CN115786246B - Construction method of zebra fish immune non-responsive heart failure model - Google Patents
Construction method of zebra fish immune non-responsive heart failure model Download PDFInfo
- Publication number
- CN115786246B CN115786246B CN202211627686.8A CN202211627686A CN115786246B CN 115786246 B CN115786246 B CN 115786246B CN 202211627686 A CN202211627686 A CN 202211627686A CN 115786246 B CN115786246 B CN 115786246B
- Authority
- CN
- China
- Prior art keywords
- zebra fish
- gfp
- myl
- heart
- heart failure
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000252212 Danio rerio Species 0.000 title claims abstract description 75
- 206010019280 Heart failures Diseases 0.000 title claims abstract description 31
- 238000010276 construction Methods 0.000 title claims description 4
- QFOHBWFCKVYLES-UHFFFAOYSA-N Butylparaben Chemical compound CCCCOC(=O)C1=CC=C(O)C=C1 QFOHBWFCKVYLES-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 24
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 210000002257 embryonic structure Anatomy 0.000 claims abstract description 8
- 238000011161 development Methods 0.000 claims abstract description 4
- 210000002216 heart Anatomy 0.000 claims description 33
- 210000002540 macrophage Anatomy 0.000 claims description 21
- 230000017531 blood circulation Effects 0.000 claims description 16
- 230000002107 myocardial effect Effects 0.000 claims description 10
- 230000007935 neutral effect Effects 0.000 claims description 9
- 230000009261 transgenic effect Effects 0.000 claims description 9
- 238000013518 transcription Methods 0.000 claims description 7
- 230000035897 transcription Effects 0.000 claims description 7
- 238000011529 RT qPCR Methods 0.000 claims description 6
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- 230000002757 inflammatory effect Effects 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- JRBJSXQPQWSCCF-UHFFFAOYSA-N 3,3'-Dimethoxybenzidine Chemical compound C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 JRBJSXQPQWSCCF-UHFFFAOYSA-N 0.000 claims description 4
- 108020001621 Natriuretic Peptide Proteins 0.000 claims description 4
- 102000004571 Natriuretic peptide Human genes 0.000 claims description 4
- 230000009067 heart development Effects 0.000 claims description 4
- 239000000692 natriuretic peptide Substances 0.000 claims description 4
- 238000010186 staining Methods 0.000 claims description 4
- 101800000407 Brain natriuretic peptide 32 Proteins 0.000 claims description 3
- 102400000667 Brain natriuretic peptide 32 Human genes 0.000 claims description 3
- 101800002247 Brain natriuretic peptide 45 Proteins 0.000 claims description 3
- 238000010226 confocal imaging Methods 0.000 claims description 3
- 210000003743 erythrocyte Anatomy 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- HPNRHPKXQZSDFX-OAQDCNSJSA-N nesiritide Chemical compound C([C@H]1C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CO)C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 HPNRHPKXQZSDFX-OAQDCNSJSA-N 0.000 claims description 3
- 238000000799 fluorescence microscopy Methods 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 4
- 230000000747 cardiac effect Effects 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 210000000440 neutrophil Anatomy 0.000 description 7
- 241000700159 Rattus Species 0.000 description 6
- 230000007423 decrease Effects 0.000 description 6
- 238000004904 shortening Methods 0.000 description 5
- 230000002861 ventricular Effects 0.000 description 5
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 229940067596 butylparaben Drugs 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000005086 pumping Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 230000036770 blood supply Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 2
- 206010030113 Oedema Diseases 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229940009456 adriamycin Drugs 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- 238000013537 high throughput screening Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000000270 postfertilization Effects 0.000 description 2
- 108010008359 protein kinase C lambda Proteins 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 229930184857 Asterin Natural products 0.000 description 1
- YWGAKIGNXGAAQR-UHFFFAOYSA-N Astin C Natural products N1C(=O)C(CO)NC(=O)C(CC)NC(=O)C2C(Cl)C(Cl)CN2C(=O)C(CC)NC(=O)CC1C1=CC=CC=C1 YWGAKIGNXGAAQR-UHFFFAOYSA-N 0.000 description 1
- 102000002723 Atrial Natriuretic Factor Human genes 0.000 description 1
- 101800001288 Atrial natriuretic factor Proteins 0.000 description 1
- 101800001890 Atrial natriuretic peptide Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000002330 Congenital Heart Defects Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101150050438 NPPA gene Proteins 0.000 description 1
- 101150114487 NPPB gene Proteins 0.000 description 1
- 206010037423 Pulmonary oedema Diseases 0.000 description 1
- 229920002334 Spandex Polymers 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 108010051204 asterin Proteins 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- NSQLIUXCMFBZME-MPVJKSABSA-N carperitide Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 NSQLIUXCMFBZME-MPVJKSABSA-N 0.000 description 1
- 230000007248 cellular mechanism Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- YTMNONATNXDQJF-UBNZBFALSA-N chrysanthemin Chemical compound [Cl-].O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC=C(O)C(O)=C1 YTMNONATNXDQJF-UBNZBFALSA-N 0.000 description 1
- 208000035850 clinical syndrome Diseases 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000003090 exacerbative effect Effects 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 230000005831 heart abnormality Effects 0.000 description 1
- 210000002837 heart atrium Anatomy 0.000 description 1
- 210000002064 heart cell Anatomy 0.000 description 1
- 210000003709 heart valve Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006450 immune cell response Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 210000003516 pericardium Anatomy 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 208000005333 pulmonary edema Diseases 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 239000004759 spandex Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for constructing a zebra fish immune non-responsive heart failure model, which utilizes butyl p-hydroxybenzoate to induce zebra fish embryos so as to obtain the zebra fish immune non-responsive heart failure model. Specifically, selecting a zebra fish embryo with normal development of 5-6hpf, transferring the zebra fish embryo into a zebra fish embryo culture solution added with 1.2-2.0mg/L of butyl p-hydroxybenzoate, culturing at 28-28.5 ℃, changing the solution every 22-24h, and culturing to 3-3.5dpf to obtain the zebra fish immune non-responsive heart failure model. The model test period constructed by the invention is short, and the method is simple to operate.
Description
Technical Field
The invention relates to the field of animal models, in particular to a method for inducing and generating a zebra fish immune unresponsive heart failure model by utilizing butyl p-hydroxybenzoate.
Background
Heart failure is a clinical syndrome of cardiac insufficiency due to various heart diseases, and is the final destination of most cardiovascular diseases, and its main symptoms are manifested by dyspnea, fatigue, pulmonary edema, etc. Heart failure disease can result in the heart failing to pump enough blood to meet the metabolic needs of the body, and serious heart failure disease can be life threatening. Heart failure syndrome may be caused by lesions at any level of the heart, including heart muscle, cardiovascular system, pericardium, heart valves, and heart abnormalities, among others.
Compared with the traditional animal models (mice, rats, rabbits, etc.), the zebra fish has the advantages of short growth cycle, high reproductive capacity, transparent zebra fish body in embryo period, easy observation, convenient realization of high throughput screening, and the like. In addition, the genome sequence of zebra fish has homology as high as 87% with human, and the developed molecular and cellular mechanisms are very similar to those of mammal, so that it can effectively simulate the pathological characteristics of human diseases. The heart of zebra fish consists of one ventricle and one atrium, resembling the 3-week human puppy heart, has a heart morphology, physiology, molecules and pathological features that are highly similar to humans, and is the first organ for zebra fish to develop and function, thus zebra fish is an ideal animal model for studying heart disease.
The current methods for modeling heart failure mainly include: pressure overload method, volume load method, myocardial ischemia/myocardial infarction method, and drug method. However, these methods are basically established based on animal models such as mice, rats, rabbits, etc., and mainly have the disadvantages of long test period, complex method, difficult operation, etc. For example, patent document 1 (Bayong, wei Yihong, asterin cloud, etc.) the shen clam san has heart protection effect and mechanism research on pressure-loaded heart failure rats [ J ]. Chinese medicine academic periodical, 2022 (004): 040.) and a pressure-loaded method is adopted to build a rat heart failure model. For example, in document 2 (Wang Siyuan, CHEN WEIDAN, study of Chen xinxin. Adriamycin-induced heart failure model in rats of low age [ J ]. Ind. J.Guangzhou medical school, 2019,047 (001): 10-12, 16.), an Adriamycin pharmaceutical method was used to construct a heart failure model in rats.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: provides a method for constructing a zebra fish immune non-responsive heart failure model with short period and simple method.
The technical scheme of the invention is as follows: the method for constructing the zebra fish immune non-responsive heart failure model utilizes butyl p-hydroxybenzoate to induce zebra fish embryos, so that the zebra fish immune non-responsive heart failure model is obtained.
Further, selecting a zebra fish embryo with normal development of 5-6hpf, transferring the zebra fish embryo into a zebra fish embryo culture solution added with 1.2-2.0mg/L of butyl p-hydroxybenzoate, culturing at 28-28.5 ℃, changing the solution every 22-24h, and culturing to 3-3.5dpf to obtain the zebra fish immune non-responsive heart failure model.
Further, the addition amount of the butyl p-hydroxybenzoate is 1.8mg/L.
Further, 5hpf normal-developing zebra fish embryos were selected.
Further, the culture temperature was 28.5 ℃.
Further, culturing to 3dpf resulted in a zebra fish immune non-responsive heart failure model.
Further, the liquid is changed every 24 hours.
Further, in order to facilitate the visual observation of the physiological condition of zebra fish, the zebra fish employs transgenic labeled zebra fish lines such as Tg (myl: GFP), tg (myl: GFP) by Tg (gata 1: dsred) hybrid zebra fish, tg (myl: GFP) by Tg (mpeg 1: mcherry), tg (myl: GFP) by Tg (lyz: dsred) hybrid zebra fish, and the like.
In the present invention, hpf is an abbreviation of hor post-fertilization, which means that zebra fish fertilize for several hours. Dpf is an abbreviation for day post-fertilization, which refers to zebra fish after fertilization for several days.
The invention also discloses a method for evaluating the zebra fish immune non-responsive heart failure model, which comprises one or more of the following (a) - (c):
(a) The heart development of the transgenic Tg (myl: GFP) zebra fish model was observed using a fluorescence microscope, and the end systole and end diastole images were obtained by the tracker software, further using video recordings.
(B) Observing the quantity, blood volume and blood flow velocity of red blood cells at the heart of the hybridized zebra fish model by fluorescence confocal, integral fluorescence microscopy shooting and combining o-dianisidine staining to observe Tg (myl 7:GFP) x Tg (gata 1: dsred); and detecting endocardial blood flow response gene and natriuretic peptide and brain natriuretic peptide transcription level by qPCR.
(C) The numbers of macrophages and neutral particles in myocardial tissue of the hybridized zebra fish model were observed by fluorescence confocal imaging for Tg (myl: GFP). Times.Tg (mpeg 1: mcherry), tg (myl: GFP). Times.Tg (lyz: dsred); shooting the quantity conditions of whole macrophages and neutral grains of the transgenic zebra fish model with a split fluorescent microscope according to Tg (mfap 4:GFP) and Tg (lyz: dsred); and detecting transcription level of the inflammatory factors related to the heart region by qPCR.
Compared with the prior art, the invention has the following beneficial effects:
1. The test period is short, and the method is simple to operate.
2. The dynamic change of the heart area can be observed in real time.
3. High throughput screening can be performed for drugs that rescue macrophage defects or immune non-responsiveness from causing or exacerbating heart failure.
Drawings
FIG. 1 is a graph showing the effect of different concentrations of butyl parahydroxybenzoate solution on zebra fish heart development in an example; wherein A is a heart phenotype chart under a white light and fluorescent microscope; b, a heart rate change chart of zebra fish; c is a mortality change chart of zebra fish; d is a zebra fish heart cell area chart; e is a graph of zebra fish arteria-venous sinus distance or atrioventricular distance.
FIG. 2 is a graph showing the effect of different concentrations of butyl paraben on the ability of a zebra fish embryo to contract heart in an example; wherein A is a cardiac schematic of zebra fish, and end systole and end diastole schematic; b ventricular area (size); c shortening the fraction; d, pumping blood fraction; e stroke volume.
FIG. 3 is a graph showing the effect of varying concentrations of butyl paraben exposure on blood supply to the heart of zebra fish and blood flow rate in an example embodiment; wherein A is a representative graph of cardiac blood cell number; b cardiac blood volume representative map; c, heart blood cell intensity statistical graph; d, a tail vein blood flow velocity statistical schematic diagram; e, a tail vein blood flow velocity statistical graph; f endocardial blood flow response gene kfl a transcript levels; e heart failure is well known to indicate transcript levels.
FIG. 4 is a graph showing the effect of varying concentrations of butyl paraben exposure on macrophages and neutrophils in zebra fish myocardial tissue in an example; wherein A is a representative graph of the number of macrophages in myocardial tissue; b myocardial tissue neutrophil count representation; c, counting the number of macrophages in myocardial tissue; d, counting a neutral grain number in myocardial tissue; e, representative graph of whole-body macrophage number of zebra fish; f, representative graph of whole body neutral particle number of zebra fish; g, a whole-body macrophage quantity statistical graph of zebra fish; h, counting the total neutral particle number of the zebra fish; i transcription level of inflammatory factors in heart region.
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.
1. Experimental method
1. Normally farmed zebra fish are placed in mating cylinders in a ratio of 1:1 or 1:2 for male and female and separated by a spacer. 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 ℃.
2. Zebra fish embryos with normal development of 5-6hpf are selected under a microscope, transferred into butyl p-hydroxybenzoate solutions (0.6 mg/L, 1.2mg/L, 1.8 mg/L) with different concentration gradients prepared by embryo culture water, and simultaneously a control group (the same volume of embryo culture water is used for incubation) is arranged and cultured in an incubator at 28-28.5 ℃ for changing liquid every 22-24 hours.
3. The transgenic Tg (myl 7:7 GFP) after treatment was observed for the cardiac development of zebra fish using a fluorescence microscope (Lycra M205 FA, germany). And meanwhile, video recording is further adopted, and images of the systole end and the diastole end are obtained through tracker software. The ventricular area (size), fractional shortening, fractional pumping, stroke volume (shown in fig. 2) were obtained with the short axis length a and the long axis length b, respectively.
Ventricular area = pi x ab/2;
ventricular volume = a2×b×0.523;
Fractional shortening= (end diastole area-end systole area)/end diastole area;
pump fraction = (end diastole volume-end systole volume)/end diastole volume;
Stroke volume = end diastole volume-end systole volume.
4. Hybridization of the zebra fish with Tg (myl: GFP) multiplied by Tg (gata 1: dsred), shooting by a fluorescence confocal microscope, and observing the number of red blood cells, the blood volume and the blood flow velocity of the zebra fish at the heart by combining o-dianisidine staining; and detecting endocardial blood flow response gene and natriuretic peptide and brain natriuretic peptide transcription level by qPCR.
5. The numbers of macrophages and neutral particles in the myocardial tissue of the hybridized zebra fish were observed by fluorescence confocal imaging for Tg (myl: GFP). Times.Tg (mpeg 1: mcherry), tg (myl: GFP). Times.Tg (lyz: dsred); and shooting the quantity conditions of whole-body macrophages and neutral grains of the transgenic zebra fish Tg (mfap 4:4) and Tg (lyz: dsred) by using a body fluorescence microscope; and detecting transcription level of the inflammatory factors related to the heart region by qPCR.
2. Experimental results
(A) Effect of different concentrations of butyl p-hydroxybenzoate solution on the ability of zebra fish to contract heart.
The results are shown in FIG. 1, and the initial phenotype pericardial edema, cardiac linearization, heart rate decline (A, B in FIG. 1) was observed after butyl parahydroxybenzoate treatment. And as the concentration of butyl parahydroxybenzoate and the treatment time increased, the survival rate, heart rate of zebra fish decreased (B, C in fig. 1), and the state of pericardial edema, heart linearization became more serious (A, D, E in fig. 1). Further video recordings were made of end systole and end diastole images by the tracker software as shown in fig. 2 (a in fig. 2), and statistics have found that butyl parahydroxybenzoate solutions resulted in significant decreases in zebra fish ventricular area (size) (B in fig. 2), fractional shortening (C in fig. 2), fractional pumping (D in fig. 2), and stroke volume (E in fig. 2) with increasing concentration. Wherein the fractional shortening and the fractional pumping are key indicators of the ability of the heart to contract, and insufficient ability of the heart to contract satisfies one of the key features of heart failure.
(B) Effects of different concentrations of butyl paraben solutions on blood supply capacity and blood flow rate of zebra fish heart.
As a result, as shown in FIG. 3, it was further found that the combination of fluorescence confocal and o-dianisidine staining resulted in a decrease in blood supply to the heart (decrease in blood flow to the heart) (A, B, C in FIG. 3). And was found to cause blood circulation disorders (decrease blood flow rate) (D, E in fig. 3). The decrease in cardiac blood flow was further confirmed by endocardial blood flow response gene detection (F in fig. 3). I.e. phenotypically fulfilling the key features that heart failure has. And by increasing the transcript levels of atrial natriuretic peptide (nppa), cerebral natriuretic peptide (nppb) (G in fig. 3), key molecular indicators leading to heart failure were validated.
(C) Butyl parahydroxybenzoate solutions resulted in macrophage defects and immune cell response defects at the zebra fish heart.
As shown in fig. 4, the cardiac region macrophages and neutrophils were recorded by fluorescence confocal (A, B in fig. 4) and found that the butyl p-hydroxybenzoate treated myocardial tissue and cardiac region did not recruit more macrophages and neutrophils (C, D in fig. 4) and resulted in a loss of the number of cardiac resident macrophages (B in fig. 4); and results in abnormal changes in macrophage morphology and size (arrow a in fig. 4). Imaging of whole-body macrophages and neutrophils of zebra fish by a whole-body fluorescence microscope revealed that butyl p-hydroxybenzoate did not significantly affect whole-body macrophage and neutrophil status (E, G, F, H in fig. 4); however, by QPCR on isolated hearts for detection of inflammatory factor transcript levels (I in fig. 4), significant inflammatory response was found in heart regions; the results indicate that butyl parahydroxybenzoate inhibits the recruitment of macrophages and neutrophils to myocardial tissue and cardiac regions and leads to defects in resident macrophages in the heart.
Claims (9)
1. The method for constructing the zebra fish immune non-responsive heart failure model is characterized by selecting 5-6 hpf normal-development zebra fish embryos, transferring the zebra fish embryos into zebra fish embryo culture solution added with 1.2-1.8mg/L butyl p-hydroxybenzoate, culturing at 28-28.5 ℃, changing the solution every 22-24 h, and culturing until the temperature reaches 3-3.5 dpf to obtain the zebra fish immune non-responsive heart failure model.
2. The construction method according to claim 1, wherein the addition amount of butyl parahydroxybenzoate is 1.8mg/L.
3. The method of claim 1, wherein 5 hpf normal-developing zebra fish embryos are selected.
4. The method according to claim 1, wherein the culturing temperature is 28.5 ℃.
5. The method of claim 1, wherein culturing to 3dpf results in a zebra fish immune non-responsive heart failure model.
6. The method of claim 1, wherein the liquid is changed every 24 hours.
7. The method of claim 1, wherein the zebra fish is a transgenic tagged line.
8. The construction method according to claim 7, wherein the transgenic marker line is one or more of Tg (myl: GFP), tg (myl: GFP) x Tg (gata 1: dsred), tg (myl: GFP) x Tg (mpeg 1: mcherry) or Tg (myl: GFP) x Tg (lyz: dsred).
9. A method of assessing a model of zebra fish immune non-responsive heart failure as claimed in any one of claims 1-8, comprising one or more of the following (a) - (c):
(a) Observing the heart development condition of the transgenic Tg (myl 7:GFP) zebra fish model by using a fluorescence microscope, and obtaining images of the end systole and the end diastole of the heart by using video recording and tracker software;
(b) Observing the quantity, blood volume and blood flow velocity of red blood cells at the heart of the hybridized zebra fish model by fluorescence confocal, integral fluorescence microscopy shooting and combining o-dianisidine staining to observe Tg (myl 7:GFP) x Tg (gata 1: dsred); detecting endocardial blood flow reaction genes and the transcription level of the natriuretic peptide and the brain natriuretic peptide by qPCR;
(c) The numbers of macrophages and neutral particles in myocardial tissue of the hybridized zebra fish model were observed by fluorescence confocal imaging for Tg (myl: GFP). Times.Tg (mpeg 1: mcherry), tg (myl: GFP). Times.Tg (lyz: dsred); shooting the quantity conditions of whole macrophages and neutral grains of the transgenic zebra fish model with a split fluorescent microscope according to Tg (mfap 4:GFP) and Tg (lyz: dsred); and detecting transcription level of the inflammatory factors related to the heart region by qPCR.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211627686.8A CN115786246B (en) | 2022-12-16 | 2022-12-16 | Construction method of zebra fish immune non-responsive heart failure model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211627686.8A CN115786246B (en) | 2022-12-16 | 2022-12-16 | Construction method of zebra fish immune non-responsive heart failure model |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115786246A CN115786246A (en) | 2023-03-14 |
| CN115786246B true CN115786246B (en) | 2024-09-06 |
Family
ID=85425528
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211627686.8A Active CN115786246B (en) | 2022-12-16 | 2022-12-16 | Construction method of zebra fish immune non-responsive heart failure model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115786246B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115261306B (en) * | 2022-07-19 | 2023-07-14 | 宜宾五粮液股份有限公司 | Zebra fish cardiovascular disease model, construction method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116369249A (en) * | 2023-01-13 | 2023-07-04 | 四川轻化工大学 | Construction method of zebra fish enteritis model |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4630989B2 (en) * | 2005-11-11 | 2011-02-09 | 国立大学法人三重大学 | Zebrafish with heart failure symptoms |
| EP3534924A4 (en) * | 2016-11-04 | 2020-07-01 | Groupe Santé Devonian Inc. | Composition for the prevention and/or treatment of cardiovascular diseases |
| CN106667982B (en) * | 2017-02-24 | 2019-07-26 | 北京中医药大学 | A method of preparing zebra fish thrombus model |
| US20220081691A1 (en) * | 2018-08-07 | 2022-03-17 | Dana-Farber Cancer Institute, Inc | Modulating ptpn2 to increase immune responses and perturbing gene expression in hematopoietic stem cell lineages |
| CN112271000A (en) * | 2020-10-28 | 2021-01-26 | 东南大学 | Construction and detection method of zebra fish heart failure model |
-
2022
- 2022-12-16 CN CN202211627686.8A patent/CN115786246B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116369249A (en) * | 2023-01-13 | 2023-07-04 | 四川轻化工大学 | Construction method of zebra fish enteritis model |
Non-Patent Citations (3)
| Title |
|---|
| Co-Exposure to Bisphenols, Parabens, and Antimicrobials and Association with Coronary Heart Disease: Oxidative Stress as a Potential Mediating Factor?;Zhang D第;《Environ Sci Technol》;20221219;第57卷(第1期);第531-538页 * |
| Toxicological assessment and developmental abnormalities induced by butylparaben and ethylparaben exposure in zebrafish early-life stages;Merola C 等;《Environ Toxicol Pharmacol》;20200904;第80卷;摘要，第1页第1段，第2.2.节，第2.3节，第3.1节，第3.3节，第4页最后1段，图4 * |
| ystolic heart failure induced by butylparaben in zebrafish is caused through oxidative stress and immunosuppression;Zhu H第;《Ecotoxicol Environ Saf》;20231118;第268卷;第1-11页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115786246A (en) | 2023-03-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Luongo et al. | The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability | |
| Dyson et al. | Atrial-like phenotype is associated with embryonic ventricular failure in retinoid X receptor alpha-/-mice. | |
| Jensen et al. | The hypertrabeculated (noncompacted) left ventricle is different from the ventricle of embryos and ectothermic vertebrates | |
| Porter Jr et al. | Bioenergetics, mitochondria, and cardiac myocyte differentiation | |
| Sedmera | Function and form in the developing cardiovascular system | |
| Navankasattusas et al. | The netrin receptor UNC5B promotes angiogenesis in specific vascular beds | |
| Moore | Postnatal lethality and cardiac anomalies in the Ts65Dn Down syndrome mouse model | |
| CN115786246B (en) | Construction method of zebra fish immune non-responsive heart failure model | |
| Lee et al. | Glycogen-branching enzyme deficiency leads to abnormal cardiac development: novel insights into glycogen storage disease IV | |
| Lu et al. | Perinatal angiogenesis from pre-existing coronary vessels via DLL4–NOTCH1 signalling | |
| Chin et al. | How insights from cardiovascular developmental biology have impacted the care of infants and children with congenital heart disease | |
| CN106492232A (en) | A kind of method for evaluating myocardial damage derivant toxicity and therapeutic agent effect with zebra fish | |
| Lin et al. | The protective effect of HIF-1α in T lymphocytes on cardiac damage in diabetic mice | |
| Ziegler et al. | Comparative morphology of the axial complex and interdependence of internal organ systems in sea urchins (Echinodermata: Echinoidea) | |
| Kirschbaum et al. | Intragenus (Campylomormyrus) and intergenus hybrids in mormyrid fish: physiological and histological investigations of the electric organ ontogeny | |
| Shrestha et al. | Using zebrafish to analyze the genetic and environmental etiologies of congenital heart defects | |
| Kim et al. | Lrrc10 is required for early heart development and function in zebrafish | |
| Arolkar et al. | Dedifferentiation and proliferation of artery endothelial cells drive coronary collateral development in mice | |
| Sankar et al. | Effect of hyperglycemia on tbx5a and nppa gene expression and its correlation to structural and functional changes in developing zebrafish heart | |
| Huang et al. | The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development | |
| CN115105528B (en) | Application of Prussian blue coordinated cobalt nano-enzyme in promoting heart regeneration | |
| Song et al. | Conditionally targeted deletion of PSEN1 leads to diastolic heart dysfunction | |
| Zhu et al. | Systolic heart failure induced by butylparaben in zebrafish is caused through oxidative stress and immunosuppression | |
| Zhang et al. | Artificial infection pathway of largemouth bass LMBV and identification of resistant and susceptible individuals | |
| Park et al. | Effect and application of cryopreserved three‐dimensional microcardiac spheroids in myocardial infarction therapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |