Composition for relieving heart development defect or resisting oxidative stress and application thereof

Abstract

The invention provides a composition for relieving heart development defects or resisting oxidative stress and application of the composition. The composition comprises L-selenomethionine and a PI3K / AKT signal channel inhibitor, the invention further provides a heart development defect animal model and an establishment method and application thereof.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy; more specifically, the present invention relates to a composition for alleviating cardiac developmental defects or resisting oxidative stress and its use; the present invention also relates to an animal model of cardiac developmental defects, a method for establishing the same and its use. Background Art

[0002] Cardiovascular disease is one of the most harmful diseases in the world and the leading cause of death from disease. There are many types of heart disease, which can be roughly divided into congenital heart disease and acquired heart disease. Congenital heart disease is mainly caused by abnormal fetal development during the embryonic period, while acquired heart disease is closely related to infection, drugs, and lifestyle habits. The severity is mainly related to the degree of disease progression. Among them, the more critical heart diseases include tetralogy of Fallot, acute heart failure, ventricular fibrillation, extensive anterior wall myocardial infarction, severe valvular stenosis, acute cardiac tamponade, etc. Patients may suffocate and die in a short period of time, and timely intervention is required.

[0003] The classification of heart disease is as follows: (a) Congenital cardiovascular disease: tetralogy of Fallot, atrial and ventricular septal defects, patent ductus arteriosus, patent foramen ovale, etc.; (b) Heart failure: When heart failure attacks acutely, the acute course is usually more serious and can be life-threatening in a short period of time; (c) Arrhythmia: including sinus, atrial, atrioventricular junctional, ventricular arrhythmias, and heart block, among which third-degree atrioventricular block and ventricular fibrillation are more serious; (d) Coronary atherosclerotic heart disease: including chronic myocardial ischemic syndrome and acute coronary syndrome. Acute coronary syndrome includes myocardial infarction. The larger the area of ​​myocardial infarction, the more dangerous the condition. (e) Hypertensive heart disease: heart disease caused by increased cardiac load due to increased blood pressure; (f) Myocardial disease: including dilated, hypertrophic, restrictive cardiomyopathy, and myocarditis; (g) Valvular heart disease: such as mitral valve, aortic valve stenosis and regurgitation, multivalvular disease, rheumatic heart disease, etc. The more severe the stenosis or regurgitation, the more serious the condition; (h) Pericardial disease: including acute pericarditis, pericardial effusion and cardiac tamponade, constrictive pericarditis. Acute cardiac tamponade can cause acute circulatory failure and shock; (i) Infective endocarditis: including native valve endocarditis, prosthetic valve endocarditis, etc.

[0004] In the field of heart disease, drug treatment is widely used, for example, antihypertensive, lipid-lowering, vasodilator, and antiarrhythmic drugs are used to improve the clinical symptoms of patients and relieve heart disease.

[0005] Some heart diseases present symptoms such as pericardial edema, enlarged pericardial area, reduced atrial wall thickness, reduced ventricular wall thickness, increased oxidative stress levels, increased ROS content, or cell apoptosis in the ventricles, which are related to the oxidative stress level of the tissue. The myocardial oxidative stress injury mechanism refers to the process in which myocardial cells are damaged under oxidative stress. Oxidative stress refers to an oxidant present in the intracellular and extracellular environment, such as free radicals and peroxides, which can react with biological molecules in the cell and cause cell abnormalities. Oxidative stress is also closely related to heart failure.

[0006] Although there have been many studies on heart diseases in this field, there is still a need to screen and develop new alleviating / therapeutic drugs, and there is also a need for typical heart disease models. Summary of the invention

[0007] The purpose of the present invention is to provide an animal model of heart disease, a method for establishing the model and an application thereof.

[0008] In a first aspect of the present invention, a PI3K / AKT signaling pathway inhibitor and L-selenomethionine are used to prepare a composition (pharmaceutical composition) for alleviating or treating cardiac developmental defects; or a composition (pharmaceutical composition) for resisting oxidative stress.

[0009] In one or more embodiments, the cardiac development defect is a cardiac development (including early development) defect; more preferably, the cardiac development defect is an embryonic cardiac development defect; more preferably, the cardiac development defect is a cardiac development defect caused by downregulation (such as mutation) of the SH2E gene.

[0010] In one or more embodiments, the PI3K / AKT signaling pathway inhibitor includes: AKT inhibitor VIII, LY294002, and Wortmannin.

[0011] In one or more embodiments, the heart development defect is a heart development defect presenting the following symptoms: pericardial edema, increased pericardial cavity area, decreased atrial wall thickness, decreased ventricular wall thickness, increased oxidative stress level, increased ROS content, and increased cell apoptosis in the ventricular part; the L-selenomethionine exerts its effect by reducing pericardial edema, shrinking pericardial cavity area, increasing atrial wall thickness, increasing ventricular wall thickness, reducing oxidative stress level, reducing ROS content, and reducing cell apoptosis in the ventricular part.

[0012] In another aspect of the present invention, a pharmaceutical composition is provided, comprising a PI3K / AKT signaling pathway inhibitor and L-selenomethionine, and a pharmaceutically acceptable carrier; the pharmaceutical composition is used to alleviate or treat cardiac developmental defects, and / or resist oxidative stress.

[0013] In one or more embodiments, the dosage form of the pharmaceutical composition is: injection, infusion, tablet, capsule, pill; preferably injection.

[0014] In another aspect of the present invention, a medicine kit is provided, comprising a PI3K / AKT signaling pathway inhibitor and L-selenomethionine; the medicine kit is used for alleviating or treating cardiac developmental defects and / or anti-oxidative stress.

[0015] In one or more embodiments, the pharmaceutical kit contains the pharmaceutical composition.

[0016] In one or more embodiments, the medicine kit further comprises: instructions for use, describing methods for alleviating or treating cardiac developmental defects and / or anti-oxidative stress.

[0017] In one or more embodiments, the molar ratio of the PI3K / AKT signaling pathway inhibitor to the L-selenomethionine is: (8-500):1 (such as 12:1, 20:1, 24:1, 30:1, 50:1, 60:1, 80:1, 100:1, 150:1, 200:1, 250:1, 350:1, 400:1, etc.); preferably (10-300):1; more preferably (15-200):1.

[0018] In one or more embodiments, in the medicine kit, the PI3K / AKT signaling pathway inhibitor and the L-selenomethionine are placed in different containers (such as syringes) / packages in the medicine kit.

[0019] In one or more embodiments, in the drug kit, the PI3K / AKT signaling pathway inhibitor and the L-selenomethionine are mixed in the same container / package.

[0020] In another aspect of the present invention, a method for screening potential substances that promote L-selenomethionine to alleviate or treat cardiac developmental defects and / or resist oxidative stress (i.e., potential substances that can be used in combination with L-selenomethionine to alleviate or treat cardiac developmental defects and / or resist oxidative stress) is provided, the method comprising: (1) treating an expression system with a candidate substance, the system having a PI3K / AKT signaling pathway; and (2) detecting changes in the PI3K / AKT signaling pathway in the system; if the candidate substance statistically inhibits the PI3K / AKT signaling pathway, it indicates that the candidate substance is a potential substance that promotes L-selenomethionine to alleviate or treat cardiac developmental defects and / or resist oxidative stress (such as a substance that inhibits the signaling pathway that has not been disclosed so far).

[0021] In one or more embodiments, the system containing the PI3K / AKT signaling pathway is selected from: a cell (culture) system, a subcellular (culture) system, a tissue (culture) system or an animal system.

[0022] In one or more embodiments, the increase or promotion is a statistical increase or promotion, such as an increase or promotion of 10% or 20% or more, preferably an increase or promotion of 30% or 50% or more, and more preferably an increase or promotion of 80% or 90% or more, compared to a control or substrate.

[0023] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules designed for the PI3K / AKT signaling pathway, pathway proteins or their upstream or downstream proteins or genes (such as but not limited to upregulators, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands)), CRISPR constructs, small molecule compounds, and compounds from a compound library.

[0024] In another aspect of the present invention, a method for preparing an animal model of cardiac developmental defects is provided, comprising targeting the SH2E gene in the zebrafish genome and downregulating the target to obtain an animal model of cardiac developmental defects.

[0025] In one or more embodiments, the downregulation is performed by gene knockout / silencing, gene interference.

[0026] In one or more embodiments, the down-regulation or removal is performed by gene knockout / silencing using gene editing or homologous recombination methods.

[0027] In one or more embodiments, the SH2E gene is knocked out by a CRISPR / Cas (such as Cas9) method, including using a specific gRNA to target the SH2E gene.

[0028] In one or more embodiments, the gRNA targets exon 1 of the SH2E gene.

[0029] In one or more embodiments, the sequence targeting the SH2E gene is shown as SEQ ID NO:2 and / or SEQ ID NO:3.

[0030] In one or more embodiments, after being treated with CRISPR / Cas (such as Cas9), the protein encoded by the SH2E gene mutates at position 59 and / or position 82, causing the translation of the SH2E protein to terminate prematurely.

[0031] In one or more embodiments, by injecting gRNA and mRNA of Cas (such as Cas9) protein or DNA that can form the mRNA into zebrafish fertilized eggs, a fertilized egg transformed by CRISPR / Cas (such as Cas9) is obtained; further, the fertilized egg is cultivated into an animal embryo or an animal.

[0032] In one or more embodiments, the animal embryo or animal comprises: 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 20 days, 30 days, 45 days, 60 days after fertilization to the adult stage such as 90 days.

[0033] In one or more embodiments, the animal embryo or animal comprises: embryonic, larval, juvenile and adult stages.

[0034] In one or more embodiments, the cardiac development defect is a cardiac development (including early development) defect.

[0035] In one or more embodiments, the cardiac development defect is an embryonic cardiac development defect.

[0036] In one or more embodiments, the cardiac developmental defect presents a phenotype including the following (or is a cardiac developmental defect presenting the following symptoms): decreased SH2E expression level (including decreased mRNA transcription level), pericardial cavity edema, increased pericardial cavity area, decreased atrial wall thickness, decreased ventricular wall thickness, increased oxidative stress level, increased ROS content, and increased cell apoptosis in the ventricular area.

[0037] In one or more embodiments, the increase, improvement, increase, or multiplication is a statistically significant or notable increase, improvement, increase, or multiplication; for example, an increase, improvement, increase, or multiplication of 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 80%, 90%, 100% or more.

[0038] In one or more embodiments, the reduction is a statistically significant or significant reduction (or decrease, weakening, etc.); for example, a reduction of 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 80%, 90%, 100% or more.

[0039] In one or more embodiments, the oxidative stress level is detected using a reactive oxygen species fluorescent probe.

[0040] In another aspect of the present invention, there is provided an application of an animal model of cardiac developmental defects obtained by the method, which is used as an animal model for screening candidate drugs or therapeutic agents for alleviating or treating cardiac developmental defects; or as an animal model for studying cardiac developmental defects. Preferably, the cardiac developmental defect is an early cardiac developmental defect; more preferably, the cardiac developmental defect is an embryonic cardiac developmental defect.

[0041] In one or more embodiments, the methods or applications are non-diagnostic or therapeutic methods or applications, and do not have the diagnosis or treatment of a disease as a direct purpose.

[0042] In another aspect of the present invention, a method for screening candidate drugs or therapeutic agents for alleviating or treating heart developmental defects is provided, the method comprising: (1) preparing an animal model of heart developmental defects using the method described above; (2) administering a candidate substance to the animal model of (1) to observe whether the candidate substance has an alleviating or therapeutic effect on the heart developmental defect; if it is observed that the heart developmental defect symptoms of the animal model are alleviated, the candidate substance is a substance for alleviating or treating the heart developmental defect.

[0043] In one or more embodiments, the cardiac development defect is a cardiac development (including early development) defect; more preferably, the cardiac development defect is an embryonic cardiac development defect.

[0044] In one or more embodiments, the observation of whether the candidate substance has an alleviating or therapeutic effect on cardiac developmental defects includes (but is not limited to) analyzing the following phenotypes: SH2E expression level, pericardial edema, pericardial area, atrial wall thickness, ventricular wall thickness, oxidative stress level, ROS content or ventricular cell apoptosis; wherein, if the decrease in SH2E expression level, pericardial edema, increase in pericardial area, decrease in atrial wall thickness, decrease in ventricular wall thickness, increase in oxidative stress level, increase in ROS content or increase in ventricular cell apoptosis are reversed, then the candidate substance is a substance that alleviates or treats cardiac developmental defects.

[0045] In one or more embodiments, the test further includes providing a control animal, wherein the control animal is not administered with the candidate substance.

[0046] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 , SH2E gene deletion causes heart development defects in zebrafish embryos;

[0048] (A) Schematic diagram of CRISPR target design in zebrafish SH2E gene;

[0049] (B) Two SH2E gene mutants, G59R and S82R, and their genomic sequences;

[0050] (C) The SH2E gene of the SH2E mutant has a frameshift mutation;

[0051] (D) Agarose gel electrophoresis confirmed the genomic mutations of the SH2E mutant;

[0052] (E) Detection of SH2E messenger RNA transcription levels with normal nucleotide sequence length in mutant embryonic tissues;

[0053] (F) Detection of SH2E messenger RNA transcription levels with normal nucleotide sequence length in the heart tissue of mutant zebrafish;

[0054] (G) Pericardial cavity morphology in SH2E mutant embryos;

[0055] (H) Measurement of pericardial cavity area in SH2E mutants;

[0056] (I) Hematoxylin-eosin staining of cardiac tissue sections of SH2E mutants and wild-type littermates;

[0057] (J) Atrial wall thickness measurements of SH2E mutants and wild-type littermate controls;

[0058] (K) Ventricular wall thickness measurements of SH2E mutants and wild-type littermate controls.

[0059] Figure 2 , Se-Met reduces oxidative stress in the pericardium, thereby alleviating cardiac development defects caused by SH2E gene defects;

[0060] (A) The reactive oxygen species fluorescent probe DCFH-DA was used to detect the content of reactive oxygen species in embryonic tissues of SH2E mutants and littermate controls after Se-Met treatment;

[0061] (B) Quantitative statistics of reactive oxygen species content in whole tissues of SH2E mutant and littermate control embryos after Se-Met treatment;

[0062] (C) Quantitative statistics of reactive oxygen species content in the pericardial cavity of SH2E mutant and littermate control embryos after Se-Met treatment;

[0063] (D) Correlation between the content of reactive oxygen species in the pericardial cavity and the pericardial cavity area of ​​SH2E mutant embryos after Se-Met treatment;

[0064] (E) Propidium iodide was used to detect cell apoptosis in embryonic tissues of SH2E mutants and littermate controls after Se-Met treatment;

[0065] (F) Number of apoptotic cells in whole tissues of SH2E mutant and littermate control embryos after Se-Met treatment;

[0066] (G) The number of apoptotic cells in the pericardial cavity of SH2E mutant and littermate control embryos after Se-Met treatment;

[0067] (H) Correlation between the number of apoptotic cells and the pericardial cavity area in SH2E mutant embryos after Se-Met treatment.

[0068] Figure 3 The NF-κB signaling pathway may be the downstream pathway of cardiac development defects caused by SH2E gene defects;

[0069] (A) The reactive oxygen species fluorescent probe DCFH-DA was used to detect the ROS content in the pericardial cavity of SH2E mutant embryos after treatment with PD184352, BAY11-7082, and AKTinhibitor VIII, respectively;

[0070] (B) Quantitative statistics of reactive oxygen species content in the pericardial cavity of SH2E mutant embryos after PD184352 treatment;

[0071] (C) Quantitative statistics of reactive oxygen species content in the pericardial cavity of SH2E mutant embryos after BAY11-7082 treatment;

[0072] (D) Quantitative statistics of the content of reactive oxygen species in the pericardial cavity of SH2E mutant embryos after treatment with AKT inhibitor VIII.

[0073] Figure 4 , combined with AKT inhibitor VIII, it can enhance the inhibitory effect of Se-Met on pericardial edema in zebrafish embryos;

[0074] (A) Compared with DMSO (0.5%), Se-Met (1.25 nM), and AKT inhibitor VIII (0.03 μM or 0.10 μM), simultaneous treatment with Se-Met (1.25 nM) and AKT inhibitor VIII (0.03 μM and 0.10 μM) significantly inhibited the pericardial edema in SH2E mutant embryos;

[0075] (B) Quantitative statistics of pericardial cavity area after SH2E mutant embryos were treated with DMSO (0.5%), Se-Met (1.25 nM), AKT inhibitor VIII (0.03 μM or 0.10 μM), and Se-Met (1.25 nM) and AKT inhibitor VIII (0.03 μM and 0.10 μM) simultaneously. DETAILED DESCRIPTION

[0076] After in-depth research, the inventors found that inhibitors that target the PI3K / AKT signaling pathway have a synergistic effect with L-selenomethionine, which can significantly alleviate cardiac developmental defects. The combined use of PI3K / AKT signaling pathway inhibitors and L-selenomethionine (Se-Met) has an extremely excellent effect on inhibiting cardiac developmental defects or resisting oxidative stress.

[0077] In the present invention, an animal model capable of presenting symptoms of cardiac developmental defects is constructed using zebrafish, the state of the animal model is stable and controllable, the disease symptoms are typical, and it is intuitive and easy to observe. In the present invention, the animal model is obtained by targeted downregulation of the SH2E gene in the zebrafish genome, and a target suitable for targeting and a suitable gene manipulation position (gRNA target) in the SH2E gene are found, which provides support for the successful establishment of the animal model. The present invention also provides an effective therapeutic drug obtained through screening: L-selenomethionine, its mode of action and its application.

[0078] Combination application of PI3K / AKT signaling pathway inhibitor and Se-Met

[0079] In the present invention, the "PI3K / AKT signaling pathway inhibitor" includes inhibitors of the activity or function of the PI3K / AKT signaling pathway, and also includes antagonists, inhibitors, blockers, blocking agents, etc. of pathway proteins, as long as they can downregulate the expression level of the PI3K / AKT signaling pathway and inhibit the activity or function of the PI3K / AKT signaling pathway. They can be compounds, small chemical molecules, and biological molecules. The biological molecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

[0080] The PI3K / AKT signaling pathway inhibitor can be a variety of substances that can reduce the activity of the PI3K / AKT signaling pathway, reduce the stability of the PI3K / AKT signaling pathway, down-regulate the expression of the PI3K / AKT signaling pathway, and reduce the effective action time of the PI3K / AKT signaling pathway. These substances can be used in the present invention as substances useful for down-regulating the PI3K / AKT signaling pathway, so as to alleviate or treat heart developmental defects, and / or resist oxidative stress. For example, the PI3K / AKT signaling pathway inhibitor can be: a compound, a nucleic acid inhibitor, a protein inhibitor, an antibody, a ligand, a nuclease, a nucleic acid binding molecule, etc., provided that it can down-regulate the expression of the PI3K / AKT signaling pathway or pathway proteins, and inhibit its activity or function. The nucleic acid inhibitor includes: shRNA, antisense nucleic acid, small interfering RNA, microRNA, which targets the coding gene or its transcript of the PI3K / AKT signaling pathway as an inhibitory or silencing target, or a construct that can express or form the shRNA, antisense nucleic acid, small interfering RNA, microRNA.

[0081] As a preferred embodiment of the present invention, the PI3K / AKT signaling pathway inhibitor is a small molecule compound that specifically inhibits the PI3K / AKT signaling pathway, including: AKT inhibitor VIII, LY294002, and Wortmannin. After a large number of screening and comparisons, the inventors found that the small molecule compound has a particularly ideal effect when used in combination with Se-Met.

[0082] In the present invention, the small molecule compound may be a compound in pure form, or a compound with a purity greater than 85% (preferably greater than 90%, such as greater than 95%, 98%, 99%).

[0083] The small molecule compound can be obtained by chemical synthesis if its chemical structure is known. The present invention also includes the precursor of the compound, and the "precursor" refers to the precursor of the compound that undergoes metabolism or chemical reaction in the patient's body to transform into the active compound after being taken by an appropriate method.

[0084] As another optional mode of the present invention, the PI3K / AKT signaling pathway inhibitor is an interfering molecule that specifically interferes with the expression of genes in the PI3K / AKT signaling pathway. RNA interference technology is a technology for silencing gene expression. The principle of RNA interference technology is that a longer double-stranded RNA is cut and processed by a specific nuclease Dicer into a 21-23nt small interfering RNA consisting of a sense and antisense strand. The small interfering RNA then forms a silencing complex (RNA-induced silencing complex, RISC) and unwinds into a single strand. The antisense strand guides the silencing complex to specifically bind to the target mRNA through base pairing, causing the mRNA to decompose. Small hairpin RNA (short hairpin RNA, shRNA) is an RNA sequence that forms a sharp turn structure and can silence genes via RNA interference. The interfering molecule that specifically interferes with the expression of genes in the PI3K / AKT signaling pathway can be an shRNA molecule targeting the PI3K / AKT signaling pathway, or it can be an siRNA molecule targeting the PI3K / AKT signaling pathway.

[0085] As another optional mode of the present invention, the PI3K / AKT signaling pathway inhibitor is an inhibitor targeting mutation, gene editing or gene recombination of PI3K / AKT signaling pathway genes. As a more specific embodiment, by any of the above methods, the PI3K / AKT signaling pathway is converted into its mutant so that it no longer works. As a more specific embodiment, the CRISPR / Cas9 system is used for gene editing. Suitable sgRNA target sites will bring higher gene editing efficiency, so before embarking on gene editing, suitable target sites can be designed and found.

[0086] The present invention also includes isomers, solvates, precursors, or salts thereof of the compound Se-Met.

[0087] The present invention provides a method for combined drug administration, comprising a method for combining an inhibitor targeting a PI3K / AKT signaling pathway with Se-Met.

[0088] When administering, the PI3K / AKT signaling pathway inhibitor can be used to downregulate the expression or activity of the PI3K / AKT signaling pathway first, and then Se-Met can be used to inhibit it; or both can be performed simultaneously. It should be understood that a variety of administration methods are included in the present invention.

[0089] The present invention also provides a mixture of small molecule compounds, comprising: a small molecule compound AKT inhibitor VIII that specifically inhibits the PI3K / AKT signaling pathway and Se-Met as active components. Preferably, in the mixture, the mass ratio of the small molecule compound AKT inhibitor VIII that specifically inhibits the PI3K / AKT signaling pathway to the Se-Met is: (8-500):1; preferably (10-300):1; more preferably (15-200):1.

[0090] The present invention provides a pharmaceutical composition comprising: (a) an effective amount of a small molecule compound that specifically inhibits the PI3K / AKT signaling pathway; (b) an effective amount of Se-Met; and (c) a pharmaceutically acceptable carrier or excipient.

[0091] In the present invention, the term "comprising" means that various components can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting mainly of" and "consisting of" are included in the term "comprising".

[0092] In the present invention, "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio.

[0093] In the present invention, "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or excipient used to deliver the active ingredient of the present invention to animals or humans. "Pharmaceutically acceptable carrier" can be liquid or solid.

[0094] The pharmaceutical composition or mixture of the present invention can be prepared into any conventional preparation form by conventional methods. The dosage form can be various, as long as it can effectively allow the active ingredient to reach the body of a mammal. For example, it can be selected from: injection, infusion, tablet, capsule, pill. The active ingredient can be present in a suitable solid or liquid carrier or diluent.

[0095] The mixture or pharmaceutical composition of the small molecule compound that specifically inhibits the PI3K / AKT signaling pathway and Se-Met of the present invention can also be stored in a sterilized device suitable for injection or infusion. Generally, in the pharmaceutical composition of the present invention, the small molecule compound AKT inhibitor VIII that specifically inhibits the PI3K / AKT signaling pathway and Se-Met as active ingredients can account for 0.01-20% of the total weight of the pharmaceutical composition, and the rest can be a pharmaceutically acceptable carrier.

[0096] The effective dose of the small molecule compound that specifically inhibits the PI3K / AKT signaling pathway and Se-Met used may vary depending on the mode of administration and the severity of the disease to be treated. When necessary, the small molecule compound AKT inhibitor VIII and Se-Met that specifically inhibits the PI3K / AKT signaling pathway may also be administered in combination with other active ingredients or drugs.

[0097] The present invention also includes isomers, solvates, precursors, or pharmaceutically acceptable salts thereof of L-selenomethionine, as long as they also have the same or substantially the same function as L-selenomethionine. The "pharmaceutically acceptable salt" refers to a salt formed by the reaction of a compound with an inorganic acid, an organic acid, an alkali metal or an alkaline earth metal. These salts include (but are not limited to): (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium or magnesium) in the form of esters, carbamates, or other conventional "prodrugs". The compound has one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereoisomer mixtures, cis or trans isomers.

[0098] The "precursor of the compound" refers to a compound, or a salt or solution composed of L-selenomethionine, which is converted into L-selenomethionine by metabolism or chemical reaction in the patient's body after being taken by an appropriate method.

[0099] Those skilled in the art should understand that, after knowing the structure of the compound of the present invention, the compound of the present invention can be obtained by a variety of methods well known in the art, using well-known raw materials, such as chemical synthesis or extraction from organisms (such as animals or plants) or modification based on extraction, and these methods are all included in the present invention.

[0100] The compounds of the present invention can be synthesized by known methods; the synthesized compounds can be further purified by column chromatography, high performance liquid chromatography, etc. In addition, the compounds of the present invention can also be obtained by commercial means.

[0101] The present invention also provides a composition (eg, a pharmaceutical composition) containing an effective amount of L-selenomethionine, or its isomers, solvates, and precursors.

[0102] In the present invention, the term “contains”, “has” or “includes” includes “comprises”, “mainly consists of”, “substantially consists of”, and “consisting of”; “mainly consists of”, “substantially consists of” and “consisting of” are subordinate concepts of “contains”, “has” or “includes”.

[0103] As a preferred embodiment, the composition is a pharmaceutical composition, which further contains a pharmaceutically acceptable salt and / or a pharmaceutically acceptable carrier or excipient.

[0104] As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. A "pharmaceutically acceptable carrier" is a pharmaceutically or food-acceptable solvent, suspending agent, or excipient for delivering the compounds of the present invention to animals or humans. The carrier can be a liquid or a solid.

[0105] In the present invention, the pharmaceutical composition contains 0.001-50% of L-selenomethionine or a pharmaceutically acceptable salt thereof by weight. Preferably, the pharmaceutical composition contains 0.05-30% of L-selenomethionine or a pharmaceutically acceptable salt thereof by weight; more preferably, the pharmaceutical composition contains 0.01-20% of L-selenomethionine or a pharmaceutically acceptable salt thereof by weight. It should be understood by those skilled in the art that other weight ratios are also feasible according to actual clinical needs or drug design methods in pharmacy.

[0106] The dosage form of the pharmaceutical composition of the present invention can be varied, as long as it is a dosage form that can effectively enable the active ingredient to reach the mammalian body. For example, it can be selected from: gel, aerosol, tablet, capsule, powder, granule, syrup, solution, or suspension. According to the type of disease treated by the compound of the present invention, those skilled in the art can choose a dosage form that is convenient to use. From the standpoint of easy preparation and administration, the preferred pharmaceutical composition is a solid composition, especially a tablet and a solid-filled or liquid-filled capsule. The compound of the present invention or its pharmaceutical composition can also be stored in a sterile apparatus suitable for injection or instillation. The effective dosage of L-selenomethionine as an active ingredient can vary with the mode of administration and the severity of the disease to be treated.

[0107] The present invention also provides a medicine kit for treating tumors, wherein the medicine kit comprises: a container 1, and a PI3K / AKT signaling pathway inhibitor AKT inhibitor VIII placed in the container 1; and a container 2 and Se-Met placed in the container 2.

[0108] The PI3K / AKT signaling pathway inhibitor and Se-Met are both small molecule compounds, so the drug kit may also contain a mixture of the PI3K / AKT signaling pathway inhibitor and Se-Met, wherein the contents of the PI3K / AKT signaling pathway inhibitor and Se-Met are as described above.

[0109] In addition, the medicine box may also contain some auxiliary medication materials, such as injection syringes, etc.

[0110] In addition, the medicine kit may also contain instructions for use, explaining the method of using the combined drug method of the present invention to alleviate or treat cardiac developmental defects and / or resist oxidative stress.

[0111] The technical solution of the present invention is to use a PI3K / AKT signaling pathway inhibitor and Se-Met in combination. Although when used alone, the actual effects of the two drugs alone are negligible and require high doses. However, in the present invention, it is found that the combined use of the two has an excellent effect, can significantly reduce the dosage of the two (that is, only a low amount of drugs is needed to achieve excellent effects), and plays an ideal role in alleviating heart developmental defects, which greatly exceeds the expectations of general personnel / physicians in this field.

[0112] Preparation and application of animal models

[0113] The inventors are committed to the establishment of an animal model of cardiac developmental defects. In the early research work, they conducted multi-target and multi-faceted analyses, and finally located the SH2E gene in the zebrafish genome as a target, targeted downregulating the target, and obtaining an animal model of cardiac developmental defects. In a preferred embodiment, the inventors further combined CRISPR / Cas technology to prepare an animal model.

[0114] As used in the present invention, the "gRNA target" refers to the target region in the gene of interest in the present invention that is suitable for gene editing operations. For example, regulation can be implemented by introducing exogenous gene editing reagents (such as gRNA and Cas mRNA) targeting the target.

[0115] As used in the present invention, "animal" is preferably zebrafish.

[0116] As used in the present invention, "exogenous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or refers to the relationship between a protein / gene and a host. For example, although the host itself may also contain the corresponding gene or produce the corresponding protein, when a synthetic / recombinant gene / protein is introduced into a host cell by a genetic engineering method, it is "exogenous" with respect to the host cell. The "exogenous" includes "heterologous". A "heterologous" nucleic acid or protein is usually not present in the host cell itself.

[0117] The present invention discloses for the first time that the SH2E gene in the zebrafish genome is used as a regulatory target for down-regulation.

[0118] Downregulation of a gene includes "deletion / deletion" or "inactivation" or "inhibition", meaning that the enzyme or protein encoded by the gene or coding region is not produced, or is produced in an inactive form in the host cell, or is produced in the host cell at a level lower than that found in the wild-type form of the host cell under the same or similar growth conditions. This can be achieved, for example (but may not be limited to), by one or more of the following methods: homologous recombination, RNA interference-based technology, ZFN and TALEN, CRISPR / Cas system, etc.

[0119] As a preferred method of the present invention, the CRISPR / Cas (such as Cas9) system can be used for targeted gene editing, thereby transforming the SH2E gene in the target area. Common knockout methods include: co-transferring gRNA or a nucleic acid that can form the gRNA, Cas mRNA or a nucleic acid that can form the Cas mRNA into the target area or target cell. After determining the target site, known methods can be used to introduce gRNA and Cas into the cell.

[0120] In a specific embodiment, the CRISPR / Cas is CRISPR / Cas9 technology, which includes: introducing a specific gRNA targeting the SH2E gene in zebrafish, and simultaneously introducing Cas9 mRNA to enable smooth gene editing.

[0121] After using the CRISPR / Cas9 system for gene editing as the basis for constructing an animal model, a suitable gRNA target site will bring higher gene editing efficiency. In the preferred embodiment of the present invention, a preferred target site is designed and found, and gRNA is designed based on this. The inventors have repeatedly analyzed and studied the SH2E gene and experimentally verified it, and determined that in the preferred scheme, targeted mutation is performed on exon 1 of the SH2E gene. Preferably, the sequence targeted to the SH2E gene is shown in SEQID NO:2 and / or SEQ ID NO:3. Preferably, after treatment by the CRISPR / Cas (such as Cas9) method, the protein encoded by the SH2E gene mutates at position 59 and / or position 82, so that the translation of the SH2E protein is terminated prematurely.

[0122] After the target is determined, gRNA or a nucleic acid capable of forming the gRNA, Cas9 mRNA or a nucleic acid capable of forming the Cas9 mRNA are co-transferred into an animal fertilized egg to obtain a gene-edited animal. As an option, the nucleic acid capable of forming the gRNA is a nucleic acid construct or an expression vector, or the nucleic acid capable of forming the Cas9 mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into cells to form active gRNA and Cas9 mRNA in the cells. In addition, Cas9 mRNA and gRNA can also be obtained by in vitro transcription.

[0123] As an optional method, homologous recombination can be used to specifically target the SH2E gene to cause expression defects or lack of expression. Cre and loxp methods can also be used to selectively knock out related genes in the cell genome, reduce expression or inactivate them.

[0124] As an alternative, RNAi can be applied to the downregulation or removal (such as siRNA, shRNA, miRNA and other interfering RNA molecules) of the SH2E gene. According to the information of the SH2E gene provided in the present invention, such interfering RNA molecules can be prepared. The interfering RNA can be delivered into the cell by using an appropriate transfection reagent, or can also be delivered into the cell by using a variety of techniques known in the art.

[0125] The animal model constructed by the present invention can be used for screening and testing of specific drugs. In drug screening, candidate drugs or therapeutic agents refer to substances that are known to have certain pharmacological activities or are being tested and may have certain pharmacological activities, including but not limited to nucleic acids, proteins, carbohydrates, chemically synthesized small molecules or macromolecular compounds, cells, etc. The administration of candidate drugs or therapeutic agents can be oral, intravenous, intraperitoneal, subcutaneous, spinal or direct intracerebral injection.

[0126] The animal model constructed by the present invention can be used as a powerful tool for scientific research and new drug evaluation.

[0127] Those skilled in the art are aware that it is difficult to obtain an animal model showing typical disease symptoms due to the complexity of the body's genes, the impact of multiple signal pathways on the disease, the body's own compensation or repair mechanism, and other factors. The present invention overcomes such technical difficulties through optimized design.

[0128] In terms of disease mechanism research, the animal model that can stably present heart development defects constructed by the present invention is used to conduct disease mechanism research, explore the key factors that lead to common inhibition to heart development defects, and explore the intermediate mechanism to prevent or delay the development of such diseases. The model system of the present invention helps to better understand heart development defects and is used to explore / identify candidate drugs / therapeutics that can prevent, delay or reverse the disease process.

[0129] In terms of preclinical drug in vivo testing, the animal model that can stably present cardiac development defects constructed by the present invention is expected to be used for preclinical drug metabolism, toxicity and efficacy testing. The animal model is physiologically close to the human body and supports long-term sampling, detection and tracking, which is conducive to the development of new drugs. In the present invention, the type of candidate drug used for drug testing is not particularly limited and can be obtained from a variety of sources, including synthetic or natural compound libraries. For example, there are many ways to randomly and directed synthesize a variety of organic compounds and biomolecules, including expressing random oligonucleotides and oligopeptides; or, natural compound libraries in the form of bacterial, fungal, plant and animal extracts can be obtained or easily produced. In addition, libraries and compounds produced by natural or synthetic methods can be easily modified by conventional chemical, physical and biochemical methods, and can be used to produce combinatorial libraries. Known pharmacological agents can be subjected to directed or random chemical modifications (such as acylation, alkylation, esterification, amidation, etc.) to produce structural analogs.

[0130] The method for preparing the animal model of the present invention is simple to operate, the obtained animal model has good stability, can well simulate the heart defect phenotype of human / animal body, the disease symptoms caused by it are very typical, and the phenotypic changes are easy to observe. The animal model of the present invention provides a new approach for the study of the pathogenic mechanism of heart development defects, drug screening and clinical treatment.

[0131] Based on the method of the present invention, the present invention also provides a kit for preparing an animal model of heart developmental defect disease, the kit comprising: gRNA for targeting downregulating the SH2E gene based on the CRISPR / Cas system; preferably, the gRNA targets exon 1 of the SH2E gene; more preferably, the sequence targeting the SH2E gene is shown in SEQ ID NO:2 and / or SEQ ID NO:3; and, mRNA of the Cas protein or DNA that can form the mRNA.

[0132] The kit may also include instructions for preparing the animal model of the present invention to facilitate use by those skilled in the art.

[0133] Drug Screening

[0134] After obtaining the animal model of the present invention, the model can be used to screen for substances of interest that can (or potentially can): alleviate or treat cardiac developmental defects. After screening, truly useful drugs can be found from the substances of interest.

[0135] Therefore, the present invention also provides a method for screening potential substances, the method comprising: (1) preparing an animal model of cardiac developmental defects using the method or kit described above; (2) administering a candidate substance to the animal model of (1) to observe whether the candidate substance has an alleviating or therapeutic effect on the cardiac developmental defect; if it is observed that the symptoms of the cardiac developmental defect in the animal model are alleviated, the candidate substance is a substance that alleviates or treats the cardiac developmental defect.

[0136] According to the typical heart development defect symptoms presented by the animal model of the present invention, the observation of whether the candidate substance has an alleviating or therapeutic effect on heart development defects includes (but is not limited to) analyzing the following phenotypes: SH2E expression level, pericardial cavity edema, pericardial cavity area, atrial wall thickness, ventricular wall thickness, oxidative stress level, ROS content or ventricular cell apoptosis; wherein, if the decrease in SH2E expression level, pericardial cavity edema, increase in pericardial cavity area, decrease in atrial wall thickness, decrease in ventricular wall thickness, increase in oxidative stress level, increase in ROS content or increase in ventricular cell apoptosis are reversed, then the candidate substance is a substance that alleviates or treats heart development defects.

[0137] In a preferred embodiment of the present invention, during screening, a control group may be set up in order to more easily observe changes in the symptoms of cardiac developmental defects. The control group may be the animal model without the addition of the candidate substance.

[0138] As a preferred embodiment of the present invention, the method further comprises: conducting further cell experiments and / or animal experiments and / or clinical experiments on the obtained potential substances to further select and determine truly useful substances.

[0139] On the other hand, the present invention also provides potential substances of interest obtained by the screening method. These initially screened substances can constitute a screening library, so that safe and relatively ideal substances that can truly alleviate or treat heart developmental defects can be screened out.

[0140] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0141] Materials and methods

[0142] gRNA design

[0143] The zebrafish SH2E genome sequence is as follows ( Underlined codons The corresponding sites are the 59th and 82nd positions of the protein encoded by this gene):

[0144] CATCTAGACAGTCGCCAGGAGTGGATATACCGGGAATGGGATCTTCAGCGAGAAAAGAAAAACTTCAAATTCTTCCGCGTTTATGCCCTTCAGAGGCTCCTCTTTATCTTGCGGGAATAATTGGGAGCAAACTAAAACAAGACAATACATTCCA ATGTGAAGAATCGTTGAAAGTGTAAGAAGTTTCCTGAAAAGAAATACGAATGGGATAAATTCTGTAAACAAGATTATATAAAAGGCATTTTAATTTTCGGAACCTTTCCCTAAATCAATTGTTTTTGTTCAATTAAATAAATTTATTATTATTAGCC TATTGTTTTAAACAAACCGCACGTTAAAGTGCAGGTAAAACATCAATAATAGGGGCCACTTTGAGTGAAATTCAAAAGTTTTCTCCAGCAGTTGCGCGTGTGTATGGAGAGTGGCGCAGACACCTGTGCGTATTCGTCATGGCGAAGTGGTTTA AAGATTTTCCAACTAGTTTGAAGACTGGATCCGATAGGATCCGTTCAGCTTCAGAATCGGGAACTCAGTCCCGCCCCAAGCCGGTTCTGACAGTCGGTACTGGTTCCAAGGGGAGTCTGCGTAAAAATTCTGGAGCGGAAAACGGTGGCGGTGGTGG CGG CGTGGGCTCGTTGCTTCACGGCAGAAATCGTAAAAATTCTGCCATCGAGTTAGGACGGAACCACTCC AGC

[0145] TAGATTCTTGCACAAGCTCCACTAATGAAATTGTTTGTCATATTATGCAAGATCTCAGTCAATAGCAC

[0146] AATAGGCCAGTAATTGCAAACATAATTGTGTCCTTATTTGCAGCTCTTTTATTGAGTTTGAGCAAAAATT

[0147] GTGAATTTACCGCAAAATGACCCAAATTATTAAATTGTCTTAATCGTACTATTCCATAATTATTCT

[0148] GAGTTTGCATCTCAAAATTGCAAGTCAATGTGTAACTCTGTCTTTATTTATTTAACTATATTTTTAATAA

[0149] CAAGCAAGTTTTTCAGTAATGATTAAATGAAACAGTTTATGCATTTGGATGCAGCATACAGCATTTC

[0150] AGGGGTTTAAAAAACCCAAATGCTTTAAAGATTGCGTTTTTAAAAAATGTACCACCTTTATCTATGAA

[0151] AATATGATTTAHELPAAATGTCAGAGGCAGGGCCAGACATTTTGCTGTTTCTGCAGAGAATTTTATA

[0152] AAACAATCTGCGGACTTTGCTGATTATTAGAGTATCATAACTAAAACATGGTAAACAAAGCAAG

[0153] TCTCTCATATATATACTTATAAAGTGGATTGTAAAATAAATCA

[0154] ATGAACATTCATTACTTATTTATTTATTATHELPTATTATTTGTGAATGAGTTTAAAAACTTATATA

[0155] ATTAAAATGTCCATACACAGCAAATTCATCAGAGTTAAATTCTCGGTGTTGAAGCATTTGAGAGTAAAG

[0156] TGTTGACGTTAAAAGAGTTAGATTTTGATAAATGAATATAATAAGAGTTAGAATTGATTTTATTCAGTGCG

[0157] AAATCAAGGAGTTATCTTTTCAACACTTGGTGGTGTTATTTCCAACATCCGGGAATTCATGCAGCCCCAG

[0158] TCACTGAAGAATTTTCCAGACGCCATTTTCCATCTGAGGTTTAGAACAGCAACTGGTGAGTCAAACTAC

[0159] CTAAATGTTGGTATTTTACTGACTTTCTTTTAGGAAATACAGTTGTAAACATGTTGTTAAGTTAATAACT

[0160] TTAGAATGGAGTGACAATTTTAAACTTCTGCGGTTCATTCATGGTCGTTTGTGTATCTAGCTTAACTGCA

[0161] TTACTATTGACTTCTTTTCAAGAATGTTTTTATATATGCTCACGCATACATAGTGCATAAAAACATCAAA

[0162] TGTACATGTTCAACACATTTCTGTCACGCTTAATGCCATTTTGTGCGTTGTTACCCATTTGTAAAAATAAA

[0163] ATCGACACTTCTCCTTTAATTCCAAGCAAACTAGCGAGGGAATCCCTGGAGCCGCCTAACGTTAGCCTA

[0164] CTCTGCCCGGATGCTAACGGCAACACAGGACGGTTTCCATAAGCTCTGGAGAGGTTTCTAAAACATATCT

[0165] GGTGAGTAAATGAACATATGCTTACTTGTAAAAGGCTTCCTGACTGAAACAAATGATTGTTTGTTATTCG

[0166] TGTACGTTAATTTGGTTATTTTAAACACCGCGGCCCTTTTAAACTGGTTCATTCATGGCCGTCCATATAC

[0167] CGTTAGTCCATAACTTAGACTCGTGTGATAACGTACGCTTGAATTAACGTTATATTAAGTGTTGGCAACC

[0168] ATTAAAGTCGGAATGTTAACATTAATGTCTTTAAATGACCGCGTTTGCACTTTATCAGACATTTAACGTT

[0169] ACAGAAGTCTCCCGGAAGTTGCGGGACTAACGTTAACGTTATCCCGCAAATGCATAGAGACTCCCAGAT

[0170] GCCCAAGTAGATGCCCGCAAATGAATGATAATCTCCCGGAAATCGCGCGTCTCCCGCCCGGTCATTAAA

[0171] CACTGCACGCCCCTCTCCACCGCATCCCTCCCAGCTCTTCAGGTACGTCGCGCGCAAGTCACCCCCACCG

[0172] CTCTTCAGGTACGTCGCGCGCAACTCATCCCATTGCTCTTTAGGTACTCATCTCTCCCGCTACCTCCCGC

[0173] AAATCAACTCTGTATCCCCTGAAAATGACTTTTCCCAACTCGGGATGTCTGACGTTAGTAAGTTAGGCTA

[0174] TTTCTGTTGTCAGGAACATCTTAGTCAAGCTTTTTCTCCCGCATGATATTGTGCTTAAAACTATAGGCTA

[0175] AACTTAGCATGTAACGTTACTGTACACAACATTACCGTTTTATTAGTGCGTTGTAAAAAGCCACCTGTCT

[0176] GTAAAATTAATCAACTGATTCATATTTGAGCAGCCTAATGATGATACAGGTTTGATTTGTAGATTTATTA

[0177] TCAATAATCAACATCTGAATCAAAAGATATGTGAAAATGTGTGAGGCATACTTTGTGATGTTCATCTCTC

[0178] TTTTTTATCTGAAGGTTATGCATATGTGTTAGATACTAATGTGTTGTTGTTTTTTAACAGCCACAGTTCTC

[0179] TGTACAATAACACAAGACCCTGTGACGGTGGCTGATTAGAGACGGATGGTGTATTTCCAGAGTTGGTGA

[0180] AATGACCAAGCACACCTTCTTGGTCATATGTGGTGATAATAATGAAGGTATGTTTGATAAAGTTCTGTAT

[0181] TTGTTTCACATAAGCTGAGAGGGTATTAGAGCATTAGAGAATAACAAATGACATGAAGGTGATAAGTTT

[0182] TGTATTGTGGGTGAACTGTTACTTAAATAATGTTTTGCTCTTTGCTCTGCACTTTTTCTCTTCTGCTTATCC

[0183] TAATATTTTAGATTATATTTGGACCTGAAACATCTGCTAGACTCCAGGAAAGGTGGCGTCAATACTGGA

[0184] CCTGAAAGGGCCTAACAAGTTGAATCTGGTAAAGAAACACGCACACACACACACACACACACACACAC

[0185] GCACGCACATGCTTACAGAAAACTGTAAGTTTATGAATATTTTTTACACTGTGGCCAATATTTTGGGGAA

[0186] GTCAGCATCACTAAATGATATAAAAAAAATTTTAAGCTCCTCTAAATTTTGCTCCAAGTGTTGTGTTAAA

[0187] TTCAGGATTCTGGACAGACAGCTGCCTGTTTTTCCAGTTCTAGTCATAGTGGTTAACCTAATTGTTTTTG

[0188] GAGATCCAACAGTGATGTATGTGTTTTTTCTACTTTTCCATATCCTGTCACCTCAACCAGCTGGGAGAGT

[0189] CAAGTTGTGGATCATCATGAAGATTTTCAAAAAGGTGAGTTTAGATTTCTTGAAAGTTTTATTTACTTTAA

[0190] ATGTTCTTTAAATATATCATAGAATTTGATTGCAATTTGTTTTCAAACATTTTTCTTAGTCATGCTGGAG

[0191] TCTTGAAGAGTTCTTAGATGAGCATCACCCTACAGTATATCCGTGCATCAGGAACCACCAGACGAGCGA

[0192] TCAGCAGCTACTACTGTACATCGTCATGGATAAAGGTCATCCTGTGGCTGGGAAGCACATCTTTCAC

[0193] AGGACATGCTTGATGAGATTTCCTAGCTCCAGTTTGTTTTCACACAAGTTTACAGCATTTATACCTTCAT

[0194] TTTAACTGCTGTGTTTGATATGGAGGACCAAGGAAACACCAGAAGTGAAAGATTTGCACGCCAAGTTGT

[0195] TTAAAATGTAATTTCAGAAGAAAACTATGCGTTCAGAAGAAAACTATGAGTGACGATATATTGAACTTT

[0196] CAAATTGCTCTGATGAAACATCAAGTTTGGACATCATATTTTGTTGATACTGTCTGTACTAATAAAACAT

[0197] TTAAAGCATATTTGACATTGTTGCATTTTAAAAACTGAATGAATTGTTGAAGTTGTGAATGTTTTCAAAT

[0198] AAAATGTTTTTTATTTTGTAATTTACAGTCTATTTGACCTTTTTACCATATTTACACTCAAGAGAGTTAAA

[0199] TAAAATAACAAATTATTACACCAGGTGGTGTTAAATATAGTAACATTTTTTAACTCTGCCCCTGAGTTAAA

[0200] ATTAACCCTTACTTCGGTGTCAATGTGCCAACCCTTTTGAAAGTGTTGATTTAAGTCTGTTTTGAATGGA

[0201] CCCCATGAGACACTTTCAAAGTGTTGAAATTAACACCCATAGAGTTGTTTTGGGTCCCCATATTTTGCTG

[0202] TGTACTTTTTACTTTTAAGTCAATGCATATACAGTTGAAGTCAGTTATAAGCCCCCTTTTGATTGTTTAAC

[0203] AGGGCAAGGACATTTTCACAGTATGTCTGAATTTTTTAATTTGTGGAGAAAGTCTTAATTGTTTTATTT

[0204] CAGCTAGAATAAAAGCAGTTTTTAATTTTTAAGCCCATTTTAAGGTCAAAATTATTAGCCCCTTTAAGC

[0205] TACATTTATTTTTCTATAGTCTACAGAACAAACTATCATTATGCAATAACTTGCCTAATTACCCTAACCT

[0206] GTCTAGTTAATCTAATTAACCTAGTTACGCCTTTAAATGTCACTTTAAGCTGTATAGAAGTGTCTTGAAA

[0207] AAATATCTAGTCAAATTTTATTTACTGTTATCATGGCAAAGATAAAATAAGTCAGTTATTAGAAAAGAGTT

[0208] ATCAAAACTATTATGTTTAGAAATGTGCTGAAAAAACAGCAGTATCATACATTATGTCCACGTTTCTTTC

[0209] TTTTCTTTCTTTCTTTCTTTCTCAATTTCCAGTAAATCTTACATAAGTTCTAAAACGTATTTAGTGGTATATT

[0210] AAGAAATTTAATATAAGTTTTTAAGATGTTTCTAAAATACTATCACACTACTTTCTAGACAGGGAAAAC

[0211] GTATTTGAATGTGTGAAGTTTCAGACAAGTTCCAAATATATTTTTCTATGACAAATCTGTTATAAATG

[0212] ACAACAAACTAAAACAAACGTCAAAGATCAGATGTACAAAACAAATAATGGAAAATGATTTTAGCCTT

[0213] AAATACTCTATAAATAAATAGTAGTGAATCTTTTTTTAGATGTGATTTCATAGTTGGTCCCAGAGAGGGT

[0214] TCATTTGCAACTATTTCCCATTACTGAGAGTTTATTACACAAAAGATACAAATGTTGCAATACTGAAAAA

[0215] TGTCTGAATTCAGATAAACCAACTTGCAAACTACAAGAAAAACTCTTAAATGTAAGATATAAAACTTCC

[0216] AGTTTTATTCAGAGGCTGAAAGAAACTTACCACAAACCAAACCTTAAATAATAAAGTAGTTACCGT

[0217] TTTAATAATAGAATATTCACATGTCATTTTCTGTCTTGTTTAAACTGTATAAGGAGTGTTGTATCATAACT

[0218] GTGAAAAAAACATAAAAAACATACTTTTTTGCTTAAACAGATTAAAATAACTAAATAAAAGATAATATG

[0219] ATTCATACAGTATTTGTGCTTTTTAATTGAACTTTTCTGTTCAAAACATCTCAACTGTCAGTGAATATCGT

[0220] TCATAAACTTTCCCTCAACGTGACGCAGGGCAAAAGGCAGCTACATAGTTGCTAATAGTGTAAATAAGG

[0221] CTTTTAAACTCCCCAAGCTTCTTGAAAACTTTTCTGAGGTTGCTACGCACAGATGAGTGGGCTTCAAAA

[0222] GATTCACCATTTTCTAACAATTCAAATGTCTTGCTGCAATCTTTCTTTCTGTTTTTATAAATTTTAAGTA

[0223] TCATACCTCTATATTTATGAGATATGAGTCATATTTCTCTTATATTTCTCATATTTGTGTGTGTGTGTGTG

[0224] TGTAATATGCCAAAAATGCTAATAATCTCAAACTCTAATATATATAATTTAACTAAAGAGGCTGTATGC

[0225] TTTTAGGCCTGAACACATACTGTATCAATGATATCGTGTTTAATTCATATTCTCATCTCACTGTGAACAG

[0226] ATCATCGTAGAGGATTATGCCGACCCATTTGATGCTGAGAAAACCAGAGAACAGAAAGAAGCTGAAAG

[0227] AGAAGGAGAGAACGATGGCTACATGGAGCCTTATGATGCCCAGCTCATTATTACAGGTACACTGTTTTT

[0228] TTCTAGTCACCCTAACGCTTTCTCAAAAGCAGCATTTCTTGGTATATCAAGCTGATTTTCAGGGATTTTG

[0229] AAAGGTATGAGAAATCACAGCTCCCCTCATATGGTGGTTTCCACAGTGACAGTGTTGTGCTGGGACAAT

[0230] GGGTGTAGTTTCCTCTTTCTGCTTCCTGTTTCCTGTCTGTGTTTCCTTTGCCCCATTCTGTGGACAGGAAG

[0231] TTGACCCCTGCCGTCCCCCTTAGAAAAGCCTGGCTATGTGAGAAAGACCCCTCCAACACCTCCTACTCCC

[0232] CCCAATCACTTCCCAGAACTCAGGAAGTGGCCTGCTCAGCCAGCCAATCGCAGACGAGCTTTGACTTTC

[0233] CCCTGGTATGACCATTGTTTTCACACACAATAGGCCAGACACAAAGTGACAGCAAAGAAACAGCAAGT

[0234] GAGGGGAGAACGATGGAGCTCAGGGTACATTTTTTTGTTATTGATGATGAACTTTAAATCATGAGGATT

[0235] GAATCGGTGCATAAATCGAGAAACGCAGAGAAATTTTGTCACTACTGACTGAAAATGCAGTAATAATTT

[0236] CTATGCGTGATGATTTTTTGTTTTAAAAGGACAAATATTACATCTTAAGTTTGAATTCATTAAAAAAATAA

[0237] TTCCTTTATGTGTGCAAGTTGTCCTGAATTTCTTACTGCGTATCTGAGTTGAAAACTTATTTAAACATT

[0238] TACTTTAAATGTAAAATCTAAATATTATTTTAAAAACAGTTCACCCTTGTGTACGGTTCAAATTTACTAC

[0239] CCTTTTTTAATGTTAGGGATGAAAACATTAACTGAATTAAACTGCTGTAAAAGTGCATCAGATAATTTTT

[0240] TTTTTGCATATAAATCTGTTAATCTACCTCATCAAAACTACTAAATTGTTCAGAAAATTACAGGATTGTA

[0241] AAAACACACTACTACTACTACTATTACCACTACTACTACTGCTACTACTACTACTACTACTACTGCTACTAGT

[0242] ACTACTTCTACTACTACTACTACTACTACTACTACTAATAATAATAATAATAATGCAAACCCAACAACTA

[0243] GTACCAGGTTGGACCTCCTTTTACTGCCATAATTCTTTGTGGCATGGATTCAATAAAACATTCATCAGAA

[0244] ATTTTTTCCACACTGGCATGATAAAATTATGCTGTTGCTGCAGATTTGTCACCTACATACTGTATCTAC

[0245] GACGTGAATCTCCCATTTCAGCACATTCCATAATTGCTCTATTGCAGGGGTGTCCCAACTCGGTCCTGGA

[0246] GGGCCGGTGTCCTGCATACTTTAGGTCCAACTTCCTTCAACACACCTGCCTGGAAGTTTCTAGTATACCT

[0247] AAAAAGAGCGTGATTAGCTGGGTTCAGGTGTGTTTATTTGGGGTTGGAACTATAATAATTGCAGGTCACCGA

[0248] CCCTCCAGGAGCGTGTTTGGACACCCATGCTCTATTGGGTTGAGATCTTTAATGCAAGGCAGAATGGAT

[0249] TTTATTTCATTGTGAAAATGTAAAAAAAAAATAGACTCATTTAGATTTTTTTATTAATGTTTGTTTATGGT

[0250] CAATTCTGACATTTACATTACAGATGTTGCAGCAGAAAATGAGATGCATCAGTTTATGCAAATTTTTTAA

[0251] TCTTTCTATTGGCCGTGTGGTCTATTGGTAAGCCTGTGTGAATTGTAGCCTCTGTCGTCTTTTCTTCTTAG

[0252] CTGACAGGAATGTTGCCAGTGTTTTCTTTTGCTTGAAGGTTCAGTGTGTTGTGTGTTCAGAGATGCTCTTT

[0253] TTTTAAGACATTAGATTTAATGAGTCGTTATTTCAGTAGTATCTGACTGAAGACTTGCGCATGCAGTCTC

[0254] AGACATGCAATCTCAGACACAAAGCTAGTGACATCCCTGTGAGGGGTAGGATTAGGAGTGGAGTTCGGT

[0255] GTGTGCATTAAAAGCATTGCATGCAGCTCAGTTTGCATCTGCACCCGATCTGCAGCAACACCCTTCACTT

[0256] AAAAATATATATTTTTGCTGCTTGTTCAAACTTATTTACAATGTGCAGAATTAACACAATTCCTGAGATT

[0257] TTGTTTTGGGACAACTTGTTTTATGTTTAATCTACCTAAAATTGTTAACAAAGGTGCAGTAGGTGATTGT

[0258] CTTCAGAAACATTTTTTTTCTGCTGGTTGAAAGTCTCTTCATATTCCAATAGTAATGATTAAAGTAAATG

[0259] ATTTAAAAGTGTTTATATGTATTTTTATATTCTGGGTAAGGCATAAAACTAAAAATGTTTACCCAATTAA

[0260] ATACTGTCAGGCCGGCATCTCCCATAATTCTGATCAAACTGTGAACAAATGCAGATTTGGGCTTTTGCGC

[0261] ATCTCTGTTCATGCATATCCACCATTAGCGTGTGCACGGCTAGATGAGTGCCATCCATTCACGACACATG

[0262] CACACGAGACTCACAGTCGAGGCAAAATCAAAATACTGAATTGAAACTTTTTAAAATCCTGAATCAATA

[0263] TTGGAGTGATCTTTGCACACTGGAGGAAGAAAGACACCATGACTGAAGTATTTCTGTTAGATAGGTAAT

[0264] GTTCTGTTTTAAAACTGTCTAAGCTTCACACAGAGCTGATGCAGATGTTGTTACAAATGGGTTATATGGC

[0265] GCCACTGAAATCTCCCGATCTCTGAAATCTCCCGGTGAAAGATTAATGTGACTATTTTCAGTTTCATAAG

[0266] TTCCTTTTACAGCATCAATAATGTGGATTTATATTTAGTTTAACAACTAAACATAAGTCATTAGCGCTAT

[0267] TCTGCCTAGCCTGCTCTACTAAGCTAAAGTTGCTTTTATTTTCACATTGGGTCATTTCTGAACGATGACA

[0268] GCCTGTTACGCGCTCCTGATTTTGTTGACCATGCTACTCTGAATATTGGCTCTGAAAAAGCATGTAAGTG

[0269] TGGCTTGGTAATAAGTGAAATTCCTGCGCGCTGCCAGGCCAATGACATGAACTAGTGGGTTGCCTGCTG

[0270] TCGTGTGTCGCGGTGTGTGCGGTCGCGATTTTGGAAGGCGTGGCTTTGAAACACAGTCCCTCATCCAAA

[0271] GTTATTATGCTAACCGATTAGCATTTTGGCAGATCACTTACTGCACCTTTAATCAATTAAAACATATTAA

[0272] TATTATCAACTACATTATTAACTTGATCAACTTGTGTTGGGACAACATGATTTGTGTGAACTCTTGAAAC

[0273] AATGGGAATGTTGACAGGTATGAAAAAATGGCCAGTGTTCTCTTGGTGTATGAGATAAATTATAAATGA

[0274] TTATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATA

[0275] TATATATATATATATATATATATATATATATATATATATATATATGTAATTC

[0276] TTTTAAAGTCAACTATTTAATTCATTTATATTTGCAGTATTTTATGAATTTGATAAAGAAAGGCTAGAG

[0277] ATTGACAAGAAACTAGAGAAGGATTGATGTTGACAGTTTGTTCTGCTGTTTAGCACATGGGTTATGCAC

[0278] TTGAAAGTGGAACATCTTAAGCTCCTTCTAAATTCTAAGTCACTTTTATGAACATTTAAGGTGATCTT

[0279] TTCCTACTTTTAATGAAGCTAAATATAGTTAAATCTGTCTTAGTAATAAAATGAAGCAAGTTACTTGCTG

[0280] CTCAGAGTGTTTTATGTAACAGTTTCATACCAATGTGCTATAATGATGTATGTGAAAAAAAACTAAGGTT

[0281] AAAAAACTAAATGTAGTTTAAAACTTGAGGTCAGATTTACATCTGTTCTGCAAAATTGCTCCACATG

[0282] GATTTTACATTATATATAAGTTGATCATGCAAATTTGCTGCCACAAACCAAGAGAAATTTGCATAGATTG

[0283] AAAGTGACTTTTGTGCTTATGCATCAATAAACTGTTGACAAATTGTTTTGTTTTGCCTGACAAATATTGT

[0284] GAGCAGACTAGCTCAATAGGGAGAGATTTAACAGATATCAAACACTAATTTTTTTTTATGAAAGATTTC

[0285] TCTCACATGTAAGTATATGCTCTTGTGCAGAGATTCGCAGACGAGGCTCGAAGGATCTGCTGAAAGTGT

[0286] GTGTAGTGTTGAACGGAGGCGAAGGTGAGGGGTCTGGACCCCCTCAGATCTATGATGTCCCATATGAGG

[0287] AGGTTCTGGAGGGAATTCCGTTGACCCGACCTGAGTCCGACTCCAGACCGCCGGCAGAGTATGAGCTGC

[0288] CCTGGGAGTGGAAGAAAGAGCAGATTGTTAGAGCCCTGTCAGGTAACTCCCCTTGCATTCATATTAGTA

[0289] ACAAACATCTTCTTTATTTAATATATAGACACAGTATAATGGGAAAAAACTATTTAAAAGTCATTTGCTG

[0290] TTTGGTTTTAATGACATATTATTGAGGGAATTCTCATGAGCAGTTTCTCCAAATGTCTGAAATAACTAGA

[0291] GTCATTATAACTGACAGATGACAAATCTAAAATCTATGCGACTTAAATCTGTAGTGTTTAATGTTTCTTT

[0292] CTGTGCACAGCTCAGTTTGATGGAGCCGAGCGCATGTCGAAAGAAGAATCAGGTTCTCCTACTATAAAA

[0293] CAGCAGACTCTAAGACATAAAAGCTGGACGACCAAATCTCTACGACCGAATCCCCCCTCCCCAACCTCC

[0294] CCCTCTTCCCCCAGCATCACCCCAGACAGTGAGACCCCAATTGTTGATCCCACCCTGCCATTGGAGAAA

[0295] CAGAGGTGTGTGTTTGTGTCTTAAAATGTGTTTTGGAGAGTTACTTTAAATCTAAAAATTAAACTAAAAC

[0296] AATTTAGAGGCAATATTATTAAGAAGGTTGTGAAAAGGGTATGTTTTGGTCAAAATTTGCACTAAAGTT

[0297] TGGTATTTAAATATTTACAGTATGTTTCACTGAAAGCTTGCACACTTAATATTTTTATTATAGATACAGT

[0298] ATACTCACTGGCCCTTTTATTAGTGACACATGTTCATTTACGTTCATTTAGCAGATATTTTTATACAAAGT

[0299] GACTTACAAGTGGGGATAAAGTAGTTTAACTTTTTTCTCTCCATATCAAACCCACTCACCTGTGTGAAGT

[0300] TCAGTCAGATTGCGCAATGCTTCCCCCAAAAATGGGGTGTTATTAAAAAAAATGATTTGTTTTTCTGATA

[0301] ATTACTTAATTGAATAACAAAACATGAATAAATAAAAATAGCTGGTTTCTTTCCAGCCAGCTATAGAGA

[0302] AACAACAGAGAATGGCTATGTCGTGTAGAAGCTTAGAATTTTGAGCTGAGAACATGCTTTATTTGTAGA

[0303] AAATCCCATTTTTTTCATATATCCTACTTTGATTATATACAGCTACTCAGCTAACATGAAAAGGACTGTC

[0304] ATATTTCGTAGTTCCTCTGAAGACCCTCCCTCAAGAGGCTCTGATTAGTCAACTAACATAATGTTCTGTG

[0305] ATTTGCAGATCAGCTCCACATCACCAAGAAGTCTCTGTTATAACATAACAGCTATTCTGTCCACGCCCAG

[0306] AAGGGATTATGACATTATTACCCAGTAGTCAAATGTTTACAAATATAAGCGCAGCTGTTTAGAGGTAAT

[0307] TTCTGGTTAATAATGTCAGAATTTACCGCTATTTTGGAATGGATGTGTAATCTGTCTTTTCCGGAAAAAT

[0308] TCTGCAACGTTCTTGCCTGGTTAAACAACGCTTTTGAAATTACCTGTAAAAGTTGTTCCGGAATTTTTTT

[0309] GCATATTTACCAGTATCACTGTGTGAAAGAGGCTAATGTAGAGGTGGGCACTTACATCTGGCCCGGGAG

[0310] TTTTTAAAATACTGCTTCTGAGTAACTGTGCGTTCACACCAAAAGCGGTGAGAGCGTCCAAGGTCACTCT

[0311] GGCCGCCCTGGCAACAACGCTGTCTGCCTTCAGCTCCGGCGGTGAGAGCGTCAAATTCACCACATTGA

[0312] TCTCATATTTAAAGGGGCCGTTGTAGCATATCAGTTACATTCCTGCATAAAACATGGTTTCTAGCGTGAA

[0313] AATTTTGCGCATGAATTTAACAATGGAAGATCAAGTTGCATGTGGTGTGGCTTTGCTCCACTTAATTATC

[0314] ATGTGTCTATATGTCTGAAATATCCTAAAGCAGCAGTACTATTTTGTACTGTCGCATGAGATTCCCGCTT

[0315] TTTTAAAGCAAAATATCTATAACGTTAATGCACATCAGTAACTCGCCAGTAATTTTTTAATGTATGTTC

[0316] CTATAAGAAAACATTGTAAATAATTGAAAATCCGGCGACTGCAATAATCAAACCCCTGGGGAACAACTG

[0317] TGGCGGTACAGTTAGTGCAACCAGTAGATTTAGAACACGCTCTCGGATATTAAAATGCCCTCGACTTAA

[0318] AATGAGTCAGGTAAAAAAATGACTCTGAATGTTGTGTGTTTAAGGAAGAATGGACAACAAAATACCTCA

[0319] CCAATATCTGACAGAAAGCAGTAGGTCTAATATGCCAAGAAAGTATTGCTTTTTTTAAAGACTACAACC

[0320] TAAGGGGCTGAGCACACAGCTAGGTGCGCAGTGCGCTTTTTTATGTCAGGCAACGACTGAATCAGCTAG

[0321] GTATTTTGCGAGAGTGTTGCTGTTGATATTAATGTAACTTTATTTTCATACTGTTCACATTAACACACTTA

[0322] TAATAACATGACTTGGAATAACAAGGATTAAGATTTTTCCGTTTTATCTCATAGTTTTATAAGGATTGAT

[0323] ATAAATAGCAGTGCAAATTCATTTGACTTGTAAATTTGATTCAATAATAACTTTAAAAAAAAAATCTGTT

[0324] TTGTGTTAATGTGTTAATGTGACATATGTTTCATGCATACTGCCACTCTAAATAAAAAAATATTTTTTGA

[0325] AGGATTTGACATGTGGCCCTTCACTAGGTTTGCAAAACTTGATGTGGCCCTCGGGTCTAAAAAGTTTGCC

[0326] CACCACTGGGCTAATGACCAAAAACAAAAATCATCTTAAACTATATTTTTCCACTACGCTTAAATGACCT

[0327] CCATAGTCCCAGATCTCAGTTCAATAGAGCACCTTTGGGATAAGGAGAATGTCGTTTTAAAGCTTAAAT

[0328] ATATGATCTGTCCATTCAGGTTCATTCATGTTTATAGTTGTCTGTACATCATTCTCAGTTTATTCTGATTC

[0329] CTTTCTTCTGTAATCTATACTCGATTAAATATGTTTTATAAGCACTGTTCTTTTTACATAATCATACATTC

[0330] TCTCTCTCTCTCTCTTTAGCTGGTATCATGGCAGTGTGAGTCGTCAGGAGGCAGAGGCTCAGCTCCAGCA

[0331] GTGTAGGGAGGCCAGTTTCCTGGTGCGAGACAGTGAATCAGCCACCAGCAAGTACTCCATTGCCCTCAA

[0332] GTAAGTGTGTGTTTGTTCAAGAGAACAAGAAACGAGTTAGCAGTCCTGAAAAGCTGACACGATGATATA

[0333] AGATCCTTGTTTTCCTCTCATTACAGGACAAGTCAAGGATGTGTGCACATCATCGTAGCGCAGACTAAA

[0334] GAGTGTGGCTACACACTGGAAAAGAGCAGCTGTGTGTTTCCAAGCATCCCAGAAGTGGTACACCACTAC

[0335] TGTACCCAGCGCCTCCCTTTTACAGGGGCTGAACACATGACCCTGCAGCACCCCGTGCCACGCACACACTGAACCCACACACACACACACACACACAAAAAA (SEQ ID NO: 1)

[0336] After in-depth research and analysis, the inventors selected two sequences in the zebrafish SH2E gene sequence to design corresponding gRNAs, where the gRNA target sequences are as follows (the bases in brackets refer to the bases corresponding to the PAM sequence):

[0337] The gRNA1 target sequence is (SEQ ID NO: 2): ggtggcggtggtggcggcgt[ggg];

[0338] The gRNA2 target sequence is (SEQ ID NO: 3): ggacggaaccactccagcac[cgg].

[0339] Microinjection

[0340] The expression of SH2E gene was knocked out by microinjecting gRNA and Cas9 protein mRNA into fertilized eggs. Under a stereomicroscope, gRNA and Cas9 mRNA were co-injected into the cytoplasm of 1-cell fertilized eggs using a microinjector.

[0341] During injection, adjust the pressure of the microinjector and inject 1 nL of a mixed solution containing approximately 100 pg of gRNA and approximately 200 pg of Cas9 mRNA into each fertilized egg.

[0342] The injected fertilized eggs were cultured in culture water at 28.5°C and pH 7.0 according to conventional methods.

[0343] Embryo genome extraction

[0344] Genome extraction was performed using a genome extraction kit produced by Beijing Quanshijin Biotechnology Co., Ltd. 10-15 embryos of the corresponding phase were collected in a 200 μL centrifuge tube, the liquid was discarded, and after oven drying, 10 μL AD1 and 2.5 μL AD2 were added to each tube, and then slightly blown and centrifuged.

[0345] Then, digestion was performed using a PCR instrument at 55°C for 20-30 minutes and 95°C for 3 minutes. After digestion, 10 μL of AD3 was added to each tube, and the tube was slightly blown and centrifuged, and the supernatant was used as a template for polymerase chain reaction (PCR) amplification to identify the genotype.

[0346] PCR amplification

[0347] The SH2E gene fragment was amplified using Ex Taq DNA polymerase produced by Takara Biotechnology Co., Ltd. of Japan according to the instructions.

[0348] First, prepare the reaction system: add 0.1 μL of Ex Taq DNA polymerase solution, 0.8 μL of forward primer and reverse primer, 1.6 μL of dNTP mixture, 2 μL of 10×Ex Taq buffer, and 2 μL of genomic DNA solution into a 200 μL centrifuge tube, and then make up to 20 μL with distilled water.

[0349] The PCR instrument was set to the following reaction conditions: 94°C for 5 minutes in the first stage; 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 40 seconds in the second stage, with 31 cycles; and 72°C for 10 minutes in the third stage. The primers used are shown in Table 1.

[0350] Table 1

[0351]

[0352] Enzyme digestion to identify genotype

[0353] After amplifying the fragment containing the CRISPR / Cas9 target site in the SH2E gene by PCR, the genomic mutation was confirmed using restriction endonuclease Fnu4FI produced by New England Biolabs in the UK, Cfr10I produced by Takara in Japan, and agarose gel electrophoresis.

[0354] The reaction system for digestion of genomic DNA PCR products with Fnu4FI endonuclease was as follows: 1 μL of Fnu4FI solution, 1 μg of PCR product, 1×rCutSmart buffer, and distilled water to 50 μL, and the reaction was carried out at 37°C overnight. The reaction system for digestion with Cfr10I endonuclease was as follows: 1 μL of Cfr10I solution, 1 μg of PCR product, 2 μL of 10×Cfr10I buffer, and distilled water to 20 μL, and the reaction was carried out at 37°C overnight. The next day, the digestion system was taken out, and the nucleic acid band cutting was detected by gel electrophoresis, and the samples that failed to be cut were selected for sequencing to confirm the mutation type.

[0355] Fluorescence quantitative PCR

[0356] The SYBR Green fluorescent quantitative PCR kit produced by Takara, Japan, was used to detect the relative expression of full-length SH2E messenger RNA in zebrafish embryos and heart tissues. The reaction system was prepared as follows: 2×SYBR Green Master Mix 10 μL, 10 μM forward primer and reverse primer 0.4 μL each, cDNA extracted and reverse transcribed from the corresponding tissue 2 μL, and distilled water was added to 20 μL. The reaction conditions set by the fluorescent quantitative PCR instrument were: pre-denaturation at 95°C for 3 minutes; PCR reaction at 95°C for 15 seconds, 60°C for 1 minute, and the reaction was carried out for 40 cycles; melting curve generation at 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds, and 60°C for 15 seconds. The primers used are shown in Table 2.

[0357] Table 2

[0358]

[0359] Histological examination

[0360] SH2E knockouts (embryo cardiac development defect model) and littermate controls were collected into culture dishes 4 days after fertilization, anesthetized with 0.04% MS-222, and each fry was cut off from the center of the trunk with a clean blade.

[0361] The front end with the heart was fixed with 4% paraformaldehyde overnight, and the back end was used for genotyping. The next day, the fixed larval fish tissue was taken out and placed in a 15mL centrifuge tube containing 10% sucrose solution until it sank to the bottom. Then, the tissue was taken out and placed in a 15mL centrifuge tube containing 20% ​​sucrose solution until it sank to the bottom. After that, the tissue was taken out and placed in a 15mL centrifuge tube containing 30% sucrose solution until it sank to the bottom. Take out the tissue, dry the surface moisture on absorbent paper, place it in a mold containing OCT embedding agent and carefully adjust the position of the tissue sample with a needle. Place the mold containing the tissue in liquid nitrogen, wait for the OCT embedding agent to completely solidify, fix it on a freezing microtome, and slice it at a thickness of 4μm.

[0362] Place the frozen sections of zebrafish tissue at room temperature for equilibrium, draw the sample area with a water-blocking pen, and fix it with 4% paraformaldehyde for 10 minutes. Discard the paraformaldehyde, wash with distilled water for 2 minutes, and stain with hematoxylin staining solution for 5-10 minutes (staining time can be adjusted as needed). Immerse the sections in slightly flowing tap water to rinse off excess staining solution for 10 minutes, and quickly wash once with distilled water. Immerse the sections in eosin staining solution for 30 seconds to 2 minutes (staining time can be adjusted as needed). Soak the sections in 70%, 80%, 90% and anhydrous ethanol for 10 seconds respectively, then make them transparent in xylene for 5 minutes, and then change to fresh xylene for 5 minutes. Seal the sections with neutral gum, observe and take pictures under a microscope.

[0363] Selenomethionine (Se-Met) treatment

[0364] After Se-Met powder was completely dissolved in distilled water, it was added to the culture water of zebrafish fertilized eggs to make the final concentrations 2.5 μM and 5 μM, respectively, and the group without Se-Met solution was 0 μM. The fertilized eggs of each group were placed in a biological culture cabinet for routine culture for 72 hours, and then taken out and observed or used for subsequent experimental detection.

[0365] Reactive oxygen species (ROS) detection

[0366] The ROS fluorescent probe DCFH-DA (Shanghai Biyuntian Biotechnology Co., Ltd.) was used to detect the generation of ROS in zebrafish larvae. First, the larvae were collected into a 1.5 mL centrifuge tube, the culture water was discarded, and the larvae were rinsed twice with phosphate buffered saline (PBS) and discarded. Add 1 mL of DCFH-DA solution diluted with PBS to make the final concentration of DCFH-DA 5 μg / mL. The larvae were placed in an incubator in the dark for 1 hour and then discarded, and quickly rinsed twice with PBS. Discard the PBS and add 1 mL of PBS containing 0.04% MS-222 for anesthesia. The larvae were placed in methylcellulose, photographed under a microscope, and quantified using Image J.

[0367] Propidium iodide (PI) staining

[0368] Use whole PI staining to detect cell apoptosis in zebrafish larvae. First, collect the larvae into a 1.5mL centrifuge tube, discard the culture water, rinse twice with PBS and discard. Add 1mL of PI solution (BD Company, USA) diluted with PBS to make the final concentration of PI 5μg / mL. Place the larvae in an incubator in the dark for 30 minutes and then discard, and quickly rinse twice with PBS. Discard PBS and add 1mL of PBS containing 0.04% MS-222 for anesthesia. Place the larvae in methylcellulose, take pictures under a microscope and count them using ImageJ.

[0369] Signaling pathway inhibitor treatment

[0370] The MAPK / ERK signaling pathway inhibitor PD184352, the NF-κB signaling pathway inhibitor BAY11-7082, and the PI3K / AKT signaling pathway inhibitor AKT inhibitor VIII powders were completely dissolved in DMSO and added to the zebrafish fertilized egg culture water to make the final concentrations of 80mM and 160mM, 4nM and 8nM, 0.03μM, 0.10μM and 1.00μM, respectively, and the group adding 0.5% DMSO solution was used as the control group. The fertilized eggs of each group were placed in a biological culture cabinet for routine culture for 72 hours, and then taken out and observed or used for subsequent experimental detection.

[0371] Example 1: SH2E gene deletion causes cardiac developmental defects in zebrafish embryos

[0372] In order to verify the role of SH2E in early heart development, the present inventors used the CRISPR / Cas9 system to knock out the SH2E gene in zebrafish embryos, and then observed the changes in heart development of zebrafish embryos after SH2E knockout.

[0373] like Figure 1 As shown in A, the inventors designed two CRISPR targets on exon 1 of the zebrafish SH2E gene. By injecting gRNA and Cas9 protein mRNA into fertilized eggs, two SH2E mutants G59R and S82R ( Figure 1 B), their SH2E genes all contain frameshift mutations, and the transcribed mRNA can lead to premature termination of SH2E protein translation.

[0374] Figure 1 C is a schematic diagram of the SH2E protein peptide of the SH2E mutant. It can be seen that only a small fragment of the SH2E peptide chain of the mutant is retained. The homozygous SH2E mutant was tailed, the SH2E genomic fragment was cloned, and the SH2E mutant genomic mutation was confirmed using restriction endonucleases Fnu4FI, Cfr10I and agarose gel electrophoresis ( Figure 1 D).

[0375] 2 days after fertilization ( Figure 1 E) and 2 months old ( Figure 1 F) In the heart tissue of mutant zebrafish, the transcription level of SH2E messenger RNA with normal nucleotide sequence length was significantly reduced.

[0376] On days 4 and 5 after fertilization, SH2E mutants showed significant pericardial edema ( Figure 1 G). The pericardial cavity area was measured and it was found that the pericardial cavity area of ​​the mutant was significantly increased ( Figure 1 H).

[0377] Hematoxylin-eosin staining of heart tissue sections of SH2E mutants and wild-type littermates 4 days after fertilization showed that the atrial and ventricular walls of SH2E mutants were significantly thinner than those of wild-type littermates ( Figure 1 I).

[0378] The thickness of the atrial and ventricular walls of SH2E mutants and wild-type littermates was measured 4 days after fertilization. Figure 1 J) and ventricular wall ( Figure 1 K) were significantly lower than those of wild-type littermates.

[0379] Therefore, a zebrafish animal model of embryonic heart development defects was successfully established, showing typical symptoms of heart development defects.

[0380] Example 2: Se-Met reduces oxidative stress in the pericardium, thereby alleviating cardiac developmental defects caused by SH2E gene defects

[0381] In this example, the zebrafish heart development defect model established in the above-mentioned Example 1 was used to analyze the effect of the oxidative stress level of the tissue in the pericardial cavity on the early development of the heart.

[0382] like Figure 2 As shown in Figure 1, the present inventors used the reactive oxygen species fluorescent probe DCFH-DA to detect the content of reactive oxygen species (ROS) in the tissues of the embryonic heart development defect model (SH2E knockout body) and the littermate control body, and found that the ROS content in the tissues of the embryonic heart development defect model was significantly increased ( Figure 2 B), the increase in ROS content in the peritoneal cavity was particularly significant ( Figure 2 C) This suggests that the loss of the SH2E gene may cause pericardial edema by increasing the level of oxidative stress in the pericardial tissue of zebrafish embryos.

[0383] The inventors then added selenomethionine (Se-Met; final concentrations were 2.5 nM and 5 nM) to the culture water to treat the fertilized eggs of the embryonic heart development defect model, and found that Se-Met treatment can reduce the ROS content in the tissue of the embryonic heart development defect model in a concentration-dependent manner, and at the same time reduce the area of ​​the pericardial cavity ( Figure 2 A). In the 5 nM Se-Met treatment group, the pericardial cavity area was positively correlated with the relative quantification of ROS in the pericardial cavity ( Figure 2 D). This suggests that Se-Met can effectively reduce the ROS content in the pericardial cavity of SH2E mutant zebrafish embryos, thereby alleviating the pericardial edema caused by oxidative stress.

[0384] Increased oxidative stress levels can lead to cell apoptosis. Figure 2 As shown in E, the present inventors used propidium iodide to detect cell apoptosis in the embryonic heart development defect model and the littermate control tissues, and found that although there was no significant change in cell apoptosis in the embryonic heart development defect model tissues ( Figure 2 F), but the apoptosis of cells in the ventricle increased significantly ( Figure 2 G). This further suggests that SH2E gene deficiency may cause ventricular myocyte apoptosis by increasing oxidative stress in pericardial tissues, thereby leading to heart development defects.

[0385] Se-Met (final concentrations of 2.5 nM and 5 nM, respectively) was added to the culture water to treat the fertilized eggs of the embryonic heart development defect model. It was found that Se-Met treatment could reduce the apoptosis of the ventricular cells in the embryonic heart development defect model in a concentration-dependent manner and reduce the area of ​​the pericardial cavity ( Figure 2 E. Figure 2 G). In the 5nM Se-Met treatment group, the pericardial cavity area was positively correlated with the number of apoptotic cells in the ventricle ( Figure 2 H).

[0386] The results indicate that Se-Met can effectively reduce apoptosis of ventricular myocytes in SH2E mutant zebrafish embryos, thereby alleviating cardiac development defects caused by oxidative stress.

[0387] Therefore, the established zebrafish heart development defect model can be used to screen substances that effectively exert alleviating effects.

[0388] Example 3: Analysis and application of downstream pathways of SH2E gene defects causing cardiac developmental defects

[0389] In order to further explore the molecular mechanism of zebrafish embryonic heart development defect caused by the embryonic heart development defect model, the inhibitor of MAPK / ERK signaling pathway PD184352 (final concentrations of 80mM and 160mM, respectively), the inhibitor of NF-κB signaling pathway BAY11-7082 (final concentrations of 4nM and 8nM, respectively), and the inhibitor of PI3K / AKT signaling pathway AKT inhibitor VIII (final concentrations of 0.03μM and 0.10μM, respectively) were added to the culture water to treat the fertilized eggs of the embryonic heart development defect model, and then compared with the control group added with DMSO (final concentration of 0.5%) ( Figure 3 A).

[0390] The results showed that PD184352 treatment could increase the ROS content in the pericardial cavity of the embryonic model of cardiac development defects in a concentration-dependent manner and increase the area of ​​the pericardial cavity ( Figure 3 AB).

[0391] BAY11-7082 treatment can reduce the ROS content in the pericardial cavity of the embryonic model of cardiac development defects in a concentration-dependent manner and reduce the area of ​​the pericardial cavity ( Figure 3 A, C).

[0392] However, AKT inhibitor VIII treatment had no significant effect on the ROS content in the pericardial cavity of the heart development defect embryo model, and the area of ​​the pericardial cavity did not change significantly ( Figure 3 A, D).

[0393] The results indicate that the MAPK / ERK signaling pathway plays an antagonistic role in tissue oxidative stress and edema in the pericardial cavity of zebrafish embryos with cardiac development defects, and that the NF-κB signaling pathway is the downstream pathway of tissue oxidative stress and edema in the pericardial cavity of zebrafish embryos caused by SH2E mutation. However, the pathological process of tissue oxidative stress and edema in the pericardial cavity of zebrafish embryos caused by SH2E mutation does not involve the PI3K / AKT signaling pathway.

[0394] Example 4: Combination with AKT inhibitor VIII can enhance the inhibitory effect of Se-Met on pericardial edema in zebrafish embryos

[0395] In order to explore whether the combination of PI3K / AKT signaling pathway inhibitor AKT inhibitor VIII and Se-Met would affect the inhibitory effect of Se-Met on pericardial edema in zebrafish embryos, a small dose of Se-Met (final concentration of 1.25 nM), AKT inhibitor VIII (final concentrations of 0.03 μM and 0.10 μM, respectively) and Se-Met (final concentration of 1.25 nM) and AKT inhibitor VIII (final concentrations of 0.03 μM and 0.10 μM, respectively) were added to the culture water to treat fertilized eggs of embryonic heart development defect model, and then compared with the control group added with DMSO (final concentration of 0.5%) ( Figure 4 A).

[0396] The results showed that Se-Met at a concentration of 1.25 nM and AKT inhibitor VIII at concentrations of 0.03 μM and 0.10 μM, respectively, could not effectively inhibit the area of ​​pericardial edema in the heart development defect model ( Figure 4 AB). However, the simultaneous application of Se-Met (final concentration of 1.25 nM) and AKT inhibitor VIII (final concentrations of 0.03 μM and 0.10 μM, respectively) can significantly reduce the area of ​​pericardial edema in the heart development defect model ( Figure 4 AB).

[0397] The results indicate that inhibiting PI3K / AKT signaling pathway inhibitors can enhance the inhibitory effect of Se-Met on pericardial edema in zebrafish embryos, thereby reducing the dose of Se-Met used to alleviate pericardial edema in zebrafish embryos.

[0398] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. Use of PI3K / AKT signaling pathway inhibitors and L-selenomethionine for: Preparation of a composition for alleviating or treating cardiac developmental defects; or A composition for resisting oxidative stress is prepared.

2. The use according to claim 1, characterized in that The cardiac development defect is a cardiac development defect; more preferably, the cardiac development defect is an embryonic cardiac development defect; more preferably, the cardiac development defect is a cardiac development defect caused by down-regulation of the SH2E gene.

3. The use according to claim 1, characterized in that The PI3K / AKT signaling pathway inhibitors include: AKTinhibitor VIII, LY294002, and Wortmannin.

4. The use according to claim 1, characterized in that The heart development defect is a heart development defect presenting the following symptoms: pericardial edema, enlarged pericardial area, reduced atrial wall thickness, reduced ventricular wall thickness, increased oxidative stress level, increased ROS content, and increased cell apoptosis in the ventricular part; the L-selenomethionine exerts its effect by reducing pericardial edema, shrinking pericardial area, increasing atrial wall thickness, increasing ventricular wall thickness, reducing oxidative stress level, reducing ROS content, and reducing cell apoptosis in the ventricular part.

5. A pharmaceutical composition comprising a PI3K / AKT signaling pathway inhibitor and L-selenomethionine, and a pharmaceutically acceptable carrier; the pharmaceutical composition is used to alleviate or treat cardiac developmental defects, and / or resist oxidative stress.

6. A medicine kit comprising a PI3K / AKT signaling pathway inhibitor and L-selenomethionine; the medicine kit is used to alleviate or treat cardiac developmental defects and / or resist oxidative stress.

7. The use according to any one of claims 1 to 4, the pharmaceutical composition according to claim 5 or the kit according to claim 6, characterized in that: The molar ratio of the PI3K / AKT signaling pathway inhibitor to the L-selenomethionine is: (8-500):1; preferably (10-300):1; more preferably (15-200):

1.

8. A method for screening a potential substance that promotes L-selenomethionine to alleviate or treat cardiac developmental defects and / or resist oxidative stress, the method comprising: (1) treating an expression system with a candidate substance, the system having a PI3K / AKT signaling pathway; and (2) detecting changes in the PI3K / AKT signaling pathway in the system; If the candidate substance statistically inhibits the PI3K / AKT signaling pathway, it indicates that the candidate substance is a potential substance that promotes L-selenomethionine to alleviate or treat heart developmental defects and / or resist oxidative stress.

9. A method for preparing an animal model of cardiac developmental defects, comprising targeting the SH2E gene in the zebrafish genome, and downregulating the target to obtain an animal model of cardiac developmental defects.

10. The method according to claim 9, characterized in that The down-regulation is performed by gene knockout / silencing, gene interference methods; Preferably, the down-regulation or removal is performed by gene knockout / silencing by gene editing or homologous recombination; More preferably, the SH2E gene is knocked out by a CRISPR / Cas method, including using a specific gRNA to target the SH2E gene.

11. The method according to claim 10, characterized in that The gRNA targets exon 1 of the SH2E gene; Preferably, the sequence targeting the SH2E gene is shown in SEQ ID NO: 2 and / or SEQ ID NO: 3; Preferably, after being treated by the CRISPR / Cas method, the protein encoded by the SH2E gene mutates at position 59 and / or position 82, so that the translation of the SH2E protein is terminated prematurely.

12. The method according to claim 10, characterized in that By injecting gRNA and Cas protein mRNA or DNA that can form the mRNA into zebrafish fertilized eggs, fertilized eggs modified by CRISPR / Cas are obtained; further, the fertilized eggs are cultivated into animal embryos or animals.

13. The method according to any one of claims 9 to 12, characterized in that: The cardiac development defect is a cardiac development defect; Preferably, the cardiac development defect is an embryonic cardiac development defect; Preferably, the cardiac developmental defect presents the following phenotypes: decreased SH2E expression level, pericardial edema, increased pericardial area, decreased atrial wall thickness, decreased ventricular wall thickness, increased oxidative stress level, increased ROS content, and increased cell apoptosis in the ventricle.

14. Use of the cardiac development defect animal model obtained by the method according to any one of claims 9 to 13 for: As an animal model for screening candidate drugs or therapeutic agents that alleviate or treat cardiac developmental defects; or Used as an animal model for studying cardiac developmental defects; Preferably, the heart development defect is an early heart development defect; more preferably, the heart development defect is an embryonic heart development defect.

15. A method for screening candidate drugs or therapeutic agents for alleviating or treating cardiac developmental defects, the method comprising: (1) Preparing an animal model of cardiac developmental defect using the method described in any one of claims 9 to 13; (2) administering the candidate substance to the animal model of (1) to observe whether the candidate substance has an alleviating or therapeutic effect on cardiac developmental defects; If it is observed that the symptoms of cardiac developmental defects in the animal model are alleviated, the candidate substance is a substance that alleviates or treats cardiac developmental defects.

Images