CN104274464A - Establishing method and application of dilated cardiomyopathy and zebrafish disease model - Google Patents
Establishing method and application of dilated cardiomyopathy and zebrafish disease model Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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Abstract
The invention relates to an establishing method and application of a dilated cardiomyopathy and zebrafish disease model. A disease model simulating human congenital dilated cardiomyopathy (CDCM) is firstly established; and the disease model can be used as a drug screening model to be applied to screening of drugs capable of relieving symptoms of heart failure caused by CDCM. The method provided by the invention is an economic and rapid high-content screening method; and the obtained compound has relatively high specificity and druggability.
Description
Technical field
The invention belongs to medical art, more specifically, the present invention relates to foundation and application thereof that a kind of sarcomere defect causes the zebra fish model of dilated cardiomyopathy.
Background technology
Expanding cardiomyopathy (Congenital Dilated CardioMyopathy, CDCM) is the main congenital cardiomyopathy of a class, and clinical manifestation is left ventricle or bilateral ventricular dilatation, declines with positive inotropic.In child's prognosis 5 years, fatality rate reaches 80%, and survivor also can suffer from rear something lost disease or need heart transplantation.In adult, caused by congenital genovariation, DCM penetrance reached 95% after 40 years old, and mortality rate is very high (the rear mean survival time male of morbidity 1.7 years, women 3.2 years) still, and its terateger rate of disabling also occupy the first in various disease and syndrome.
Myocardial cell sarcomere defect is one of main pathogenic factor of CDCM.Titin (TTN) is the necessary albumen of assembling sarcomere and stricture of vagina shape flesh, provides positive inotropic, regulates systolic pressure.TTN albumen is one maximum in human protein group, and it contains about 33,000 aminoacid, is the third-largest stricture of vagina shape myogen of content.Under physiological status, two TTN molecules combine the sarcomere forming about 2 μm wide, are anchored to Z-line and M-line.The research being published in New England Journal of Medicine for 2012 reports at 312 routine dilated cardiomyopathy (Dilated CardioMyopathy, DCM) find in that TTN sudden change occurs 72 examples, and show TTN truncated-type sudden change be the extremely important cause of disease of heritability DCM, it accounts for 25% at familial dilated familial incidence, and Sporadic cases is about 18%.TTN gene can carry out variable sheer in transcription, produces different transcripts, and wherein containing transcript specifically expressing in myocardial cell of N2B domain, visible N2B domain occurs to play certain effect in the normal development of cardiac muscle and pathology.Specify the variation of TTN function to contribute to improving DCM Pathophysiology understanding, thus help DCM patient can obtain early diagnosis, intervention and treatment.
At present, the pathology of DCM and Therapy study are made slow progress owing to lacking suitable animal model.Although have part in the world for the mouse model of heart failure research, mainly concentrate on beta-adrenoceptor activity regulation aspect, the sign of this essential myocardial disease of preexisting myocardial muscular strength defect cannot be simulated.
Therefore, a kind of animal model for DCM research is urgently developed in this area, so that the therapeutic scheme that further investigated is new, substitutes heart transplantation to a certain extent or improves prognosis survival rate.
Summary of the invention
The object of the present invention is to provide the foundation and application of dilated cardiomyopathy Brachydanio rerio disease model.
In a first aspect of the present invention, provide a kind of method of screening the potential drug for the treatment of dilated cardiomyopathy or heart failure, described method comprises:
(1) Brachydanio rerio titin-/-mutant embryos is provided;
(2) according to after fertilization Time Calculation, from after fertilization embryo with drug candidate process (1) 20 ± 2 hours;
(3) to embryo fertilization 32 ± 2 hours, 48 ± 2 hours, the phenotype of zebrafish embryo is observed respectively;
Wherein, if 48 ± 2 hours heart collecting moving frequencies significantly increase after embryo fertilization, then described drug candidate is the potential drug for the treatment of dilated cardiomyopathy or heart failure; Or
If after embryo fertilization 32 ± 2 hours, pericardium enlargement degree is significantly alleviated, the early stage diastole of heart, contractility are significantly strengthened or heart reduces degree reduction or blood circulation significantly improves, then described drug candidate is the potential drug for the treatment of dilated cardiomyopathy or heart failure.
In a preference, in described method, step (2) comprising: in test group, from after fertilization embryo with drug candidate process (1) 20 ± 2 hours; And/or
Step (3) comprising: the phenotype of observation zebrafish embryo, and compares with matched group, wherein said matched group be need not described drug candidate process, synchronous Brachydanio rerio titin-/-mutant embryos of growing;
If compared with matched group, after test group embryo fertilization, 48 ± 2 hours heart collecting moving frequencies significantly increase, then described drug candidate is the potential drug for the treatment of dilated cardiomyopathy or heart failure; Or
If compared with matched group, after test group embryo fertilization 32 ± 2 hours, pericardium enlargement degree is significantly alleviated, the early stage diastole of heart, contractility are significantly strengthened or heart reduces degree reduction or blood circulation significantly improves, then described drug candidate is the potential drug for the treatment of dilated cardiomyopathy or heart failure.
In another preference, described heart collecting moving frequency significantly increases and is: compared with matched group, the pollex number of times of test group embryo heart significantly increases, as per minute higher than 140 times in being increased to; Preferably higher than 150 times; Or
Described pericardium enlargement degree is significantly alleviated: compared with matched group, and test group embryonic epicardial enlarged degree significantly reduces; Or
Described blood circulation significantly improves: compared with matched group, and test group embryo has blood circulation.
In another preference, described drug candidate includes, but is not limited to: micromolecular compound, peptide.
In another preference, described matched group only comprises the solvent of drug candidate; Preferably, described solvent is DMSO; And/or also positive drug control group is set; Preferably, described positive drug contrast is medicine Digitoxin.
In another preference, described method also comprises: carry out further cell experiment and/or animal experiment to the potential drug obtained, to select further from drug candidate and to determine for treatment dilated cardiomyopathy or the useful material of heart failure.
In another preference, described dilated cardiomyopathy is expanding cardiomyopathy.
In another aspect of this invention, a kind of screening model of the potential drug for screening treatment dilated cardiomyopathy or heart failure is provided, described screening model is Brachydanio rerio titin-/-mutant embryos, and it is the offspring produced by Brachydanio rerio titin+/-saltant type heterozygote copulation.
In another aspect of this invention, provide the purposes of described screening model, for screening the potential drug for the treatment of dilated cardiomyopathy or heart failure.
In another aspect of this invention, provide compound or the purposes of its pharmaceutically acceptable salt in the pharmaceutical composition preparing treatment dilated cardiomyopathy or heart failure, described compound is selected from:
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, titin-/-Brachydanio rerio defect model.
The genetics characteristics of A, model Brachydanio rerio.Being positioned at No. 9 chromosomal titina gene NB2 domains is termination codon (T-> A) truncated-type titin-/-gene mutation.
The phenotype (A: atrium, V: ventricle) of B, wild type (wild-type) and gene mutation fish.
Fig. 2, the heartbeat rhythm and pace of moving things detect.Beats counting in per minute to heart, titin-/-catastrophic model beats after 48hpf obviously reduces.MU represents homozygous mutant; WT represents wild type.
Fig. 3, drug screening strategy.Add corresponding embryo in 96 orifice plates and carry out drug treating containing the E3 embryo medium of medicine, process 10 hours from zebrafish embryo after fertilization, after embryo fertilization 32 hours, the then phenotype of light Microscopic observation zebrafish embryo.
Fig. 4, the compound effect judgement being Testing index with heartbeat frequency.DMSO C is normal control, and DMSO M is negative control, and Digitoxin (digoxin) is positive control.Palmic rate returns to the compd A 1,6,7 of more than 150 times for candidate compound.
Detailed description of the invention
The present inventor is through deep research, sudden change Brachydanio rerio based on TTN Gene truncation type is, construct a kind of simulating human expanding cardiomyopathy (Congenital Dilated CardioMyopathy first, CDCM) disease model, described disease model can be used as sieve medicine model, is applied to screening and can improves heart failure symptoms medicine caused by CDCM.Method of the present invention is a kind of economy High content screening method efficiently, the specificity that the compound tool obtained is higher and druggability.
As used herein, described " significantly " refers to and change is statistically occurring.As " significantly increasing ", refer to increase by 20% or more; " significantly alleviate " refers to that disease severity significantly reduces by 20% or more, by that analogy.
The invention provides the zebra fish model that a kind of sarcomere defect causes dilated cardiomyopathy.Use titin gene mutation strain, confirm that defect is ttn protein truncation type afunction.Heterozygous mutation body is bought by Sanger Institute.A pair titin heterozygous mutation male and female adult fish in one week by generation 100 DCM offsprings (a pair fish one week in produce 400 embryo's meters, 25% offspring's morbidity).These embryos will be used to disease phenotype analysis.
The present inventor, through repeatedly testing, for the feature of Zebrafish Embryo, summarizes every Testing index of disease model:
(1) heartbeat rhythm and pace of moving things Testing index: count (Fig. 3) at the beats in per minute heart, by time duration Journal of Sex Research, is optimal detection phase time 48 after fertilizations hour (hpf).Normal beats number of times is about 170 beats/min, and model beats is about 140 beats/min.
(2) cardiomorphology Testing index: more easily observe cardiac phenotype during 32hpf, comprise visceral pericardium and expand, the early stage diastole of heart, shrinks unable.During 72hpf, heart compared with normal is little, and possible apoptosis causes caused by myocardial cell loss.
(3) blood circulation Testing index: successfully model embryo in 32hpf without blood circulation, visible red cell accumulation.
After obtaining sieve medicine model of the present invention, this model can be applied screen the potential medicine for the treatment of dilated cardiomyopathy or heart failure.
Therefore, the invention provides a kind of method of screening the potential material for the treatment of dilated cardiomyopathy or heart failure, described method comprises: (1) provides Brachydanio rerio titin-/-mutant embryos; (2) according to after fertilization Time Calculation, from after fertilization embryo with drug candidate process (1) 20 ± 2 hours; (3) to embryo fertilization 32 ± 2 hours, 48 ± 2 hours, the phenotype of zebrafish embryo is observed respectively;
Wherein, if 48 ± 2 hours heart collecting moving frequencies significantly increase after embryo fertilization, then described drug candidate is the potential drug for the treatment of dilated cardiomyopathy or heart failure;
If after embryo fertilization 32 ± 2 hours, pericardium enlargement degree is significantly alleviated, the early stage diastole of heart, contractility are significantly strengthened or heart reduces degree reduction or blood circulation significantly improves, then described drug candidate is the potential drug for the treatment of dilated cardiomyopathy or heart failure.
In optimal way of the present invention, when screening, in order to be easier to the change observing disease condition, also can arrange matched group, described matched group can be the disease model not adding described drug candidate.
As optimal way of the present invention, described method also comprises: carry out further cell experiment and/or animal experiment to the potential material obtained, to select further and to determine for treatment dilated cardiomyopathy or the really useful material of heart failure.
On the other hand, present invention also offers the potential drug for the treatment of dilated cardiomyopathy or the heart failure adopting described screening technique to obtain.The material that these Preliminary screening go out can form a screening storehouse so that people finally can therefrom filter out can for treatment dilated cardiomyopathy or the useful material of heart failure.
As the preferred embodiments of the present invention, use the screening technique of screening model of the present invention as follows:
1) select the micromolecular compound of each class formation in drug reservoir, with DMSO as solvent, be made into the storage liquid of 0.5mM; When phenotypic screen, often kind of compound embryo medium is diluted to 5 μMs; Wherein contrast uses DMSO to be blank, and Digitoxin is positive control drug.
2) offspring that produces of heterozygote (ttn+/-) copulation of ttn sudden change, wherein have the offspring of 1/4 to have mutant phenotype, will be developed to the offspring embryo that 23-somite homozygous mutation produces, 10/hole joins in 96 orifice plates.Then in each 96 orifice plates, add the corresponding E3 embryo medium containing medicine carry out drug treating.
3) to 32hpf after embryo fertilization, the improvement situation of observation model Testing index under light microscopic, 48hpf counts cardiac rhythm, judges whether small-molecule chemical thing can be used as lead compound.
4) toxicity of compound judges: check the configuration of embryo and the body segment number of corresponding phase, the development toxicity of observation analysis compound, and record is in order to analyzing effect toxicologic index.
The invention provides the purposes of some compounds or its pharmaceutically acceptable salt, for the preparation of the pharmaceutical composition for the treatment of dilated cardiomyopathy or heart failure; Described compound is selected from:
Described compound or its pharmaceutically acceptable salt and compositions thereof are by oral and intravenous, intramuscular or the administration such as subcutaneous; Preferably oral administration, as long as the characteristic of applicable active component and required specific administration mode all can be applied.In pharmaceutical compositions, normally used adjuvant also can advantageously be included, and such as flavoring agent, pigment, antiseptic and antioxidant are as vitamin E, vitamin C, BHT and BHA.
The effective dose of compound of the present invention can change with the order of severity of the pattern of administration and disease to be treated.But, usually when compound of the present invention gives with the dosage of about 1-300mg/kg the weight of animals every day, gratifying effect can be obtained, preferably give with the dosage that 1-3 time is separated every day, or with sustained release forms administration.For most of large mammal, the accumulated dose of every day is about 5-1000mg, is preferably about 10-500mg.Be applicable to the dosage form taken orally, comprise the reactive compound with the intimately mixed about 1-200mg of solid-state or liquid pharmaceutically acceptable carrier.This dosage of scalable is replied to provide optimal treatment.Such as, by an urgent demand for the treatment of situation, the dosage that several times separate can be given every day, or dosage is reduced pari passu.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise percentage ratio and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
Embodiment 1, to determine for the Testing index screened
The present invention has prepared the Brachydanio rerio mutant of similar mankind's dilated cardiomyopathy, i.e. titin gene mutation strain, confirms that defect is ttn protein truncation type afunction (Figure 1A-B).Titin heterozygous mutation can purchased from Sanger Institute.A pair titin heterozygous mutation male and female adult fish in one week by generation 100 DCM offsprings (a pair fish one week in produce 400 embryo's meters, 25% offspring's morbidity).
Identified by sequencing technologies, the N2B domain of confirmation titina gene has the sequence change of a TAT-TAA, create termination codon, make the translation premature termination of titina albumen, the mutation type of this mutant is that truncated-type titin suddenlys change.Mutant morphologic phenotype is pericardium enlargement, and cardiac contractile force declines, and does not have blood circulation.
By the vascular endothelial cell of above-mentioned titin heterozygous mutation fish and Tg (flk1::EGFP) by the genetically engineered fish of green fluorescent label (purchased from international Brachydanio rerio resource center) copulation, observe offspring's vascular development.Find that the vascular morphology after sudden change occurs completely normal, prove that vascular development is not Testing index.First dilatancy diastole (32hpf) of heart, along with passage of time, with the defect apoptosis of various heart development, when 60hpf, heart starts atrophy.And heartbeat frequency significantly slows down after 48hpf, beats is lower than every minute hand 140 times.
Carry out observed and recorded to each phenotype, sum up relatively, in conjunction with the Clinical signs of mankind's dilated cardiomyopathy, the present inventor have selected 3 class phenotypes as Testing index, comprising:
1) heartbeat rhythm and pace of moving things Testing index: count (Fig. 3) at the beats in per minute heart, by time duration Journal of Sex Research, is optimal detection phase (Fig. 2) during 48hpf.Heartbeat frequency significantly slows down after 48hpf, and beats is lower than 140 times per minute.
2) cardiomorphology Testing index: more easily observe cardiac phenotype during 32hpf, comprise visceral pericardium and expand (pericardium enlargement), the early stage diastole of heart, shrinks unable.After 3 days, the heart compared with normal that can be observed homozygous mutation embryo is little.
3) blood circulation Testing index: model embryo is without blood circulation in 32hpf, and visible red cell accumulation is on yolk sac.
Embodiment 2, drug screening method
The method of the micromolecular compound of screening treatment DCM provided by the invention, adopt model organism sarcomere defect Brachydanio rerio, concrete steps are as follows:
1. the selection in drug screening storehouse and preparation
Be applied to the compound of screening, comprise 640 kinds and be can be used for clinical small-molecule drug, 1, the micromolecular inhibitor storehouse of 057 kind of Tocris company by FDA is approved, 1,200 kinds of Prestwick companies have the multifarious compound library of structure and activity.Often kind of micromolecular compound is all mixed with the storage liquid of 0.5mM, and solvent is DMSO.Concentration selected in the present embodiment compound library be 5 μMs as primary dcreening operation concentration, multiple sieve uses 2-25 μM of Concentraton gradient.
2. the preparation of embryo
The offspring that heterozygote (titin+/-) copulation of TTN sudden change produces, wherein have the offspring of 1/4 to have mutant phenotype, i.e. the pericardium enlargement when 32hpf, cardiac systolic pressure declines, and does not have blood circulation.First, the present inventor raises the offspring of the fish copulation generation being greater than 1000 titin+/-heterozygosis and wild type, observes Appearance of the filial generation phenotype by random pair.Choose and be greater than 100 pairs of heterozygotes pairing (titin+/-), the offspring embryo produced by heterozygote selects homozygous mutation when 23-somite, and 10/hole joins in 96 orifice plates.
3. screening compound
The Testing index formulated in embodiment 1 is as the foundation of screening.Carry out observing and detecting at corresponding time point, screening strategy is as Fig. 3.
In each 96 orifice plates, add the corresponding E3 embryo medium containing drug candidate carry out drug treating, process from zebrafish embryo after fertilization 20 hours (about 25-somite), until embryo fertilization after 48 hours, under visible light microscope, then observe the phenotype of zebrafish embryo.
If embryo still phenotype is pericardium enlargement, without blood circulation, so this medicine to dilated cardiomyopathy without therapeutic effect.
During 32hpf, if relative to the embryo's (DMSO matched group) not giving drug candidate, the embryo given after drug candidate process shows as fetal tissues phenotype or have clear improvement (as not remarkable in cardiac shape atrophy or atrophy process slows down), there is blood circulation, without pericardium enlargement, the early stage diastole of heart, contractility improve, then this compound is candidate's lead drug and enters multiple sieve.During 72hpf, if relative to the embryo's (matched group) not giving drug candidate, the embryo given after drug candidate process shows as heart and does not reduce or reduce degree alleviation, then this compound is candidate's lead drug and enters multiple sieve.
Arrange DMSO C is the contrast that fetal tissues (not mutated) only gives DMSO process simultaneously, and DMSOM is the negative control that mutant only gives DMSO process, and Digitoxin (digoxin) is positive control.
If relative to the embryo's (matched group) not giving drug candidate, give the embryo heart Beating Rate after drug candidate process to beat after 48hpf and do not slow down, or the process that slows down is slower than matched group, or beats is higher than every minute hand 140 times, then this compound is candidate's lead drug and enters multiple sieve.
Determine that the highest real active compound carries out deep research, evaluate the overall embryo health with phenotypic correlation, the effectiveness analyzed in multiple sieve by heartbeat Count Test is reappeared, and gets rid of false positive, obtains the further investigation of effective lead compound.
With heart collecting moving frequency for index, part the selection result is as Fig. 4, and it is as follows that screening obtains part active compound structure:
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (9)
1. screen a method for the potential drug for the treatment of dilated cardiomyopathy or heart failure, it is characterized in that, described method comprises:
(1) Brachydanio rerio titin-/-mutant embryos is provided;
(2) according to after fertilization Time Calculation, from after fertilization embryo with drug candidate process (1) 20 ± 2 hours;
(3) to embryo fertilization 32 ± 2 hours, 48 ± 2 hours, the phenotype of zebrafish embryo is observed respectively;
Wherein, if 48 ± 2 hours heart collecting moving frequencies significantly increase after embryo fertilization, then described drug candidate is the potential drug for the treatment of dilated cardiomyopathy or heart failure; Or
If after embryo fertilization 32 ± 2 hours, pericardium enlargement degree is significantly alleviated, the early stage diastole of heart, contractility are significantly strengthened or heart reduces degree reduction or blood circulation significantly improves, then described drug candidate is the potential drug for the treatment of dilated cardiomyopathy or heart failure.
2. the method for claim 1, is characterized in that, step (2) comprising: in test group, from after fertilization embryo with drug candidate process (1) 20 ± 2 hours; And/or
Step (3) comprising: the phenotype of observation zebrafish embryo, and compares with matched group, wherein said matched group be need not described drug candidate process, synchronous Brachydanio rerio titin-/-mutant embryos of growing;
If compared with matched group, after test group embryo fertilization, 48 ± 2 hours heart collecting moving frequencies significantly increase, then described drug candidate is the potential drug for the treatment of dilated cardiomyopathy or heart failure; Or
If compared with matched group, after test group embryo fertilization 32 ± 2 hours, pericardium enlargement degree is significantly alleviated, the early stage diastole of heart, contractility are significantly strengthened or heart reduces degree reduction or blood circulation significantly improves, then described drug candidate is the potential drug for the treatment of dilated cardiomyopathy or heart failure.
3. method as claimed in claim 1 or 2, is characterized in that, described heart collecting moving frequency significantly increases and is: compared with matched group, the pollex number of times of test group embryo heart significantly increases, as per minute higher than 140 times in being increased to; Preferably higher than 150 times; Or
Described pericardium enlargement degree is significantly alleviated: compared with matched group, and test group embryonic epicardial enlarged degree significantly reduces; Or
Described blood circulation significantly improves: compared with matched group, and test group embryo has blood circulation.
4. method as claimed in claim 1 or 2, it is characterized in that, described drug candidate includes, but is not limited to: micromolecular compound, peptide.
5. method as claimed in claim 1 or 2, it is characterized in that, described matched group only comprises the solvent of drug candidate; Preferably, described solvent is DMSO; And/or
Also positive drug control group is set; Preferably, described positive drug contrast is medicine Digitoxin.
6. method as claimed in claim 1 or 2, it is characterized in that, described method also comprises: carry out further cell experiment and/or animal experiment to the potential drug obtained, to select further from drug candidate and to determine for treatment dilated cardiomyopathy or the useful material of heart failure.
7. one kind for screening the screening model of potential drug for the treatment of dilated cardiomyopathy or heart failure, it is characterized in that, described screening model is Brachydanio rerio titin-/-mutant embryos, and it is the offspring produced by Brachydanio rerio titin+/-saltant type heterozygote copulation.
8. the purposes of screening model according to claim 7, for screening the potential drug for the treatment of dilated cardiomyopathy or heart failure.
9. compound or the purposes of its pharmaceutically acceptable salt in the pharmaceutical composition preparing treatment dilated cardiomyopathy or heart failure, described compound is selected from:
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| CN112271000A (en) * | 2020-10-28 | 2021-01-26 | 东南大学 | Construction and detection method of zebra fish heart failure model |
| CN112715483A (en) * | 2020-11-25 | 2021-04-30 | 珠海沅芷健康科技有限公司 | Mutant CNPase zebra fish model capable of reducing cardiac function and application method |
| CN112715483B (en) * | 2020-11-25 | 2023-10-27 | 科偲(山东)创业服务有限公司 | Preparation method and application method of mutant CNPase zebra fish model for reducing cardiac function |
| CN114480497A (en) * | 2022-02-28 | 2022-05-13 | 湖南师范大学 | Construction method and application method of ep400 gene knockout zebra fish heart failure model |
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