genes nppa, nppb and IL-1 beta.
The invention has the beneficial effects that:
1. the constructed model induces the heart failure of the zebra fish 72 hours after the zebra fish is fertilized, and simultaneously performs the expression analysis of cardiac development and heart failure marker genes on the animal level and the tissue level of the zebra fish, so that the construction and the analysis of the heart failure model can be quickly completed;
2. the invention has high stability and good repeatability, and can be applied to high-throughput drug screening and analysis.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a schematic representation of the expression of the zebrafish nppb luc central senescence marker gene nppb of the present invention;
FIG. 2 is a schematic representation of AA influencing the cardiac development of cmlc2 GFP zebrafish of the present invention;
FIG. 3 is a statistical plot of the survival rate of zebrafish treated with AA according to the present invention;
FIG. 4 is a graph showing the expression level of CMlc2 GFP zebrafish heart failure gene after AA treatment according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.
A zebra fish heart failure model is constructed, namely cmlc2, GFP transgenic zebra fish couples a green fluorescent protein reporter gene to the downstream of a cmlc2 promoter, so that the zebra fish heart with green fluorescence can be observed under a fluorescence microscope; the method comprises the following steps of coupling luciferase reporter genes at the downstream of an nppb promoter by an nppb transgenic zebra fish, identifying the expression level of the nppb through luciferase values, and treating the embryos of the nppb transgenic zebra fish with luc or cmlc2 and GFP by AA (aristolochic acid) to enable the expression level of the nppb marker gene of the heart failure to be increased, interfere the cardiac development of the zebra fish and reduce the survival rate of the zebra fish, so that a zebra fish heart failure model is constructed, and the construction method of the model comprises the following steps:
s1, determination of the developmental stage of the zebra fish: mating and hatching the parents of the transgenic zebra fish of the GFP strain nppb luc or cmlc2, respectively, and respectively transferring the zebra fish which develop 24hpf into corresponding microwell plates and marking and distinguishing;
s2, drug treatment: the microplate was set into two experimental groups, drug treatment group and control group respectively:
s21, drug treatment group: dissolving AA powder by using a DMSO solution to prepare 10mM AA storage concentration, adding 1uL of 10mM AA into a zebra fish micropore plate containing 1mL of fish water, and carrying out drug treatment on zebra fish, wherein the treatment concentration of the AA is 10 mu M;
s22, control group: treating the zebra fish embryos with DMSO solution with the same concentration as the drug treatment group;
adding water for culturing fish into both groups, and culturing at the same temperature;
s3, adopting a zebra fish cardiomyopathy model to analyze heart failure: after the drug treatment of nppb, luc strain zebra fish to 72hpf, adding a luciferase reagent into each hole of the microplate, incubating in a dark place, measuring the value of the luciferase, and observing the expression level of the heart failure marker gene nppb;
s4, analyzing the cardiac development of the zebra fish: drug treatment cmlc2, namely after the GFP strain zebra fish is treated to 72hpf, anesthetizing the zebra fish, and observing the heart morphology of the zebra fish under a fluorescence microscope;
s5, analyzing the survival rate of the zebra fish: drug treatment of cmlc2 GFP strain zebra fish to 96hpf, observing the heartbeat of zebra fish under a microscope, and counting the survival rate of zebra fish.
Detection method for construction of zebra fish heart failure model
T1, RNA extraction: drug treatment cmlc2, namely separating the heart of zebra fish after GFP strain zebra fish reaches 72hpf, and extracting RNA;
t2, detecting the expression of the zebra fish heart failure related markers: the extracted RNA is reversely transcribed into cDNA, and fluorescence quantitative PCR is carried out to detect the expression level of mRNA of heart failure marker genes nppa, nppb and IL-1 beta, and the primer sequences are as follows:
example 1: zebra fish breeding
Culturing the transgenic zebra fish according to a standardized scheme, wherein the water temperature is 28.5 ℃, and the ratio of the light period to the dark period is 14 h: 10h, collecting embryos one hour after the adult zebra fish spawns, and culturing the embryos in E3 hatching solution (each 1L of deionized water contains 0.2925g of NaCl, 0.01267g of KCl and CaCl2 0.03663g，MgSO4 0.0396g)，The developmental stages of embryos and young fish are expressed in hours after fertilization (hpf).
When the zebrafish embryos developed to 24hpf, 0.003% of 1-phenyl-2-thiourea (1-phenyl-2-thiourea, PTU, Sigma) was added to the hatching fluid of E3 to inhibit the production of melanin, and the zebrafish embryos were cultured to 72hpf or 96 hpf.
Example 2: detection of luciferase value of nppb by AA treatment of heart failure model zebrafish
24hpf zebrafish embryos nppb: luc were transferred to 96-well plates, 100. mu.LE 3 hatching fluid per well, AA or control DMSO was added to a final concentration of 10. mu.M, and the plates were incubated in an incubator at 28 ℃.
When the zebrafish embryos developed to 72hpf, 25 μ L of luciferase reagent was added to each well, protected from light, incubated at room temperature for 60 minutes, and then luciferase assay was performed.
Because the nppb promoter carries the firefly luciferase reporter gene downstream, expression of nppb is shown by detecting luciferase values.
The results show that: the AA-treated group resulted in a significant increase in BNP expression in zebrafish embryos compared to the DMSO control group, and the results are shown in fig. 1.
Example 3: effect of AA on cardiac development and survival rate of zebra fish
24hpf zebrafish embryos tg (cmlc2: GFP) were transferred to 12-well plates, AA (10. mu.M concentration) or control DMSO was added, the plates were incubated at 28 ℃ in an incubator, and after 48 hours, 72hpf zebrafish larvae were anesthetized with tricaine methosulnate (Sigma), embedded in 2.5% methyl cellulose (Sigma), and photographed under a fluorescent microscope.
The results show that: the effects of AA on zebrafish cardiac development are mainly manifested by pericardial edema, cardiac cyclization defects, and abnormal placement of the ventricles and atria, as shown in fig. 2.
To further confirm the effect of AA on cardiac development of zebrafish, the survival rate of 96hpf zebrafish larvae was counted.
The results show that: the AA-treated group resulted in a significant reduction in the survival rate of zebrafish compared to the DMSO control group, and the results are shown in fig. 3.
Example 4: detecting the expression of heart central failure related gene after AA treatment of zebra fish
24hpf zebrafish embryos treated with AA (10. mu.M) or DMSO (cmlc2: GFP) were collected, 72hpf zebrafish embryos were collected at 48 hours, zebrafish hearts were isolated, and green hearts were collected under a fluorescent microscope.
RNA is extracted by trizol, reverse transcription is carried out to obtain cDNA, fluorescence quantitative PCR is carried out, and the mRNA expression level of heart failure marker genes nppa, nppb and IL-1 beta is detected.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, annealing extension at 60 ℃ for 30s, 40 cycles, denaturation at 95 ℃ for 10s, annealing extension at 60 ℃ for 10s, denaturation at 95 ℃ for 15s, melting curve, and performing reaction by using Bio-rad fluorescent quantitative PCR instrument.
The results show that: the AA-treated group resulted in significantly increased mRNA expression levels of heart failure-associated genes nppa, nppb, IL-1 β in zebrafish compared to DMSO control group, and the results are shown in fig. 4.
The significant differences of the BNP expression quantity, the survival rate and the heart failure related gene expression in the zebra fish embryos treated by AA in the experimental group and the control group can be found by combining the figure 1, the figure 3 and the figure 4.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (9)
1. Construction of a zebra fish heart failure model is characterized in that cmlc2 is that a GFP transgenic zebra fish couples a green fluorescent protein reporter gene to the downstream of a cmlc2 promoter, so that the zebra fish heart with green fluorescence can be observed under a fluorescence microscope; the method comprises the following steps of coupling luciferase reporter genes at the downstream of an nppb promoter by a luciferase transgenic zebra fish, identifying the expression level of the nppb through the luciferase value, and treating zebra fish embryos with two transgenes, namely, luc or cmlc2 and GFP, by using AA to enable the expression level of heart failure marker genes nppb to be increased, interfere the heart development of the zebra fish and reduce the survival rate of the zebra fish, so that a zebra fish heart failure model is constructed, wherein the construction method comprises the following steps:
s1, determining the developmental stage of the zebra fish;
s2, drug treatment: setting the microporous plate into two experimental groups which are respectively a drug treatment group and a control group;
s3, adopting a zebra fish cardiomyopathy model to analyze heart failure;
s4, analyzing the cardiac development of the zebra fish;
and S5, analyzing the survival rate of the zebra fish.
2. The construction of the zebrafish heart failure model of claim 1, wherein parents of nppb: luc or cmlc2: GFP strain transgenic zebrafish are mated and hatched in step S1, and zebrafish developing 24hpf are selected and transplanted into corresponding microplates and labeled for differentiation.
3. The construction of the heart failure model of zebra fish as claimed in claim 1, wherein the drug treatment group is prepared by dissolving AA powder in DMSO solution to prepare 10mM AA storage concentration, adding 1 μ L10 mM AA into a zebra fish micropore plate containing 1mL fish water, and performing drug treatment on zebra fish, wherein the treatment concentration of AA is 10 μ M.
4. The method for constructing the heart failure model of zebrafish according to claim 1, wherein the control group is prepared by treating zebrafish embryos with a DMSO solution at the same concentration as that of the drug-treated group.
5. The construction of the zebrafish heart failure model according to claim 1, wherein the same volume of water for fish culture is added to both the drug-treated group and the control group, and the culture is performed at the same temperature.
6. The construction of the zebrafish heart failure model according to claim 1, wherein the heart failure analysis of the step S3 is that after nppb: luc strain zebrafish is treated to 72hpf by drugs, luciferase reagent is added into each well of a microplate, incubation is carried out in the absence of light, the value of luciferase is measured, and the expression level of the heart failure marker gene nppb is observed.
7. The method for constructing the heart failure model of zebrafish according to claim 1, wherein the step S4 is characterized in that after the heart development is analyzed by treating drug cmlc2 GFP strain zebrafish to 72hpf, the zebrafish is anesthetized and then placed under a fluorescence microscope to observe the heart morphology of the zebrafish.
8. The method for constructing the heart failure model of zebrafish according to claim 1, wherein the survival rate analyzed in step S5 is calculated by observing the heart beat of zebrafish under a microscope after drug treatment of cmlc2 GFP strain zebrafish to 96 hpf.
9. The detection method for the construction of the zebra fish heart failure model according to any one of claims 1 to 8, characterized in that the detection method comprises the following steps:
t1, drug treatment cmlc2, separating zebrafish heart after GFP strain zebrafish to 72 hpf;
t2, extracting RNA, carrying out reverse transcription to obtain cDNA, carrying out fluorescent quantitative PCR, and detecting the mRNA expression levels of the heart failure marker genes nppa, nppb and IL-1 beta.
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