Mouse myocardial fibrosis pathological model inducer

Abstract

The invention discloses a mouse myocardial fibrosis pathological model inducer. The recombinant adeno-associated virus expressing RGS4 can promote the process of myocardial fibrosis of mice by up-regulating the expression level of the RGS4 gene in myocardial tissues after being injected into the heart of the mice in situ, and can be used for inducing a myocardial fibrosis model of the mice. The invention provides a new thought and strategy for constructing a mouse myocardial fibrosis pathological model, and has a wide pharmacological research application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to an inducer for a mouse myocardial fibrosis pathological model. Background Technology

[0002] Myocardial fibrosis is a pathological state characterized by abnormal proliferation of connective tissue and excessive deposition of collagen fibers in the myocardium. It represents an overrepair response of the myocardium to injury, characterized by activation and excessive proliferation of cardiac fibroblasts, myofibroblast differentiation, and excessive deposition of extracellular matrix proteins in cardiomyocytes. Myocardial fibrosis is an essential pathological process in the development of heart failure, leading to hardening and decreased elasticity of myocardial tissue, causing systolic and diastolic dysfunction, ultimately resulting in worsening cardiac function and even sudden cardiac death. Myocardial fibrosis is an independent risk factor for assessing the occurrence, development, and prognosis of heart failure. The mechanisms of myocardial fibrosis are complex and not yet fully understood. Therefore, constructing pathological models of myocardial fibrosis to further explore its mechanisms and find effective therapeutic drugs is of great significance for improving our understanding of myocardial fibrosis-related diseases and for developing strategies for the prevention and treatment of cardiovascular diseases.

[0003] Adeno-associated virus (AAV), first discovered in the 1960s, is a single-stranded parvovirus consisting of an icosahedral protein capsid (26 nm in diameter) and a genome approximately 4.7 kb in length. Most genes in the AAV genome can be removed and replaced with foreign genes, making it suitable as a gene delivery tool. AAV has been found in various mammals, including humans and non-human primates, but has not been found to be pathogenic. Thirteen serotypes and over 100 different variants have been identified in various mammals. Among the thirteen serotypes, AAV1-9 exhibit broad tissue affinity and are used as the primary delivery vectors in gene therapy.

[0004] G protein signaling regulators (RGS) are negative regulators of the G protein-coupled receptor signaling system. The RGS protein family contains more than 20 members, many of which are involved in the regulation of myocardial fibrosis. However, previous studies have reported that many RGS proteins play a protective role in myocardial fibrosis; for example, RGS5 inhibits myocardial fibrosis by attenuating MEK-ERK1 / 2 signaling; RGS2 can also inhibit the occurrence of myocardial fibrosis; and RGS14 improves the degree of myocardial fibrosis by attenuating the development of cardiac remodeling through MEK-ERK1 / 2 signaling. Currently, no studies have confirmed the role of RGS4 in constructing pathological models of myocardial fibrosis. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a mouse myocardial fibrosis pathological model inducer to help construct a stable and easy-to-operate animal model of myocardial fibrosis, thereby providing a reliable research tool for studying the mechanism of action and drug targets of myocardial fibrosis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The first aspect of the present invention provides an inducer for a pathological animal model of myocardial fibrosis, wherein the inducer is an RGS4 gene promoter.

[0008] Furthermore, the RGS4 gene is the gene encoding the RGS4 protein with the amino acid sequence shown in SEQ ID NO:1.

[0009] Furthermore, the nucleotide sequence of the coding strand of the gene encoding the RGS4 protein is shown in SEQ ID NO:2.

[0010] In some embodiments, the RGS4 promoter described in this invention refers to any substance that can upregulate RGS4 expression, increase RGS4 activity, improve RGS4 stability, increase the effective duration of RGS4 action, or promote RGS4 transcription and translation, and such substances all fall within the protection scope of this invention.

[0011] In some embodiments, the RGS4 gene expression promoter refers to any substance that can promote RGS4 expression, including but not limited to: naturally purified substances, modified naturally purified substances, semi-synthetic substances, chemically synthesized substances, or any combination thereof that can promote RGS4 expression.

[0012] In some embodiments, specific examples of the RGS4 promoter include, but are not limited to: nucleic acid promoters, protein promoters, nanoparticles, liposomes, protein microspheres, and small molecule compounds, such as vectors or constructs thereof that overexpress RGS4, RGS4 protein or its active peptide, oligonucleotides that promote RGS4 expression, nanoparticles carrying RGS4, liposomes that encapsulate RGS4, protein microspheres that encapsulate RGS4, or small molecule compounds that promote RGS4 expression.

[0013] Furthermore, the promoter includes an RGS4 overexpression vector that promotes RGS4 gene expression.

[0014] Furthermore, the vector may include a viral vector or a non-viral vector.

[0015] Furthermore, the viral vector includes adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, poxvirus vector, vaccinia virus vector, or herpesvirus vector.

[0016] Furthermore, the non-viral vector includes liposome nanoparticles, polymer nanoparticles, protein microspheres, exosomes, polypeptide complexes, aptamers, or mRNA vectors.

[0017] Furthermore, the promoter is an RGS4 overexpression viral vector that promotes RGS4 gene expression.

[0018] Furthermore, the promoter is an RGS4 overexpressing adeno-associated virus vector that promotes RGS4 gene expression.

[0019] Furthermore, the adeno-associated virus vector includes AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV-DJ.

[0020] Furthermore, the adeno-associated virus vector is AAV9.

[0021] In some embodiments, the adeno-associated virus (AAV) is a tiny, replication-defective virus belonging to the parvovirus family, and is a non-enveloped, single-stranded linear DNA virus. AAV vectors can infect both replicating and quiescent cells, exhibit superior long-term transgene expression, and no viral pathological or toxicological reactions have been observed. Therefore, AAV has gained widespread attention in the field of gene research and is the most commonly used viral vector.

[0022] The AAV genome is a linear single-stranded DNA molecule. Its coding region is flanked by two cis-acting inverted terminal repeat (ITR) sequences, each approximately 145 nucleotides in length, which function as primers during DNA replication initiation. The AAV gene coding region consists of two main open reading frames: a non-structural replication gene (Rep) and a structural capsid gene (Cap), located between the two inverted terminal repeat sequences (L-ITR and R-ITR). The AAV genome contains approximately 4.7 kilobases (kb).

[0023] Furthermore, the animals include mice, rats, guinea pigs, hamsters, rabbits, dogs, pigs, and monkeys.

[0024] Furthermore, the animal in question is a mouse.

[0025] Furthermore, the inducing agent is administered via in situ injection into the myocardium.

[0026] In some implementations, the AAV viral vector is injected into the heart via methods including but not limited to: in situ injection into the myocardium, coronary artery injection, jugular vein injection, and tail vein injection, with in situ injection into the myocardium and tail vein injection being the most commonly used.

[0027] In specific implementations, the in situ myocardial injection involves directly injecting the carrier into the heart tissue using a syringe or similar device, often employing a multi-point in situ injection approach. Compared to intravenous injection, in situ myocardial injection offers higher specificity, enabling efficient transduction of the target region of interest.

[0028] In a specific implementation, the tail vein injection involves immobilizing the mouse and then injecting the vector into a vein on the side of the tail to deliver it to the heart. Tail vein injection is easy to perform, but its specificity is slightly lower than that of myocardial injection. Because the vector is widely distributed throughout the body, the injected agent will be diluted in the blood volume, so a higher viral titer is usually required.

[0029] In a specific implementation, the jugular vein injection involves making an incision in the neck of anesthetized mice, separating the jugular vein, and injecting the carrier into the jugular vein. This method requires surgery, demands high skill levels, and is potentially harmful to the animal.

[0030] In specific implementations, coronary artery injection is further divided into antegrade coronary artery injection and retrograde coronary artery injection. Antegrade coronary artery injection involves directly injecting the carrier through a catheter, allowing for minimally invasive delivery of cardiac-selective carriers. Retrograde coronary artery injection is a reverse infusion that regulates pressure by blocking the coronary sinuses; it is more effective than antegrade injection but is more invasive and carries a higher risk.

[0031] A second aspect of the present invention provides a method for constructing a pathological animal model of myocardial fibrosis, the method comprising using an inducer provided in the first aspect of the present invention to promote the expression of the RGS4 gene in the heart of an animal.

[0032] Furthermore, the method includes injecting the inducing agent provided in the first aspect of the present invention into an animal body by in situ injection into the myocardium to obtain a pathological animal model of myocardial fibrosis.

[0033] The third aspect of this invention provides the application of the RGS4 gene provided in the first aspect of this invention in constructing a pathological animal model of myocardial fibrosis.

[0034] The fourth aspect of the present invention provides an application of the animal model obtained by the method described in the second aspect of the present invention, the application including any one of the following:

[0035] 1) Application in the study of the pathogenesis of myocardial fibrosis.

[0036] 2) Application in screening candidate drugs for myocardial fibrosis-related diseases.

[0037] In some implementations, the candidate drug includes antibodies, DNA, RNA, and small molecule compounds.

[0038] Specifically, the antibody is an immunoglobulin produced by plasma cells differentiated from B lymphocytes in response to antigen stimulation by the body's immune system. It is capable of specifically binding to the corresponding antigen. The structure of an antibody is mainly divided into two parts: a constant region and a variable region.

[0039] Specifically, the DNA includes single-stranded DNA, double-stranded DNA, circular DNA, and linker DNA.

[0040] Specifically, the RNA includes mRNA, tRNA, rRNA, snRNA, hRNA, antisense RNA, tCRNA, dsRNA, scRNA, catalytically active RNA, and various viral RNAs.

[0041] Specifically, the sources of the small molecule compounds are selected from: newly synthesized or existing databases; wherein existing databases include, but are not limited to, general natural product databases (COCONUT, Super Natural II, NPASS), plant natural product databases (KNApSaCK, CMAUP, TriForC, Alkamid, NPACT DB, BioPhytMol), traditional Chinese medicine natural product databases (TCM@Taiwan, CEMTDD, CHDD, ETCM, TM-MC, TCMID, YaTCM), microbial natural product databases (StreptomeDB, NP Altas, ProCarDB, PAMDB, Lichen Database), marine natural product databases (MNPD, SWMD), natural product databases from different countries and regions (IMPPAT, NeMedPlant, MedPServer, TlPdb, AfroDB, ANPDB, BIOFACQUIM, NUBBEDB), and food natural product databases (FooDB, BitterDB, Phenol-Explorer, PhytoHub, SuperSweet). Databases include: toxic natural product databases (Exposome-Explorer, T3DB, Snake Neurotoxin Database, TPPT), natural product industry directories (Greenpharma, AnalytiConDiscovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol), databases deduplicated using MS data (MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS), and databases deduplicated using NMR data (NMRShiftDB, NAPROC-13), etc.

[0042] In some implementations, the candidate drug comprises various pharmaceutically acceptable salt forms.

[0043] In some embodiments, the candidate drug includes a drug or a drug composition for use alone.

[0044] Furthermore, the pharmaceutical composition refers to a combination of a candidate drug and a pharmaceutically acceptable carrier.

[0045] As used herein, the term "pharmaceutically acceptable" refers to a compound, material, composition, or dosage form that, to a reasonable medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that meets a reasonable benefit / risk ratio. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, or amino acid copolymers. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions may comprise fluids such as water, saline, glycerol, or ethanol. Such carriers may also contain excipients such as wetting agents or emulsifiers, pH buffers, etc.

[0046] The fifth aspect of the present invention provides a method for screening candidate drugs for myocardial fibrosis-related diseases, the method comprising administering a drug to an animal model obtained by the method provided in the second aspect of the present invention and / or cells derived from an animal model obtained by the method provided in the second aspect of the present invention, wherein if the drug can improve the symptoms of myocardial fibrosis-related diseases in the animal model or cells derived from the animal model, then the drug is a candidate drug for myocardial fibrosis-related diseases.

[0047] In some implementations, the improvement refers to a reduction of at least about 10%, at least about 30%, at least about 50%, or at least about 80% in symptoms of the animal model (such as cardiac ejection fraction or fractional shortening) or in cellular phenotypes derived from the animal model (such as expression levels of fibrosis markers) compared to before drug administration.

[0048] Advantages and beneficial effects of the present invention:

[0049] This invention discovers that orthotopic injection of recombinant adeno-associated virus expressing RGS4 into the mouse heart can promote the progression of myocardial fibrosis by upregulating the expression level of the RGS4 gene in myocardial tissue, and can be used to induce a mouse model of myocardial fibrosis. This invention provides cardiovascular medical researchers with a stable and simple animal model of myocardial fibrosis, offering new ideas and strategies for exploring the mechanisms of action and drug targets of myocardial fibrosis, and has broad prospects for pharmacological research applications. Attached Figure Description

[0050] Figure 1 The following figures illustrate the expression of RGS4 protein overexpression in mouse hearts: A represents the expression of RGS4 protein in mouse hearts after 7 days of overexpression; B represents the expression of RGS4 protein in mouse hearts after 14 days of overexpression; and C represents the expression of RGS4 protein in mouse hearts after 28 days of overexpression.

[0051] Figure 2 The effect of RGS4 overexpression on cardiac function in mouse hearts is shown in Figure A. A represents a representative echocardiogram image; B is a quantitative graph of cardiac ejection fraction in Figure A; C is a quantitative graph of cardiac short-axis shortening in Figure A; and D shows the results of hematoxylin-eosin (HE) staining and Masson staining of cardiac pathological sections.

[0052] Figure 3 To investigate the effect of RGS4 overexpression on myocardial fibrosis markers in mouse hearts, the following data were presented: A) Expression of α-SMA protein in mouse hearts after 14 days of overexpression; B) Expression of α-SMA protein in mouse hearts after 28 days of overexpression; C) Expression of collagen I protein in mouse hearts after 14 days of overexpression; D) Expression of collagen I protein in mouse hearts after 28 days of overexpression; E) Expression of collagen III protein in mouse hearts after 7 days of overexpression; F) Expression of collagen III protein in mouse hearts after 14 days of overexpression; and G) Expression of collagen III protein in mouse hearts after 28 days of overexpression.

[0053] Figure 4 This is the carrier structure used in this invention. Detailed Implementation

[0054] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0055] As used in this invention, the terms “having,” “comprising,” or “including,” or any grammatical variations thereof, are used in a non-exclusive manner. Thus, these terms can all refer to a situation where no other features exist in the entity described in this context besides the features introduced by these terms, and can also refer to a situation where one or more other features are present.

[0056] Furthermore, as used in this invention, the terms “preferred,” “more preferred,” “most preferred,” “particularly,” “more particularly,” “specifically,” “more specifically,” or similar terms are used in combination with optional features without limiting other possibilities.

[0057] Unless otherwise stated, all figures used in this specification and claims to represent volume, weight, temperature, time, density, parts by weight, technical effect, etc., should in any case be understood to be modified by the terms "about" or "approximately". Therefore, unless indicated to the contrary, the numerical parameters listed in the specification and appended claims are approximate values. Unless otherwise stated, the terminology used herein has its common meaning as understood by one of ordinary skill in the art, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by one of those skilled in the art.

[0058] Example 1: Construction and overexpression efficiency verification of AAV9-RGS4 adeno-associated virus vector

[0059] I. Experimental Methods

[0060] 1. Experimental materials:

[0061] (1) Mice: Male C57BL / 6 mice (20-25 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0062] (2) Antibody:

[0063] Primary antibodies: α-SMA antibody (Proteintech, 14395-1-AP), collagen-I antibody (Proteintech, 66761-1-Ig), collagen-III antibody (Proteintech, 22734-1-AP), RGS4 antibody (Santa Cruz, sc-398348), β-actin antibody (Proteintech, 660091-lg); Secondary antibodies: Anti-rabbit IgG, HRP-linked Antibody antibody (Cell Signaling Technology, 7074P2), Anti-mouse IgG, HRP-linked Antibody antibody (Cell Signaling Technology, 7076P2).

[0064] 2. Experimental Procedure

[0065] (1) AAV9-RGS4 carrier information: The carrier structure is as follows Figure 4 As shown.

[0066] RGS4 amino acid sequence:

[0067] MCKGLAGLPASCLRSAKDMKHRLGFLLQKSDSCEHSSSHSKKDKVVTCQRVSQEEVKKWAESLEENLIHHECGLAAFKAFLKSEYSEENIDFWISCEEYKKIKSP SKLSPKAKKIYNEFISVQATKEVNLDSCTREETSRNMLQPTITCFDEAQKKIFNLMEKDSYRRFLKSRFYLDLTNPSSCGAEKQKGAKSSADCTSLVSQCA (SEQ ID NO:1)

[0068] RGS4 nucleotide sequence:

[0069]

[0070] (2) AAV9-RGS4 vector injection:

[0071] C57BL / 6 mice were placed with their hearts exposed and a chest expander fixed in place. An appropriate volume of viral diluent was dispensed using an insulin needle. The insulin needle was bent to approximately a 120-degree angle using hemostatic forceps. A small arterial clamp was used to clamp the mouse's aorta, ensuring the heart was in a state of microcirculation only. The insulin needle was inserted into the left ventricle along the apex, and the viral diluent was slowly injected. The needle was then withdrawn, and the arterial clamp was removed after approximately 2-3 cardiac cycles. The chest expander was removed, allowing the serratus anterior and pectoralis major muscles to return to their physiological positions automatically. The skin was sutured with 3-0 sutures. The mice were removed from the surgical board, the ventilator was removed, and once spontaneous breathing resumed, they were returned to their cages for continued rearing. Samples were collected at 7, 14, and 28 days for later use.

[0072] (3) Western blot experiment:

[0073] Tissue blocks were homogenized, centrifuged, and BCA protein concentration was determined. Protein concentration was calculated based on the protein concentration standard curve. 5× protein loading buffer (SDS-PAGE) was added, and the mixture was heated at 100℃ for 5 min to denature. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an NC membrane. After blocking with 5% skim milk, the NC membrane was incubated with primary antibody overnight at 4℃. The next day, it was incubated with fluorescent secondary antibody to complete primary-secondary antibody hybridization. Membrane imaging was performed using an Odyssey infrared fluorescence scanning system, with GAPDH and β-actin as internal controls. Protein bands were analyzed using the Odyssey CLx2.1 image analysis software for integrated optical density values.

[0074] II. Experimental Results

[0075] like Figure 1 As shown, on the 7th day after orthotopic injection of AAV9-RGS4 into the myocardium ( Figure 1 A), 14 days ( Figure 1 B), 28 days ( Figure 1 After C), RGS4 was consistently expressed at a high level, proving that in vivo overexpression of RGS4 was successful.

[0076] Example 2: Effects of RGS4 overexpression on the progression of myocardial fibrosis in mice

[0077] I. Experimental Methods

[0078] 1. Experimental materials: Same as in Example 1.

[0079] 2. Experimental steps:

[0080] (1) Establishment of an animal model of myocardial infarction:

[0081] Before starting the experiment, C57BL / 6 mice were retrieved from the animal center and placed in the animal housing for acclimatization. After three days of housing, they were subjected to a 12-hour fasting and water deprivation. One mouse to be operated on was weighed, and the anesthetic afluttin (0.1 mL / 10 g) was administered via intraperitoneal injection. After anesthesia, the mouse was placed on a surgical board, and its teeth, tail, and limbs were secured. Tracheal intubation was then performed, and the mouse was kept breathing by a small animal ventilator. The chest area was then prepared by trimming the chest hair with curved scissors and disinfecting with iodine. Make a 1-1.5 cm oblique incision in the left side of the center of the mouse's chest using straight scissors. Use curved hemostats for blunt dissection to separate the epidermis and muscle, then further separate the pectoralis major and serratus anterior muscles to expose the ribs. Make an incision in the intercostal space between the third and fourth ribs using curved forceps. Use a chest expander to open the thoracic cavity and expose the heart. Fix the expander in place. Use 8-0 sutures to ligate the left anterior descending coronary artery of the heart; successful ligation is indicated by a noticeable white discoloration of the left ventricle. Remove the expander, allowing the serratus anterior and pectoralis major muscles to return to their physiological positions. Suture the epidermis with 3-0 sutures. Remove the mouse from the surgical board, remove the ventilator, and return it to its cage for continued rearing once spontaneous breathing resumes. Harvest the mouse four weeks later.

[0082] (2) Small animal echocardiography:

[0083] Echocardiography in small animals was used to detect cardiac ejection fraction (EF%) and fractional shortening (FS%). Mice were anesthetized with aflutole, and the hair on their chests was shaved off with a power clipper, followed by deep hair removal cream to expose the chest skin. The mice were fixed supine to the operating platform with tape, and coupling gel was evenly applied to the exposed chest skin and the electrode contact points on the limbs. The probe was moved to find a suitable position for imaging. First, the long axis image of the left ventricle was saved, and then the probe was adjusted to save the short axis image of the mouse's left ventricle. The left ventricular end-diastolic internal dimension (LVIDd) and left ventricular end-systolic internal dimension (LVIDs) were measured. The ejection fraction (EF%) was calculated by the instrument's built-in software, and the fractional shortening (FS%) was calculated using the formula ((LVIDd-LVIDs) / LVIDd)×100.

[0084] The other experimental steps are the same as in Example 1.

[0085] II. Experimental Results

[0086] On days 7, 14, and 28 after injection of AAV9-RGS4, the cardiac ejection fraction and fractional shortening of mice were reduced. Figure 2 AC). Hematoxylin-eosin (HE) staining and Masson staining revealed increased RGS4 expression, increased collagen secretion, and significant collagen deposition. Figure 2 D). There was no significant difference in cardiac function or pathological status between 28 days after RGS4 overexpression and 28 days after myocardial infarction surgery.

[0087] The expression levels of myocardial fibrosis markers α-SMA, collagen I, and collagen III in myocardial tissue were detected after injection of AAV9-RGS4. Figure 3 As shown, after overexpression of RGS4, the expression of α-SMA, collagen I, and collagen III proteins all increased significantly. Among them, the markers α-SMA and collagen I began to increase significantly 14 days after injection of AAV9-RGS4. Figure 3 AD), the marker collagenne III began to increase significantly 7 days after AAV9-RGS4 injection ( Figure 3 EG).

[0088] The above results collectively demonstrate that overexpression of RGS4 can successfully construct a mouse myocardial fibrosis pathological model, and the model construction efficiency is not significantly different from that of commonly used myocardial infarction surgical construction methods in this field.

[0089] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A myocardial fibrosis pathological animal model inducer, characterized by, The inducer is a RGS4 gene promoter.

2. The inducer according to claim 1, characterized in that, The RGS4 gene is a gene encoding RGS4 protein with an amino acid sequence as shown in SEQ ID NO:

1. Preferably, the nucleotide sequence of the coding chain of the RGS4 gene encoding gene is as shown in SEQ ID NO:

2.

3. The inducer according to claim 1, wherein The promoter comprises a RGS4 overexpression vector promoting expression of the RGS4 gene. The vector comprises a non-viral vector or a viral vector. Preferably, the promoter is a RGS4 overexpression viral vector promoting expression of the RGS4 gene. Preferably, the viral vector comprises an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a poxvirus vector, a vaccinia virus vector or a herpes virus vector. Preferably, the promoter is a RGS4 overexpression adeno-associated virus vector promoting expression of the RGS4 gene.

4. The inducer according to claim 3, wherein The adeno-associated virus vector comprises AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV-DJ. Preferably, the adeno-associated virus vector is AAV9.

5. The inducer of claim 1, wherein, The animal comprises a mouse, a rat, a guinea pig, a hamster, a rabbit, a dog, a pig, a monkey. Preferably, the animal is a mouse.

6. The inducer of claim 1, wherein, The induction mode of the inducer is myocardial in situ injection.

7. A method for constructing an animal model of myocardial fibrosis pathology, characterized in that, The method comprises using the inducer of any one of claims 1-6 to promote expression of the RGS4 gene in the heart of an animal.

8. Use of the RGS4 gene of claim 2 in constructing an animal model of myocardial fibrosis pathology.

9. Use of the animal model obtained by the method of claim 7, characterized in that, The use comprises any one of the following: 1) use in research on the pathogenesis of myocardial fibrosis diseases; 2) use in screening of candidate drugs for myocardial fibrosis related diseases.

10. A method for screening a candidate drug for a myocardial fibrosis-related disease, characterized by, The method comprises administering a drug to the animal model obtained by the method of claim 7 and / or cells derived from the animal model obtained by the method of claim 7, and if the drug can improve the symptoms related to myocardial fibrosis of the animal model or the cells derived from the animal model, the drug is a candidate drug for myocardial fibrosis related diseases.

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