A method for constructing a fluorescent thrombus zebrafish model, its application and a drug for treating thrombus

Abstract

The present invention identifies the phenotype of abnormal aggregation of smarca5 mutant red blood cells, and studies the development, cell morphology, number and other characteristics of corresponding erythroid cells and myeloid cells in smarca5 mutants. It partially analyzes the mechanism of smarca5 regulating the phenotype of red blood cell aggregation, thereby constructs a fluorescent thrombus zebrafish model, establishes a venous thromboembolism disease model, and provides a method, use and drug for preventing red blood cell aggregation, or anti-thrombosis, or promoting thrombolysis, or reducing thrombosis formation in a subject, with a view to being used as a thromboembolism disease model for drug screening, which has clinical guiding significance.

Description

Technical Field

[0001] This application relates to the field of biotechnology and new pharmaceutical technologies, and specifically relates to a method for constructing a fluorescent thrombus zebrafish model and a drug for treating thrombus Background Art

[0002] Mature red blood cells are rich in hemoglobin and have the function of carrying oxygen. Because of their cell deformation ability, red blood cells can flow in all blood vessels and are crucial for maintaining the body's homeostasis (Rodriguez-Garcia et al., 2016). In addition, red blood cells are involved in the maintenance of thrombosis and hemostasis (Weisel & Litvinov, 2019). Defects in red blood cells can lead to the occurrence of various diseases, including hemoglobinopathy anemia, hemolytic anemia, and thrombosis (Kato et al., 2018; Roumenina, Rayes, Lacroix-Desmazes, & Dimitrov, 2016; Weisel & Litvinov, 2019). Among them, the occurrence of thrombosis seriously endangers human health (Wendelboe & Raskob, 2016). Studying the occurrence of thrombosis through animal disease models helps to explore the mechanism of thrombosis and find treatment methods. Nowadays, the inferior vena cava ligation model, free radical thrombosis formation model, and gene knockout model are widely used in the study of murine venous thrombosis (Diaz et al., 2019; Grover & Mackman, 2019). These models mainly induce thrombosis formation by changing blood flow, damaging the endothelium, and regulating coagulation factors. In addition, phenylhydrazine treatment can damage red blood cells, resulting in the externalization of phosphatidylserine of red blood cells and the generation of free radicals, thereby leading to the occurrence of thrombosis (Zhu et al., 2016). However, current research still lacks dynamic observation of thrombosis formation and in-depth mechanism research Summary of the Invention

[0003] Starting from the phenotype of abnormal aggregation of mutant red blood cells, this invention conducts research on thrombo-disease models in order to use them as thrombo-disease models for drug screening and explore their clinical guiding significance

[0004] This invention specifically relates to the following technical solutions

[0005] 1. A method for constructing a fluorescent thrombus zebrafish model, characterized by comprising

[0006] Obtaining zebrafish heterozygotes with smarca5 mutations through gene editing methods

[0007] Mate the zebrafish heterozygotes with the smarca5 mutation and the transgenic zebrafish with fluorescence, and screen from the offspring for fluorescent thrombus zebrafish with the smarca5 mutation phenotype and fluorescence.

[0008] 2. The method according to item 1, characterized in that the zebrafish heterozygotes with the smarca5 mutation are obtained by editing the chromosomes of zebrafish by the CRISPR / Cas9 method.

[0009] 3. The method according to item 1 or 2, characterized in that the transgenic zebrafish with fluorescence are transgenic zebrafish with different fluorescence markers, and preferably different fluorescence markers are used to label red blood cells and vascular endothelial cells respectively.

[0010] 4. The method according to any one of items 1 to 3, characterized in that

[0011] Obtain zebrafish heterozygotes with the smarca5 mutation by gene editing method and determine the zebrafish heterozygotes F0 with the smarca5 mutation;

[0012] The steps of mating the zebrafish heterozygotes with the smarca5 mutation and the transgenic zebrafish with fluorescence and screening from the offspring for fluorescent thrombus zebrafish with the smarca5 mutation phenotype and fluorescence include:

[0013] Mate the smarca5 mutant heterozygotes F0 with the transgenic zebrafish with fluorescence to obtain the smarca5 mutant F1 generation with fluorescence;

[0014] Select heterozygotes with the smarca5 mutation from the F1 generation mutants for mating between male and female to obtain F2 generation mutant embryos;

[0015] Genetically identify the homozygotes with the smarca5 mutation in the F2 generation mutants as the fluorescent thrombus zebrafish model.

[0016] 5. The method according to any one of items 1 to 4, wherein the fluorescent thrombus zebrafish model is a fluorescent thrombus zebrafish embryo.

[0017] 6. A method for anti-thrombosis or promoting thrombolysis, which includes performing gene therapy on a subject in need thereof to repair the smarca5 mutation.

[0018] 7. Use of the fluorescent thrombus zebrafish in screening drugs for promoting thrombolysis or reducing thrombosis formation or anti-thrombosis in a subject.

[0019] 8. A method for preventing red blood cell aggregation, which includes overexpressing keap1a or knocking down hmox1a in vitro or in vivo.

[0020] 9. Use of a substance that promotes the overexpression of keap1a or reduces the activation of hmox1a in the preparation of a drug for anti - thrombosis, promoting thrombolysis, or reducing thrombosis in a subject.

[0021] 10. A method for anti - thrombosis, promoting thrombolysis, or reducing thrombosis in a subject, comprising: overexpressing keap1a in a subject in need thereof or knocking down hmox1a in the subject by means of genetic manipulation.

[0022] 11. The method according to item 10, wherein the genetic manipulation includes using gene editing or gene expression.

[0023] 12. A drug for anti - thrombosis, promoting thrombolysis, or reducing thrombosis in a subject, comprising: a substance that inhibits the mutation of smarca5, a substance that promotes the overexpression of keap1a, or a substance that reduces the activation of hmox1a.

[0024] Advantages of the Invention

[0025] 1. The present invention identifies the phenotype of abnormal aggregation of smarca5 mutant red blood cells.

[0026] 2. The present invention partially analyzes the mechanism by which smarca5 regulates the phenotype of red blood cell aggregation.

[0027] 3. The present invention establishes a venous thrombosis disease model for use as a thrombosis disease model for drug screening, which has clinical guiding significance. Description of the Drawings

[0028] Figure 1 A shows the comparison of red blood cell aggregation between control group embryos and smarca5 homozygous mutant embryos. The area marked by the dotted line is the blood clot in the caudal vein.

[0029] Figure 1 B shows the comparison of the expression of the primary red blood cell marker gene scl in red blood cells between control group embryos and smarca5 homozygous mutant embryos. The area marked in the black rectangular frame is scl in the blood clot in the caudal vein (indicated by the arrow).

[0030] Figure 1 C shows the comparison of red blood cell aggregation in the tail vessels between control group embryos and smarca5 homozygous mutant embryos. The cells marked by the arrow are gata1:dsRed cells in the vascular lumen.

[0031] Figure 1 D shows the comparison of the expression of the erythroid cell marker genes gata1, ikaros, and scl in red blood cells between control group embryos and smarca5 homozygous mutant embryos.

[0032] Figure 1 E represents the expression levels of erythroid cell marker genes gata1, ikaros, hbae1, and hbbe1 in control group embryos and smarca5 homozygous mutant embryos. Data are mean ± SD. Asterisks indicate significance of differences (n.s. no significant difference). P values were calculated by two-tailed Student's t-test.

[0033] Figure 2 A shows the comparison of myeloid cell distribution in control group embryos and smarca5 homozygous mutant embryos. The green fluorescence-labeled area is the area within the circle.

[0034] Figure 2 B shows the comparison of the expression of myeloid cell marker genes pu.1 and lyz in control group embryos and smarca5 homozygous mutant embryos.

[0035] Figure 2 C represents the expression levels of myeloid cell marker genes pu.1, mfap4, and lyz in control group embryos and smarca5 homozygous mutant embryos. Data are mean ± SD. Asterisks indicate significance of differences (n.s. no significant difference). P values were calculated by two-tailed Student's t-test.

[0036] Figure 3 A shows the aggregation process of red blood cells in control group embryos and smarca5 homozygous mutant embryos.

[0037] Figure 3 B shows the blood cell aggregation phenotype in smarca5 mutant and control parabiotic embryos.

[0038] Figure 3 C shows the distribution of red blood cells (gata1:GFP + labeled) derived from smarca5 mutants and red blood cells (gata1:dsRed + labeled) derived from control group embryos in parabiotic embryos. The green fluorescence-labeled (gata1:GFP + labeled) area is the area within the circle.

[0039] Figure 3 D shows the distribution of megakaryocyte precursors (CD41:GFP high ) in control group embryos and smarca5 homozygous mutant embryos.

[0040] Figure 3 E shows the alleviating effect of drug treatment on the red blood cell aggregation phenotype.

[0041] Figure 3 F shows the red blood cell aggregation phenotype in control group embryos and smarca5 homozygous mutant embryos after treatment with the drug Argatroban.

[0042] Figure 3 G shows the statistical results of the erythrocyte aggregation phenotypes in the control group embryos and smarca5 homozygous mutant embryos after treatment with the drug Argatroban. The data are mean ± SD. Asterisks indicate the significance of the difference (**p < 0.01). The P value was calculated by Student's t-test, two-tailed.

[0043] Figure 4 A shows the distribution of hematopoietic stem and progenitor cells in the control group embryos and smarca5 homozygous mutant embryos. The green fluorescence-labeled area is the area within the circle.

[0044] Figure 4 B shows the statistical results of the number of hematopoietic stem and progenitor cells in the tail hematopoietic tissue in the control group embryos and smarca5 homozygous mutant embryos. The data are mean ± SD. Asterisks indicate the significance of the difference (n.s. no significant difference). The P value was calculated by Student's t-test, two-tailed.

[0045] Figure 5 A is a longitudinal section view of the tail artery and venous plexus in the control group embryos observed by transmission electron microscopy.

[0046] Figure 5 B is a subcellular structure diagram of erythrocytes in the control group embryos. The arrow indicates a mitochondrion with normal structure.

[0047] Figure 5 C is a longitudinal section view of the tail artery and venous plexus in the smarca5 homozygous mutant embryos observed by transmission electron microscopy.

[0048] Figure 5 D shows the erythrocyte aggregation in the mutant tail vein region.

[0049] Figure 5 E is the subcellular structure of erythrocytes in the mutant group. The arrow indicates a mitochondrion with abnormal structure. Ery, erythrocyte; EC, endothelial cell; Mito, mitochondrion; Nuc, nucleus; Cyto, cytoplasm.

[0050] Figure 6 A is the flow cytometry analysis of the proportion of erythrocytes (gata1:dsRed + ) in the control group embryos and smarca5 homozygous mutant embryos.

[0051] Figure 6 B is the flow cytometry statistical results of the proportion of erythrocytes (gata1:dsRed + ) in the control group embryos and smarca5 homozygous mutant embryos.

[0052] Figure 6C shows blood smears and Giemsa staining of control group embryos and smarca5 homozygous mutant embryos.

[0053] Figure 6 D shows the analysis of the nuclear-cytoplasmic ratio of red blood cells in control group embryos and smarca5 homozygous mutant embryos. Data are mean ± SD. Asterisks indicate the significance of differences (n.s. no significant difference). P values were calculated by two-tailed Student's t-test.

[0054] Figure 7 A shows the flow chart of the RNA-seq sequencing experiment.

[0055] Figure 7 B shows a volcano plot of gene expression changes in red blood cells after smarca5 knockout.

[0056] Figure 7 C shows differential pathways of red blood cells in control group embryos and smarca5 homozygous mutant embryos analyzed by gene set variation analysis (GSVA). The x-axis represents the t value of the GSVA score.

[0057] Figure 7 D shows the enrichment changes of the "erythrocyte homeostasis" and "inflammatory response" signaling pathways in red blood cells of smarca5 homozygous mutant embryos.

[0058] Figure 8 A shows the principal component analysis plot of RNA-seq samples of red blood cells in control group embryos and smarca5 homozygous mutant embryos.

[0059] Figure 8 B shows the expression of hbae1.1, hbae1.2, hbae1.3, hbae3, hbbe1.3, hbbe2, hbbe3, hbaa1, hbba1, and hbba2 in control group embryos and smarca5 homozygous mutant embryos.

[0060] Figure 8 C shows in situ hybridization demonstration of the expression of hbae1, hbae3, hbbe1, hbbe2, hbbe3, hbaa1, hbba1, and hbba2 in control group embryos and smarca5 homozygous mutant embryos.

[0061] Figure 8 D shows the dianisidine staining of control group embryos and smarca5 homozygous mutant embryos. Black arrows indicate blood clots in mutants.

[0062] Figure 8 E shows the expression levels of myeloid cell marker genes spi1a, spi1b, mfap4, and lyz in control group embryos and smarca5 homozygous mutant embryos.

[0063] Figure 8 F shows the aggregation phenotypes of red blood cells in smarca5 homozygous mutants and control embryos after injection of pu.1 MO.

[0064] Figure 8 G shows the statistical results of the aggregation phenotypes of red blood cells in smarca5 homozygous mutants and control embryos after injection of pu.1 MO. Data are mean ± SD. Asterisks indicate the significance of differences (n.s. no significant difference). P values were calculated by Student's t-test, two-tailed.

[0065] Figure 9 A is a Venn diagram showing the number of genes with open promoter regions and distal regulatory regions in control embryos and smarca5 homozygous mutants.

[0066] Figure 9 B shows motif screening for regions with decreased chromatin openness after deletion of smarca5.

[0067] Figure 9 C is a Venn diagram showing the number of genes with both upregulated and downregulated chromatin openness and transcription levels in the promoter regions of control embryos and smarca5 homozygous mutants.

[0068] Figure 9 D shows genes with high expression and concomitant chromatin openness in control embryos and smarca5 homozygous mutants, respectively.

[0069] Figure 10 A heatmap showing the distribution of ATAC-seq peaks in the 1 kb intervals upstream and downstream of the transcription start site (TSS). The left heatmap shows the distribution of nucleosome-free (less than 100 bp), and the right shows the distribution of mononucleosomes (180 - 247 bp).

[0070] Figure 10 B is Figure 10 A distribution map of ATAC-seq peaks near the TSS in A.

[0071] Figure 10 C shows the results of principal component analysis of ATAC-seq samples of red blood cells in smarca5 homozygous mutants and their control groups.

[0072] Figure 10 D shows the distribution characteristics of ATAC-seq peaks of mutants and their control groups across the entire genome.

[0073] Figure 10 E is a Venn diagram showing the number of genes with both upregulated and downregulated chromatin openness and transcription levels in the distal regulatory regions of the mutant group.

[0074] Figure 11A shows the ATAC-seq peaks in the keap1 promoter region of control group embryos and smarca5 homozygous mutant embryos. The black arrows mark the predicted Gata1 binding sites.

[0075] Figure 11 B shows the expression levels of keap1a in red blood cells of control group embryos and smarca5 homozygous mutant embryos.

[0076] Figure 11 C shows the expression of hmox1a, gclc, ggt1b, gsr, gstp1, gstk1, fbp1a, gsto2, prdx1, pgd and g6pd in red blood cells of control group embryos and smarca5 homozygous mutant embryos.

[0077] Figure 11 D shows the fluorescence image of EGFP expression in transgenic fish Tg(hsp70:keap1a-EGFP) at 2 days post-fertilization after heat shock treatment (top), and the aggregation phenotype of red blood cells in smarca5 homozygous mutant and control group embryos after heat shock treatment (bottom).

[0078] Figure 11 E is Figure 11 The statistical results of the red blood cell aggregation phenotype in D.

[0079] Figure 11 F shows the aggregation phenotype of red blood cells in control group embryos and smarca5 homozygous mutant embryos after injection of hmox1a MO.

[0080] Figure 11 G is Figure 11 The statistical results of the red blood cell aggregation phenotype in F.

[0081] Data are mean ± SD (B, C, E, G). Asterisks indicate the significance of differences (*p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significant difference). P values were calculated by two-tailed Student's t-test.

[0082] Figure 11 H is a schematic diagram showing that Smarca5 affects the Keap1-Nrf2 signaling pathway and the expression of downstream target gene hmox1a by regulating the chromatin openness of the keap1a promoter region.

[0083] Figure 12 A shows the phenotypes of tail vein red blood cell aggregation in control group embryos and smarca5 homozygous mutant embryos under control treatment and glutathione treatment by microscopic observation.

[0084] Figure 12 B is the statistical results of the red blood cell aggregation phenotype in 12A.

[0085] Data are mean ± SD. Asterisks indicate significance of differences (*p < 0.05, **p < 0.01). P values were calculated by Student's t-test, two-tailed. Detailed implementation manners

[0086] The following elaborates and illustrates the implementation manners of the present application through specific embodiments, but the following content should not be construed as any limitation to the present application.

[0087] Unless otherwise defined, the technical and scientific terms in this specification have the same meanings as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein can be used in experiments or practical applications, the materials and methods are described hereinafter. In case of conflict, the present specification, including its definitions, shall prevail. Additionally, the materials, methods, and examples are for illustrative purposes only and are not restrictive. The following further illustrates the present application with specific embodiments, but does not limit the scope of the present application.

[0088] Definition

[0089] In the examples described below, multiple terms are used. To provide a clear and consistent understanding of the specification and claims, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meanings as those commonly understood by one of ordinary skill in the art to which the present invention pertains. The disclosures of all publications, patent applications, patents, and other references are incorporated herein by reference in their entirety.

[0090] As used herein, unless otherwise defined, the term "ordinary scientific terms" refers to technical and scientific terms having the same meanings as those commonly understood by one of ordinary skill in the art to which the present invention pertains. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the implementation of the present invention. Indeed, the present invention is in no way limited to the methods and materials described, and the implementation of conventional techniques in the fields of molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing, and related fields is well known to those skilled in the art.

[0091] As used herein, the term "expression level" of a gene refers to the amount of RNA transcript transcribed from the gene and / or the amount of protein that can be translated from the RNA transcript, such as mRNA. For example, for a gene encoding miRNA, the expression level can be determined by, for example, quantifying the amount of RNA transcript expressed using standard methods such as quantitative PCR of mature miRNA, microarray, or RNA blot. Alternatively, the expression level can also be determined by measuring the effect of miRNA on target mRNA.

[0092] As used herein, the term "expression of a gene" refers to the process of transcribing a DNA region operably linked to a suitable regulatory region, particularly a promoter, into a biologically active, i.e., translatable into a biologically active protein or peptide (or an active peptide fragment), or (e.g., in post-transcriptional gene silencing or RNAi) an RNA that is itself active.

[0093] As used herein, the term "gene" generally refers to any DNA fragment related to a biological function. Genes encompass sequences, including but not limited to coding sequences, promoter regions, cis-regulatory sequences, non-expressed DNA fragments that are specific recognition sequences of regulatory proteins, non-expressed DNA fragments that contribute to gene expression, DNA fragments designed to have desired parameters, or combinations thereof.

[0094] As used herein, the term "gene expression" refers to the cellular process of producing a biologically active polypeptide from a DNA sequence.

[0095] As used herein, the term "phenotype" refers to at least one observable characteristic or trait of an organism or a cell of an organism, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and products of behavior. A phenotype results from the expression of genes as well as the influence of environmental factors, and the interaction between the two. Although a phenotype is a collection of observable characteristics exhibited by an organism, the term "phenome" is sometimes used to refer to the collection of traits, and the simultaneous study of them is called "phenomics".

[0096] As used herein, the terms "high-throughput sequencing", "next-generation sequencing", and "deep sequencing" refer to sequencing technologies that can generate a large number of sequence reads, typically on the order of thousands (i.e., tens of thousands or hundreds of thousands) or millions, rather than a few hundred at a time. High-throughput sequencing is distinct from and different from conventional Sanger or capillary sequencing.

[0097] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, which is located upstream of the transcription start site of the gene with respect to the transcription direction, and is structurally recognized by the presence of: a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art to directly or indirectly regulate the amount of transcription from the promoter.

[0098] As used herein, the term "heterozygote" refers to an individual with a genotype in which the two alleles at the same locus on homologous chromosomes are different. Offspring produced by mating between heterozygotes will exhibit segregation of traits.

[0099] As used herein, the term "homozygote", also known as homozygote and homozygous combination, refers to an individual with a genotype in which the alleles at the same locus on homologous chromosomes in a diploid are the same.

[0100] As used herein, the term "locus" refers to the specific position occupied by a gene on a chromosome.

[0101] As used herein, the term "sequencing" refers to determining the sequence of nucleotides (base sequence) in a nucleic acid sample such as DNA or RNA.

[0102] The terms "individual", "subject", "host", and "patient" are used interchangeably herein and refer to any mammalian subject for which diagnosis, treatment, or therapy is desired, including species such as dogs, cats, horses, cows, sheep, etc., as well as primates, specifically humans.

[0103] As used herein, the term "CRISPR / Cas9" is an adaptive immune defense formed by bacteria and archaea during long-term evolution, which can be used to combat invading viruses and foreign DNA. The CRISPR / Cas9 gene editing technology is a technology for specific DNA modification of target genes. The CRISPR / Cas9-based gene editing technology has shown great application prospects in a series of gene therapy application fields, such as blood diseases, tumors, and other genetic diseases. The technical achievements have been applied to the precise modification of the genomes of human cells, zebrafish, mice, and bacteria.

[0104] As used herein, the term "Cas9" refers to an enzyme (wild-type or recombinant) that can exhibit minimal endonuclease activity (e.g., cleave a phosphodiester bond within a polynucleotide) guided by a CRISPR RNA (crRNA) that carries a complementary sequence to a target polynucleotide. Cas9 polypeptides are known in the art and include Cas9 polypeptides from any of a variety of biological sources, including, for example, prokaryotic sources such as bacteria and archaea. Bacterial Cas9 includes Actinobacteria (e.g., Actinomyces naeslundii) Cas9, Aquificae Cas9, Bacteroidetes Cas9, Chlamydiae Cas9, Chloroflexi Cas9, Cyanobacteria Cas9, Elusimicrobia Cas9, Fibrobacteres Cas9, Firmicutes Cas9 (e.g., Streptococcus pyogenes Cas9, Streptococcus thermophilus Cas9, Listeria innocua Cas9, Streptococcus agalactiae Cas9, Streptococcus mutans Cas9, and Enterococcus faecalis Cas9), Fusobacteria Cas9, Proteobacteria (e.g., Neisseria meningitides, Campylobacter jejuni, and Campylobacter lari) Cas9, Spirochaetes (e.g., Treponema denticola) Cas9, etc. Archaeal Cas9 includes Euryarchaeota Cas9 (e.g., Methanococcus maripaludis Cas9), etc.A variety of Cas9 and related polypeptides are known and have been reviewed, for example, in Makarova et al. (2011) Nature Reviews Microbiology 9:467-477, Makarova et al. (2011) Biology Direct 6:38, Haft et al. (2005) PLOS Computational Biology 1:e60, and Chylinski et al. (2013) RNA Biology 10:726-737; K. Makarova et al., An updated evolutionary classification of CRISPR-Cas systems. (2015) Nat. Rev. Microbiol. 13:722-736; and B. Zetsche et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. (2015) Cell. 163(3):759-771.

[0105] As used herein, the term "overexpression" in the context of the present invention refers to the overexpression of a gene, i.e., the upregulation of gene expression, meaning that the gene is overly transcribed, translated, and the final gene expression product exceeds the normal level. There are mainly three ways to overexpress a gene: constructing an overexpression of a foreign gene, CRISPR SAM, and saRNA.

[0106] As used herein, when referring to the effect of RNAi on gene expression, the term "knockdown" means that the gene expression level is inhibited or reduced to a level lower than that typically observed when examined under substantially the same conditions but in the absence of RNAi.

[0107] As used herein, the term "knockout" refers to the partial or complete inhibition of the expression of an endogenous gene. This is generally accomplished by deleting a portion of the gene or by replacing a portion with a second sequence, but can also be caused by other modifications to the gene, such as introducing a stop codon, mutations in key amino acids, removal of intron junctions, etc. Thus, a "knockout" construct is a nucleic acid sequence, such as a DNA construct, which when introduced into a cell results in the inhibition (partial or complete) of the expression of the polypeptide or protein encoded by the endogenous DNA in the cell. In some embodiments, "knockout" includes mutations such as point mutations, insertions, deletions, frameshifts, or missense mutations.

[0108] As used herein, the term "mutated" refers to a change in a sequence, such as a nucleotide or amino acid sequence, from the native, wild-type, standard, or reference version of the respective sequence, i.e., the non-mutated sequence. A mutated gene can result in a mutated gene product. The mutated gene product differs from the non-mutated gene product by one or more amino acid residues. In some embodiments, the mutated gene that results in a mutated gene product can have a sequence identity of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater with the corresponding non-mutated nucleotide sequence.

[0109] As used herein, the term "constitutive expression" refers to the continuous expression of certain genes in almost all cells of an individual, which are called housekeeping genes. Housekeeping genes are less affected by environmental factors and the continuous expression in most or almost all tissues at various growth stages of an individual is regarded as constitutive gene expression. The meanings include A. continuous and constant expression in most cells, B. gene expression regulated by multiple mechanisms, C. inducible gene expression, D. spatially specific gene expression.

[0110] As used herein, the term "induced expression" refers to the activation of a corresponding gene under the stimulation of a specific environmental signal, resulting in an increase in the gene expression product. Such a gene is called an inducible gene. The process by which an inducible gene shows enhanced expression in a specific environment is called induced expression.

[0111] As used herein, the term "repressed expression" refers to the inhibition of a gene in response to a specific environmental signal. Such a gene is a repressible gene. The process by which the level of the repressible gene expression product decreases is called repressed expression.

[0112] As used herein, with respect to a particular disease condition, the term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of partially or completely curing the disease and / or adverse effects attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease or disorder in a subject, particularly a human, and includes: (a) preventing the occurrence of a disease or disorder in a subject who may be predisposed to the disease but has not been diagnosed as having the disease; (b) inhibiting the disease or disorder, i.e., arresting its development; and (c) alleviating or reducing the disease or disorder, i.e., causing regression of the disease or disorder and / or alleviating one or more symptoms of the disease or disorder. "Treatment" can also encompass the delivery of an agent or the administration of a treatment to provide a pharmacological effect, even in the absence of a disease, disorder, or disease condition. In some embodiments, the term "treatment" is used to refer to the administration of a compound of the present disclosure to alleviate a disease or disorder in a host, preferably a mammalian subject, more preferably a human. Thus, the term "treatment" can include: preventing the occurrence of a disorder in a host, particularly when the host is predisposed to the disease but has not been diagnosed as having the disease; inhibiting the disorder; and / or alleviating or reversing the disorder. With respect to the methods of the present disclosure that relate to preventing a disorder, it should be understood that the term "prevention" does not require complete prevention of the disease state. Instead, as used herein, the term prevention refers to the ability of one of ordinary skill in the art to identify a population that is predisposed to the disorder, such that the administration of a compound of the present disclosure can occur prior to the onset of the disease. The term does not imply that the disease state must be completely avoided.

[0113] As used herein, the term "vector" refers to a nucleic acid molecule capable of mediating entry (e.g., transfer, transport, etc.) of another nucleic acid molecule into a cell. The transferred nucleic acid is generally linked (e.g., inserted) to the vector nucleic acid molecule. A vector can include sequences that direct autonomous replication, or can include sequences sufficient to allow integration into the host cell DNA. It will be apparent to those of ordinary skill in the art that, in addition to the nucleic acid that mediates entry of the transferred nucleic acid, viral vectors can include various viral components. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viral vectors. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors (including lentiviral vectors), and the like.

[0114] In one aspect, the present invention provides a method for constructing a fluorescent thrombus zebrafish model, characterized by comprising:

[0115] Obtaining a zebrafish heterozygote with smarca5 mutation by gene editing method;

[0116] The smarca5 mutant zebrafish heterozygote is mated with a fluorescent transgenic zebrafish, and fluorescent thrombotic zebrafish with a smarca5 mutant phenotype and fluorescence are screened out from the offspring.

[0117] Zebrafish is a common tropical fish with 87% homology to human genes. It has a high degree of biological similarity with humans, a short reproductive cycle, no seasonal mating, many eggs, rapid embryonic development, and can complete the construction of internal organs within 24 hours after fertilization. The embryo is transparent and easy to observe. At present, zebrafish has become one of the most important vertebrate model animals for disease research in the world, and has been successfully used in new drug screening.

[0118] The present invention is to obtain zebrafish smarca5 mutant, i.e. zebrafish smarca5, by gene editing method zko1049a The mutant was developed by Liu Feng's team from the Institute of Zoology, Chinese Academy of Sciences.

[0119] The smarca5 is a key subunit member of the ISWI family. This protein can regulate the nucleosome spacing and thus affect gene expression. The smarca5 mutation inhibits the survival of zebrafish hematopoietic progenitor cells in the tail hematopoietic tissue and their differentiation into blood precursor cells of various lines. In addition, specific knockout of Smarca5 in mouse red blood cells leads to the obstruction of red blood cell maturation and development in the fetal liver, and the mutants show an increase in immature red blood cells accompanied by reduced cell proliferation and increased apoptosis.

[0120] In the present invention, the applicant observed abnormal behavior of red blood cells in the above-mentioned smarca5 mutant, that is, abnormal aggregation of red blood cells occurred in the smarca5 mutant. Based on this, the present invention constructed a fluorescent thrombosis zebrafish model for the mutant.

[0121] Fluorescent transgenic zebrafish are zebrafish that have fluorescent proteins introduced into their bodies and expressed in specific tissues and organs, making it possible to observe the development and physiological changes of specific organs under a fluorescence microscope, dynamically track the entire process of embryonic development, and the effects of exogenous substances or gene mutations on organ development.

[0122] The smarca5 mutant zebrafish heterozygote used in the present invention is obtained by editing the zebrafish chromosome through the CRISPR / Cas9 method.

[0123] The CRISPR / Cas9 method is a gene editing method that can prevent DNA methylation from interfering with gene editing through mutual recognition between nucleic acids, resulting in higher editing efficiency.

[0124] The transgenic zebrafish with fluorescence described in the present invention are transgenic zebrafish with different fluorescence labels, including but not limited to the following strains: Tg(flila:EGFP) y1 , Tg(fliila:nEGFP) y7 , Tg(fliila:EGFP-cdc42wt) y48 , Tg(mTie2:GFP), Tg(kdrl:G-RCFP), Tg(kdrl:G-memCherry) s896 , Tg(kdrl:EGFP) s843 , Tg(flila:DsRed), Tg(flila:EGFP;kdrl:ras-cherry), Tg(fltl:YFP,kdrl:mCherryRed), Tg(stabilin:YFP) hu4453 , Tg(gatal:DsRed) sd2、 , Tg(zp3:fsta,myl7:EGFP), Tg(fli1a:EGFP), Tg(myl7:GFP), Tg(kdrl:EGFP), Tg(kdrl:mCherry), Tg(kdrl:RFP), Tg(fli1a.ep:DsRedEx), Tg(hsp:vegf165);Tg(kdrl:GFP), Tg(zp3b:zar1,myl7:EGFP), Tg2(fli1a:mCherry), Tg(-5.1myl7:DsRed2-NLS), TgBAC(-36nkx2.5:ZsYellow), Tg(kdrl:GFP;gata1:dsRed), etc.

[0125] In some specific embodiments, the transgenic zebrafish with fluorescence are zebrafish that use different fluorescence labels to label red blood cells and vascular endothelial cells respectively.

[0126] In some specific embodiments, the transgenic zebrafish strain with fluorescence is Tg(kdrl:GFP;gata1:dsRed).

[0127] The method for constructing a fluorescent thrombus zebrafish model as described above specifically comprises the following steps:

[0128] Obtain zebrafish heterozygotes with smarca5 mutations by gene editing methods and determine zebrafish heterozygotes F0 with smarca5 mutations;

[0129] The steps of mating the smarca5 mutant zebrafish heterozygotes with transgenic zebrafish with fluorescence and screening for fluorescent thrombus zebrafish with smarca5 mutant phenotypes and fluorescence from the offspring include:

[0130] Mate the smarca5 mutant heterozygote F0 with transgenic zebrafish with fluorescence to obtain the smarca5 mutant F1 generation with fluorescence at the same time;

[0131] Select heterozygotes with smarca5 mutations among the F1 generation mutants for male-female mating to obtain F2 generation mutant embryos;

[0132] Genetic identification of the homozygotes with smarca5 mutations in the F2 generation mutants is the fluorescent thrombus zebrafish model.

[0133] The gene editing methods include, but are not limited to, homologous recombination (HR) technology, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease TALEN technology, clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins (CRISPR) technology. In some specific embodiments, the gene editing method used in the present invention is clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins (CRISPR) technology. Preferably, the gene editing method used is the method using the CRISPR / Cas9 system. In some specific embodiments, the method for constructing the fluorescent thrombus zebrafish model is specifically:

[0134] Take the zebrafish smarca5 mutant constructed by the CRISPR / Cas9 method (smarca5 zko1049a)(Ding et al., 2021) Adult F0 fish were mated with the fluorescently labeled transgenic fish line Tg(kdrl:GFP; gata1:dsRed) (this transgenic line was provided by the laboratory of Steve Wilson (King’s College London, London, United Kingdom)). F1 embryos were collected, and fertilized eggs that simultaneously carried the kdrl:GFP (vascular endothelial cells) and gata1:dsRed (red blood cells) fluorescence were selected under a fluorescence microscope and cultured to adult fish for tail clipping identification. The identification method was to extract genomic DNA using a DNA extraction method, then clone the DNA fragment near the target by PCR, and then identify the gene mutation type by sequencing.

[0135] The DNA extraction methods include, but are not limited to, CTAB method, glass bead method, ultrasonic method, grinding method, freeze-thaw method, guanidine isothiocyanate method, alkaline lysis method, enzymatic method, etc. In some specific embodiments, the DNA extraction method is the alkaline lysis method.

[0136] Adult fish of smarca5 mutants that simultaneously carried kdrl:GFP and gata1:deRed fluorescence were determined as F1 mutants. Heterozygotes with smarca5 mutations were selected from the F1 mutants for self-mating to obtain F2 mutant embryos. Among the collected embryos, one-fourth were homozygous mutants, namely the fluorescent thrombosis zebrafish model, and the remaining three-fourths were control embryos. The F2 smarca5 homozygous mutants showed head apoptosis 36 hours after fertilization and could be identified under a bright-field microscope.

[0137] gata1:dsRed can label red blood cells to indicate the relative position of red blood cells, and kdrl:GFP can label vascular endothelial cells to indicate the relative position of red blood cells to blood vessels.

[0138] The adult zebrafish were raised in a zebrafish fish house, where the temperature of the system water in the zebrafish fish house was between 25 and 37 °C. For example, it could be 25 °C, 25.5 °C, 26 °C, 26.5 °C, 27 °C, 27.5 °C, 28 °C, 28.5 °C, 29 °C, 29.5 °C, 30 °C, 30.5 °C, 31 °C, 31.5 °C, 32 °C, 32.5 °C, 33 °C, 33.5 °C, 34 °C, 34.5 °C, 35 °C, 35.5 °C, 36 °C, 36.5 °C, 37 °C.

[0139] In some specific embodiments, the temperature of the system water in the zebrafish fish house was 28.5 °C.

[0140] The embryos of the zebrafish are cultured in a culture medium, which can be any culture medium that enables the normal growth of zebrafish embryos. In some specific embodiments, the culture medium is prepared as follows: 70 g of NaCl, 0.5 g of NaHCO3, 2 g of CaCl 2 , 2 g of KCl are dissolved in 20 L of ddH 2 O.

[0141] In some specific embodiments, the culture medium containing the embryos is placed in an incubator. In some specific embodiments, the temperature of the incubator is between 25 and 37 °C. For example, it can be 25 °C, 25.5 °C, 26 °C, 26.5 °C, 27 °C, 27.5 °C, 28 °C, 28.5 °C, 29 °C, 29.5 °C, 30 °C, 30.5 °C, 31 °C, 31.5 °C, 32 °C, 32.5 °C, 33 °C, 33.5 °C, 34 °C, 34.5 °C, 35 °C, 35.5 °C, 36 °C, 36.5 °C, 37 °C.

[0142] In some specific embodiments, the temperature of the incubator is 28.5 °C. In some specific embodiments, the culture conditions for the zebrafish and zebrafish embryos are as follows: Adult zebrafish are raised in the system water at 28.5 °C in a zebrafish fish house, and the embryos are cultured in a culture medium (70 g of NaCl, 0.5 g of NaHCO3, 2 g of CaCl 2 , 2 g of KCl are dissolved in 20 L of ddH 2 O) and placed in an incubator at 28.5 °C.

[0143] In some specific embodiments, the fluorescent thrombus zebrafish model is a fluorescent thrombus zebrafish embryo.

[0144] In another aspect of the present invention, a method for anti-thrombosis or promoting thrombolysis is provided, which includes performing targeted gene therapy on a subject in need thereof to repair the smarca5 mutation.

[0145] In some specific embodiments, the fluorescent thrombus zebrafish model of the present invention can be used to evaluate the efficacy of potential gene therapy strategies. That is, disease-related genes or polynucleotides can be modified to inhibit or reduce the development and / or progression of the disease. In particular, the method includes modifying disease-related genes or polynucleotides to produce an altered protein. As a result, the animal or cell has an altered response. Therefore, in some methods, genetically modified animals can be compared with animals prone to disease development, so as to evaluate the effect of gene therapy events.

[0146] For the present invention, "thrombus" includes diseases occurring in the arterial and venous vascular systems and treatable by the methods of the present invention, particularly diseases in the coronary arteries of the heart, such as acute coronary syndrome (ACS), myocardial infarction with ST-segment elevation (STEMI) and without ST-segment elevation (non-STEMI), stable angina, unstable angina, re-occlusion and restenosis after coronary interventions such as angioplasty, stent implantation or aortocoronary bypass grafting, and thromboembolic or thrombotic diseases in other vessels leading to peripheral arterial occlusive disease, pulmonary embolism, venous thromboembolism, venous thrombosis, particularly in the deep veins of the lower extremities and renal veins, transient ischemic attack, and thrombotic and thromboembolic strokes.

[0147] In certain embodiments, the subject is an animal. Such an animal can be of any gender and can be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep or goat. In certain embodiments, the subject is a dog. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a dairy cow, pig, horse, sheep or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal (e.g., transgenic mouse, transgenic pig). In certain embodiments, the subject is a fish or a reptile.

[0148] The gene therapy method for repairing the smarca5 mutation can be any method capable of repairing the smarca5 mutation, including but not limited to in situ repair of genes, site-directed integration of genes, and site-directed integration of RNA interference technology, etc.

[0149] In another aspect of the present invention, there is also provided the use of fluorescent thrombus zebrafish in screening for drugs for promoting thrombus dissolution or reducing thrombus formation or anti-thrombosis in a subject.

[0150] In certain specific embodiments, the fluorescent thrombus zebrafish model of the present invention can be used to study the effects of mutations on animals or cells and the development and / or progression of diseases using measures commonly used in disease research. Alternatively, the fluorescent thrombus zebrafish model of the present invention can be used to study the effects of drug active compounds on diseases.

[0151] In one aspect of the present invention, a method for preventing erythrocyte aggregation is provided, which includes overexpressing keap1a or knocking down hmox1a in vitro or in vivo.

[0152] Through the combined analysis of ATAC-seq and RNA-seq, the present invention found that the deletion of smarca5 reduces the chromatin openness of the keap1a promoter region in erythrocytes and the transcriptional expression level of this gene. The downstream target genes of keap1a-nrf2, including hmox1a, are overactivated. Overexpressing keap1a or knocking down hmox1a in the mutant can partially rescue the erythrocyte aggregation phenotype.

[0153] In certain specific embodiments, the present invention provides a method for developing bioactive agents that regulate cell signaling events related to disease genes. The method includes contacting a test compound with cells containing a vector expressing one or more components of one or more driving systems; and detecting changes in the readout, where the readout changes indicate a decrease or increase in cell signaling events related to, for example, mutations in disease genes contained in the cells.

[0154] The method for preventing erythrocyte aggregation by overexpressing keap1a can be any way to overexpress keap1a, and the gene can be overtranscribed, translated, and the final gene expression product exceeds the normal level. The methods include, but are not limited to, constructing overexpression of foreign genes, CRISPR SAM, saRNA, etc.

[0155] In certain embodiments, overexpressing keap1a is carried out in vivo in a subject. In certain embodiments, overexpressing keap1a is carried out in vitro or ex vivo in a subject.

[0156] The method for preventing erythrocyte aggregation by knocking down hmox1a can be any way to inhibit the expression level of hmox1a, or any way to reduce it to a level lower than that usually observed when examined under substantially the same conditions but in the absence of RNAi, including but not limited to RNA interference (RNAi) technology, CRISPR technology, TALEN technology, T-DNA insertion technology, etc.

[0157] In certain embodiments, knocking down hmox1a is carried out in vivo in a subject. In certain embodiments, knocking down hmox1a is carried out in vitro or ex vivo in a subject.

[0158] The present invention further provides the use of a substance that promotes the overexpression of keapla or reduces the activation of hmox1a in the preparation of a drug for anti-thrombosis, promoting thrombolysis, or reducing thrombosis formation in a subject.

[0159] In another aspect, the present invention provides a method for anti-thrombosis or promoting thrombolysis or reducing thrombosis formation in a subject, comprising: overexpressing keap1a in a subject in need thereof by means of genetic manipulation or knocking down hmox1a of the subject.

[0160] In certain specific embodiments, the genetic manipulation includes using gene editing or gene expression.

[0161] The methods of gene editing include, but are not limited to, homologous recombination (HR) technology, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated proteins (CRISPR) technology. In certain specific embodiments, the method of gene editing used in the present invention is clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated proteins (CRISPR) technology. Preferably, the method of gene editing used is the method using the CRISPR / Cas9 system.

[0162] The ways of gene expression include, but are not limited to: constitutive expression, inducible expression, repressible expression, etc.

[0163] The levels of gene expression regulation include, but are not limited to: DNA and chromosomal levels, including, but not limited to, gene loss, gene modification, gene rearrangement, gene amplification, chromosomal structure changes; transcriptional level regulation (the main regulation mode), including, but not limited to, having an impact on transcriptional initiation, elongation, and termination. Prokaryotes rely on operons, and eukaryotes are regulated by the interaction of cis-acting elements and trans-acting factors; post-transcriptional level regulation: mainly refers to the processing of eukaryotic primary transcripts into mature mRNA, including, but not limited to, capping, tailing, methylation modification, etc.; translational level regulation: regulation of mRNA stability, regulation of the translational level by antisense RNA; post-translational level regulation: including, but not limited to, protein cleavage, chemical modification (phosphorylation, acetylation, glycosylation, etc.), transportation, etc.; regulation of mRNA degradation, etc.

[0164] The present invention further provides a drug for anti - thrombosis or promoting thrombolysis or reducing thrombosis in a subject, which comprises: a substance that inhibits the mutation of smarca5, or a substance that promotes the over - expression of keapla, or a substance that reduces the activation of hmox1a.

[0165] In certain embodiments, the drug for anti - thrombosis or promoting thrombolysis or reducing thrombosis in a subject is a substance that inhibits the mutation of smarca5.

[0166] In certain embodiments, the drug for anti - thrombosis or promoting thrombolysis or reducing thrombosis in a subject is a substance that promotes the over - expression of keapla.

[0167] In certain embodiments, the drug for anti - thrombosis or promoting thrombolysis or reducing thrombosis in a subject is a substance that reduces the activation of hmox1a.

[0168] Examples

[0169] Example 1 Identification of the abnormal aggregation phenotype of smarca5 mutant red blood cells

[0170] Take the heterozygous adult zebrafish smarca5 mutants (smarca5 zko1049a )(Ding etal., 2021) constructed by the CRISPR / Cas9 method. After self - mating, one - quarter of the obtained embryos are smarca5 homozygous mutant embryos, and the remaining three - quarters are control group embryos. Observe the development process of the two groups of embryos under a microscope. The results are as Figure 1 shown in A. The figure shows the tail images of two groups of zebrafish. It can be seen that when the smarca5 homozygous mutant embryos develop to the 2nd day after fertilization, blood cells abnormally aggregate and deposit in the tail vein. The area marked by the dotted line is the blood clot in the tail vein; while there is no phenomenon of blood cell aggregation in the control group embryos.

[0171] Use the primary red blood cell marker gene scl to label the smarca5 homozygous mutant embryos and the control group embryos. Adopt the whole - mount in situ hybridization technique (WISH) to detect the expression of primary red blood cells in the two groups of embryos. Collect pictures through a Nikon microscope (SMZ1500). The results are as Figure 1 shown in B. No blood clot is found in the control group, and the expression of the marker gene scl is enriched in the blood clot in the tail vein in the mutant, that is, shown in the black rectangular frame (the position indicated by the arrow in the enlarged figure). It shows that there are primary red blood cells in this area of the smarca5 homozygous mutant.

[0172] To better observe the blood clot, the present invention constructs a fluorescent thrombus zebrafish model. The specific method is as follows:

[0173] Take adult F0 of zebrafish smarca5 mutants constructed by the CRISPR / Cas9 method (smarca5 zko1049a )(Ding et al., 2021) and mate them with the fluorescently labeled transgenic fish line Tg(kdrl:GFP; gata1:dsRed) (this transgenic line was provided by the laboratory of Steve Wilson (King’s College London, London, United Kingdom)). Collect F1 embryos, and select fertilized eggs that simultaneously carry the fluorescence of kdrl:GFP (vascular endothelial cells) and gata1:dsRed (red blood cells) under a fluorescence microscope. Culture them to adult fish for tail clipping identification. The identification method is to extract genomic DNA using the alkaline lysis method, then clone the DNA fragment near the target site by PCR, and then identify the type of gene mutation by sequencing.

[0174] Determine that adult smarca5 mutants that simultaneously carry the fluorescence of kdrl:GFP and gata1:deRed are F1 mutants. Select heterozygotes with smarca5 mutations from the F1 mutants for self-mating to obtain F2 mutant embryos. Among the collected embryos, one-fourth are homozygous mutant, that is, the fluorescent thrombosis zebrafish model, and the remaining three-fourths are control group embryos. Head apoptosis appears in F2 smarca5 homozygous mutants at 36 hours after fertilization and can be recognized under a bright-field microscope.

[0175] gata1:dsRed can label red blood cells to indicate the relative position of red blood cells, and kdrl:GFP can label vascular endothelial cells to indicate the position of red blood cells relative to blood vessels.

[0176] Culture conditions: Adult zebrafish are raised in the system water at 28.5°C in a zebrafish fish house, and embryos are cultured in a culture solution (70 g NaCl, 0.5 g NaHCO3, 2 g CaCl 2 , 2 g KCl dissolved in 20 L ddH 2 O) and placed in an incubator at 28.5°C.

[0177] The homozygous mutant embryos and control group embryos involved in the present invention are distinguished and obtained according to this method.

[0178] Observe the aggregation phenotype of red blood cells labeled by gata1:dsRed in homozygous mutant and control group embryos through a fluorescence microscope. Take pictures with a laser confocal microscope, as Figure 1As shown in C, it was found that red blood cells (gata1:dsRed) (indicated by arrows) aggregated within the tail vessels (kdrl:GFP) of homozygous mutants, while this aggregation did not occur in control embryos.

[0179] The homozygous smarca5 mutants and control embryos were labeled with erythroid cell marker genes gata1, ikaros, and scl, and in situ hybridization was used to detect the expression of erythroid cells in the two groups of embryos. The results were as Figure 1 shown in D. By comparing with control embryos, it could be seen that the overall expression of erythroid cell marker genes gata1, ikaros, and scl was normal in homozygous smarca5 mutants.

[0180] The homozygous smarca5 mutants and control embryos were labeled with erythroid cell marker genes gata1, ikaros, hbae1, and hbbe1, and real-time fluorescent quantitative PCR (qPCR) was used to detect the expression of erythroid cells in the two groups of embryos. The results were as Figure 1 shown in E. The expression levels of erythroid cell marker genes gata1, ikaros, hbae1, and hbbe1 were similar in homozygous smarca5 mutants and control embryos.

[0181] The above results indicate that in homozygous smarca5 mutants, the genes related to the development of erythroid cells were not significantly affected.

[0182] Example 2 Effects of smarca5 knockout on the development of primary myeloid cells

[0183] Adult fish of smarca5 zko1049a heterozygous mutants were mated with transgenic fish lines Tg(mpo:GFP; gata1:dsRed) or Tg(coro1a:GFP; gata1:dsRed) with fluorescently labeled myeloid and erythroid cells (Li, Yan, Shi, Zhang, & Wen, 2012; Renshaw et al., 2006). Homozygous smarca5 mutants and control embryos with fluorescently labeled myeloid and erythroid cells were obtained according to the above method.

[0184] The aggregation phenotypes of mpo:GFP- and coro1a:GFP-labeled myeloid cells in homozygous mutants and control embryos were observed by fluorescence microscopy. Photographed by laser confocal microscopy, as Figure 2 shown in A, the distribution of myeloid cells in homozygous smarca5 mutants was normal and did not aggregate in red blood cell clots. The green fluorescence-labeled area is the area within the circle.

[0185] The myeloid cell marker genes pu.1 and lyz were used to label smarca5 homozygous mutants and control group embryos, and in situ hybridization technology was adopted to detect the expression of erythroid cells in the two groups of embryos. The results are as Figure 2 shown in B. By comparing with the control group embryos, it can be seen that the myeloid cell marker genes pu.1 and lyz are normally expressed in smarca5 mutant homozygotes.

[0186] The myeloid cell marker genes pu.1, mfap4 and lyz were used to label smarca5 homozygous mutants and control group embryos, and real-time fluorescence quantitative PCR technology was adopted to detect the expression of erythroid cells in the two groups of embryos. The results are as Figure 2 shown in C. The expression levels of the myeloid cell marker genes pu.1, mfap4 and lyz are similar in smarca5 homozygous mutants and control group embryos.

[0187] The above results indicate that in smarca5 homozygous mutants, the development of myeloid cells is not significantly affected.

[0188] Example 3 Phenotypic simulation of abnormal erythrocyte aggregation as venous thrombosis

[0189] To observe the occurrence process of erythrocyte aggregation, we used a fluorescence microscope to observe the aggregation process of erythrocytes (gata1:dsRed + ) in smarca5 homozygous mutants and control group embryos for a long time. The starting time of shooting was 36 hours after embryo fertilization, and the shooting duration was 12 hours. The results are as Figure 3 shown in A. In smarca5 homozygous mutants, erythrocytes started to aggregate at 40 hours after embryo fertilization, and finally deposited in the tail vein as the aggregation mass increased. No aggregation occurred in the control group embryos.

[0190] To further explore whether the aggregation of erythrocytes is affected by the microenvironment such as endothelium, we conducted an embryo parabiotic experiment. Embryos at the 128-cell blastula stage to 30% epiboly stage after removing the chorion were placed in methylcellulose, and the cells at the contact surface of the two embryos were stripped with the tip of a glass tube. Subsequently, the embryos were brought into contact and placed statically in an incubator until embryo fusion. The two fused embryos can exchange blood flow. Therefore, by detecting the phenotype of flowing erythrocytes, it can be judged whether the aggregation of erythrocytes is affected by the microenvironment such as vascular endothelium. The development process of the two groups of embryos was observed with a microscope. The results are as Figure 3 shown in B. After the mutant embryos and the control group embryos were parabiosed, blood clots formed in both embryos, suggesting that the aggregation of erythrocytes is largely independent of the microenvironment.

[0191] To better distinguish red blood cells in mutant embryos and control embryos, we used gata1:GFP + to label red blood cells derived from smarca5 homozygous mutants, and simultaneously used gata1:dsRed + to label red blood cells derived from control embryos. The distribution of red blood cells in the two groups of embryos was observed using a fluorescence microscope. The results are as Figure 3 shown in C. Red blood cells derived from smarca5 homozygous mutants were present in both mutant and control embryos. The region marked by green fluorescence (labeled by gata1:GFP + ) is the region within the circle. For red blood cells derived from control embryos, most of them flowed normally in mutant and control embryos, except for a few that were trapped in the blood clots. This result indicates that red blood cells lacking smarca5 can aggregate autonomously.

[0192] To investigate whether megakaryocyte progenitor cells are present in the blood clots, we crossed smarca5 zko1049a heterozygous mutant adult fish with the transgenic fish line Tg(CD41:GFP) (Lin et al., 2005) with fluorescently labeled megakaryocytes. According to the above method, smarca5 homozygous mutants and control embryos containing fluorescently labeled megakaryocyte progenitor cells (CD41:GFP high ) were obtained.

[0193] The distribution of megakaryocyte progenitor cells (CD41:GFP high ) in homozygous mutants and control embryos was observed through a fluorescence microscope. The results are as Figure 3 shown in D, and it was found that megakaryocyte progenitor cells did not appear in the blood clots.

[0194] Next, we treated smarca5 homozygous mutants with the thrombus treatment drugs Argatroban (Sigma, A0487), Aspirn (Sigma, A2093), and Heparin (Sigma, H3393). The results are as Figure 3 shown in E, which indicates that treatment with Argatroban can partially alleviate the formation of blood clots.

[0195] We continued to use the smarca5 homozygous mutants and control embryos treated with Argatroban, and observed the phenotype of red blood cell aggregation in smarca5 homozygous mutants and control embryos after treatment with the drug Argatroban under a microscope. The results are as Figure 3 shown in F, and it was found that Argatroban can partially alleviate the formation of blood clots. The statistical results are as Figure 3 shown in G. The phenotype of red blood cell aggregation in the mutants can be partially rescued, indicating that this mutant can partially mimic thromboembolic diseases and serve as a thromboembolic disease model.

[0196] Method for administering antithrombotic drug: Argatroban was dissolved in DMSO at a concentration of 2 mg / ml. At 36 hours after fertilization during embryonic development, the drug was injected into the venous plexus above the yolk sac of each embryo at a dose of 4 nl per embryo. After embryo administration, the embryos were placed in an incubator at 28.5 °C. 12 hours after administration, the erythrocyte aggregation phenotype was observed under a microscope.

[0197] Example 4: Erythrocyte aggregation does not affect the number of hematopoietic stem and progenitor cells in the caudal hematopoietic tissue

[0198] The smarca5 zko1049a Heterozygous mutant adult fish were mated with transgenic fish lines Tg(kdrl:mCherry) (Bertrand et al., 2010) and Tg(cmyb:GFP) (North et al., 2007) with fluorescently labeled endothelial cells and hematopoietic stem and progenitor cells. Homozygous mutants and control embryos containing endothelial cells and hematopoietic stem and progenitor cells were obtained according to the above method.

[0199] The distribution of cmyb:GFP + -labeled hematopoietic stem and progenitor cells in homozygous mutants and control embryos was observed by fluorescence microscopy. Photographs were taken by laser confocal microscopy, and the results are shown in Figure 4 A. The formation of blood clots does not affect the distribution of hematopoietic stem and progenitor cells (cmyb:GFP + ) in the caudal hematopoietic tissue of mutants. The green fluorescence (cmyb:GFP + )-labeled area is the area within the circle.

[0200] The statistical results are shown in Figure 4 B. The formation of blood clots does not affect the number of hematopoietic stem and progenitor cells (cmyb:GFP + ) in the caudal hematopoietic tissue of mutants.

[0201] Example 5: Observation of the subcellular morphology of smarca5-deficient erythrocytes by transmission electron microscopy

[0202] Longitudinal sections of the caudal artery and venous plexus in control embryos were observed by transmission electron microscopy. As shown in Figure 5 A, the caudal artery, venous plexus, and flowing blood cells can be seen in the figure.

[0203] The subcellular structure of erythrocytes in control embryos was observed by transmission electron microscopy. As shown in Figure 5 B, normal mitochondria can be seen inside the erythrocytes in control embryos in the figure.

[0204] The longitudinal section of the caudal artery and venous plexus in smarca5 homozygous mutant embryos was observed by transmission electron microscopy. As shown in Figure 5 Figure C, the caudal artery, venous plexus and flowing blood cells can be seen in the figure.

[0205] The mutant caudal vein region in smarca5 homozygous mutant embryos was observed by transmission electron microscopy. As shown in Figure 5 Figure D, the aggregation of red blood cells in smarca5 homozygous mutants can be seen in the figure.

[0206] To further observe whether smarca5 deficiency affects the subcellular structure of red blood cells, we performed transmission electron microscopy. The results are shown in Figure 5 Figure E. The mitochondrial cristae of red blood cells in the mutant group showed morphological abnormalities. The arrow indicates the mitochondrion with abnormal structure.

[0207] Ery, red blood cell; EC, endothelial cell; Mito, mitochondrion; Nuc, nucleus; Cyto, cytoplasm.

[0208] Example 6 Changes in the morphology and number of red blood cells in smarca5 mutants

[0209] Next, we explored whether the number of red blood cells in the mutants changed.

[0210] Flow cytometry was used to analyze the number of red blood cells in control embryos and smarca5 homozygous mutant embryos. The results are shown in Figure 6 Figure A, and the statistical results are shown in Figure 6 Figure B. The proportion of red blood cells (gata1:dsRed + ) in control embryos and smarca5 homozygous mutant embryos was similar, indicating that the knockout of smarca5 does not affect the number proportion of red blood cells in embryos.

[0211] Blood smear and Giemsa staining techniques were used to observe the morphology of red blood cells in control embryos and smarca5 homozygous mutant embryos. As shown in Figure 6 Figure C, it was found that the morphology of red blood cells was normal after the deletion of smarca5.

[0212] The nuclear-cytoplasmic ratio of red blood cells in control embryos and smarca5 homozygous mutant embryos was analyzed, that is, the maximum diameter of the nucleus was measured and divided by the maximum diameter of the cell. The results are shown in Figure 6 Figure D. This figure shows that the nuclear-cytoplasmic ratio of red blood cells was normal after the deletion of smarca5.

[0213] The above results indicate that the deletion of smarca5 does not affect the number and overall morphology of red blood cells.

[0214] Transcriptional expression analysis of red blood cells in the smarca5 mutant in Example 7

[0215] To explore the effect of smarca5 deletion on the red blood cell transcriptome, we analyzed the red blood cells of control embryos and smarca5 homozygous mutants using RNA-seq, namely transcriptome sequencing technology.

[0216] The specific steps are as follows: Collect the smarca5 homozygous mutants to be sorted and their control embryos into centrifuge tubes, and add 0.5% trypsin (diluted with PBS buffer) pre-warmed in the zebrafish incubator to the samples. Pipette and place the samples in the zebrafish incubator for digestion, pipetting 2 - 3 times during this period until digested into single-cell suspensions. Add CaCl 2 to a final concentration of 1 M and fetal bovine serum (FBS) to a final concentration of 10% to terminate trypsin digestion. After centrifugation, add an appropriate amount of PBS buffer containing 1% FBS to the cell pellet to resuspend the cells, and pass the cell suspension through a 300-mesh filter to obtain a single-cell suspension. Sort red blood cells (gata1:dsRed + ) using a flow cytometer MoFlo XDP (Beckman Coulter). Sort 50,000 cells for each sample and collect them into PBS buffer containing 1% FBS, and place them on ice. Subsequently, extract RNA using the QIAGEN RNeasy Mini Kit (Cat. No. 74104), and then perform RNA-seq library construction and sequencing. The experimental procedure is as Figure 7 shown in A.

[0217] Using DESeq2 differential expression analysis (Love, Huber, & Anders, 2014), analyze the changes in gene expression in red blood cells in smarca5 homozygous mutant embryos. The results are as Figure 7 shown in B. After smarca5 knockout, gene expression in red blood cells changes. The black dots represent genes with significant changes in gene expression (Log2(fold change)>1, adjusted P-value<0.05). This figure shows that after smarca5 knockout, 1506 genes are significantly upregulated and 633 genes are significantly downregulated in red blood cells.

[0218] Using gene set variation analysis (GSVA) technology, analyze the differential pathways in red blood cells of control embryos and smarca5 homozygous mutant embryos. The results are as Figure 7 shown in C. It is found that after smarca5 knockout, the genes with decreased transcription are mainly related to pathways such as "downstream targets of Gata1" and "red blood cell oxygen carrying". As Figure 7As shown in D, the "erythrocyte homeostasis" and "inflammatory response" signaling pathways were decreased and increased, respectively, in the erythrocytes of the mutant group. The above results indicate that the deletion of smarca5 affects the transcriptome changes of the whole erythrocytes.

[0219] Example 8 RNA-seq analysis of erythrocytes in smarca5 mutants and their control group embryos

[0220] The RNA-seq samples of erythrocytes in control group embryos and smarca5 homozygous mutant embryos were analyzed by principal-component analysis (PCA). The results are as Figure 8 shown in A. This figure shows that the mutant samples and the control samples can be clearly distinguished, that is, the deletion of smarca5 leads to overall transcriptome changes.

[0221] Erythrocytes are a type of cells rich in hemoglobin. Next, we specifically analyzed the changes in hemoglobin-encoding genes in the mutant group and the control group.

[0222] Then, RNA-seq analysis and in situ hybridization techniques were used to analyze the erythrocytes of control group embryos and smarca5 homozygous mutant embryos. The results are as Figure 8 shown in B and 8C, respectively. There were no significant differences in the expression of embryonic hemoglobin-encoding genes and adult hemoglobin-encoding genes between the mutant group and the control group, indicating that the deletion of smarca5 does not affect the expression of hemoglobin-encoding genes in erythrocytes, and the differentiation and development of erythrocytes are not affected.

[0223] Control group embryos and smarca5 homozygous mutant embryos were stained and analyzed using benzidine dimethoxyaniline. The results are as Figure 8 shown in D. It was found that the hemoglobin content in the mutant group and the control group was similar, and the black arrow indicates the blood clot in the mutant.

[0224] The expression of myeloid cells in the two groups of embryos was analyzed by RNA-seq. The results are as Figure 8 shown in E. We observed that the myeloid cell marker genes spi1a, spi1b, mfap4, and lyz were upregulated in the erythrocytes of smarca5 homozygous mutants.

[0225] Next, we attempted to knockdown the expression of pu.1, a key factor in myeloid development, in smarca5 mutants using Morpholino (MO). MO is an antisense oligonucleotide that can target a gene of interest and mainly inhibits gene expression by preventing protein translation initiation and affecting the correct splicing of RNA. The reagent to be injected was aspirated into a capillary glass tube, and the microinjector was adjusted. By adjusting the air pressure, the injection dose was judged according to the size of the ejected droplet. When injecting MO, the MO was injected into the yolk sac of embryos at the one- to four-cell stage. After observing under a microscope that 1 ng of pu.1 MO was injected, as Figure 8 shown in Figure F, we found that the aggregation phenotype of red blood cells in smarca5 homozygous mutant embryos was not significantly rescued, and the statistical results are as Figure 8 shown in Figure G, indicating that the phenotype of red blood cell aggregation is not caused by the overexpression of myeloid genes.

[0226] Example 9 smarca5 mutation affects the openness change of chromatin in red blood cells

[0227] To further explore the effect of smarca5 deletion on the chromatin accessibility of red blood cells, the ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing) chromatin openness sequencing technology was used to sequence red blood cells in smarca5 homozygous mutant and control group embryos. The ATAC-seq technology utilizes a modified Tn5 transposase to specifically recognize and label open chromatin regions. The transpos DNA was designed as an adapter sequence, so that Tn5 inserted the sequencing adapter into the open chromatin region while recognizing the open chromatin region. Subsequently, through high-throughput sequencing, the genomic sequence information of the open chromatin region can be captured.

[0228] In smarca5 homozygous mutant and control group embryos, red blood cells labeled with gata1:dsRed were sorted by flow cytometry and then subjected to ATAC-seq library construction and sequencing. The number of cells in each sample was 50,000. The analysis results are as Figure 9 shown in Figure A. The results show that there are 439 and 40 genes specifically opened in the promoter and distal regulatory regions of mutant red blood cells, respectively, while there are 256 and 20 genes specifically opened in the promoter and distal regulatory regions of red blood cells in control group embryos. This indicates that smarca5 knockout affects the openness of chromatin in the promoter region and distal regulatory region.

[0229] At the same time, we performed Motif screening on the regions where chromatin openness decreased after smarca5 deletion, and the results are as Figure 9As shown in B, the results show that in the enrichment list, the key transcription factor Gata1 that regulates erythrocyte development is included, suggesting that the deletion of smarca5 may affect the binding of Gata1 to chromatin.

[0230] Figure 9 What C shows is the number of genes with both up-regulated and down-regulated chromatin openness and transcriptional levels in the promoter region in the mutant group. It is found that compared with the control group, there are 84 genes with both increased chromatin openness and gene transcriptional expression in the promoter region in the mutant group, and 36 genes with both decreased chromatin openness and gene transcriptional expression in the promoter region, indicating that the deletion of smarca5 has an impact on both chromatin openness and gene transcriptional expression in the promoter region.

[0231] Next, we focused on analyzing the genes with both up-regulated and down-regulated chromatin openness and transcriptional levels in the mutant group. The results are as Figure 9 shown in D. The chromatin openness and transcriptional levels of genes il34, cox4i2, skap2, vclb, and acbd7 are both up-regulated in the mutant group, while the chromatin openness and transcriptional levels of genes trim2a, keap1a, skap2, acox3, igfbp1a, and ada are both down-regulated in the mutant group.

[0232] Example 10 ATAC-seq analysis of erythrocytes in smarca5 mutants and their control group embryos

[0233] The ATAC-seq data was analyzed using the R package ATACseqQC (Ou et al., 2018) (v 1.6.4). The heatmap shows the distribution of ATAC-seq peaks in the 1 kb intervals upstream and downstream of the transcription start site (TSS). As Figure 10 shown in A, the left heatmap is the distribution of nucleosome-free (less than 100 bp), and the right is the distribution of mononucleosomes (180 - 247 bp). This indicates that the ATAC-seq peak distribution is normal and the quality assessment of the library is normal.

[0234] The distribution of ATAC-seq peaks near the TSS in Figure A was analyzed using the R package ATACseqQC. The results are as Figure 10 shown in B, indicating that the ATAC-seq peak distribution near the TSS is normal.

[0235] Principal component analysis of the ATAC-seq samples of erythrocytes in smarca5 homozygous mutants and their control group. The results are as Figure 10 shown in C. The mutant samples and the control group samples can be clearly distinguished, that is, the deletion of smarca5 leads to overall changes in chromatin openness.

[0236] The bar graph drawn using ChIPseeker shows the distribution characteristics of ATAC-seq peaks of mutants and their control groups across the entire genome. The results are as Figure 10 shown in D. The deletion of smarca5 has no obvious effect on the distribution characteristics of ATAC-seq peaks across the entire genome.

[0237] Figure 10 The Venn diagram in E shows the number of genes with simultaneous upregulation and downregulation of chromatin openness and transcription levels in the distal regulatory regions of the mutant group. It is found that there are 181 genes in the mutant group with both increased chromatin openness in the distal regulatory region and gene transcription expression compared to the control group, and 55 genes with both decreased chromatin openness in the distal regulatory region and gene transcription expression, indicating that the deletion of smarca5 affects both chromatin openness in the distal regulatory region and gene transcription expression.

[0238] Example 11 smarca5 regulates the phenotype of abnormal erythrocyte aggregation through the Keap1-Nrf2 signaling pathway

[0239] Among the genes with altered chromatin openness and transcription levels after the deletion of smarca5 detected above, as Figure 11 shown in A, we found that the chromatin openness in the promoter region of keap1a decreased, and the predicted Gata1 binding site was marked by a black arrow. The expression level of keap1a in control embryos and smarca5 homozygous mutants was detected using fluorescence quantitative PCR technology. The results are as Figure 11 shown in B, indicating that the expression level of keap1a in smarca5 homozygous mutants decreased.

[0240] The expression of keap1a-nrf2 downstream target genes, including hmox1a, gclc, ggt1b, gsr, gstp1, gstk1, fbp1a, gsto2, prdx1, pgd, and g6pd, in the two groups of embryos was detected using real-time fluorescence quantitative PCR technology. The results are as Figure 11 shown in C. Most of the keap1a-nrf2 downstream target genes, including hmox1a, were overactivated.

[0241] Next, we tried to overexpress keap1a in smarca5 homozygous mutant embryos and then detect whether the aggregation phenotype could be rescued.

[0242] The full-length CDS of keap1a was cloned into the pDestTol2pA2 vector containing the hsp70 promoter and EGFP reporter system using a DNA recombination kit (NEBuilder HiFi DNA Assembly MasterMix, E2621S). The successfully constructed overexpression plasmid (50 ng / μl) was mixed with tol2 mRNA (50 ng / μl) in equal volumes, and 1 nl was injected into the one-cell stage cells of smarca5 mutant zebrafish. The injected embryos were raised to adult fish, and after mating, the embryos were collected. After heat shock treatment (42 °C for 40 minutes to 1 hour), embryos with green fluorescence expression were screened to obtain a stably inherited transgenic line: Tg(hsp70:keap1a-EGFP). This heat shock treatment can simultaneously activate the expression of the keap1a gene. The transgenic adult fish were mated, and the embryos were collected. Figure 11 Panel D (upper) shows the expression pattern of EGFP in transgenic fish Tg(hsp70:keap1a-EGFP) at 2 days post-fertilization as detected by fluorescence photography (heat shock treatment was performed 36 hours after embryo fertilization).

[0243] Heat shock treatment was performed 36 hours after embryo fertilization, and 12 hours later, the aggregation phenotype of red blood cells was observed under a microscope. The results are shown in Figure 11 Panel D (lower), and the statistical results are shown in Figure 11 Panel E, indicating that overexpression of keap1a in smarca5 homozygous mutant embryos can partially reduce the aggregation of red blood cells.

[0244] In addition, we injected hmox1a MO into control group embryos and smarca5 homozygous mutant embryos (the procedure was the same as injecting pu.1MO) to observe the aggregation phenotype of red blood cells in control group embryos and smarca5 homozygous mutant embryos. The results are shown in Figure 11 Panel F, and the statistical results are shown in Figure 11 Panel G, showing that knockdown of hmox1a can partially rescue the red blood cell aggregation phenotype.

[0245] We demonstrated the mechanism by which smarca5 affects the Keap1-Nrf2 signaling pathway and the expression of downstream target gene hmox1a by regulating the chromatin openness of the keap1a promoter region through Figure 11 a schematic diagram in Panel H.

[0246] The above results indicate that smarca5 regulates the abnormal red blood cell aggregation phenotype partially through the Keap1-Nrf2 signaling pathway.

[0247] Example 12 Generation of free radicals may play an important role in red blood cell aggregation

[0248] Treat smarca5 homozygous mutant embryos and their control group embryos with the reducing agent glutathione (Sigma, PHR1359). Glutathione was dissolved in zebrafish culture medium at a concentration of 0.5 mg / ml. The control treatment was zebrafish culture medium without drugs. The treatment time was 36 hours after embryo fertilization, and the aggregation phenotype of red blood cells was observed under a microscope 12 hours after treatment. After the control treatment and treatment with glutathione in the control group embryos and smarca5 homozygous mutant embryos, the phenotype of red blood cell aggregation in the tail vein of the control group embryos and smarca5 homozygous mutant embryos was observed microscopically. The results are as Figure 12 shown in A, and the detection time was the second day after embryo development to fertilization. The arrow indicates the blood clot in the tail vein. This figure shows that treatment with glutathione can partially alleviate the phenotype of red blood cell aggregation.

[0249] The statistical results of its red blood cell aggregation phenotype are as Figure 12 shown in B, and this figure shows that treatment with glutathione can partially alleviate the phenotype of red blood cell aggregation.

[0250] As described above, it is only the preferred embodiment of the present application, and it is not a limitation to the present application in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present application without departing from the technical solution content of the present application still belong to the protection scope of the technical solution of the present application.

Claims

1. A method for constructing a fluorescent thrombosis zebrafish model, characterized in that, comprising: obtaining zebrafish heterozygotes with smarca5 mutations by gene editing methods; mating the zebrafish heterozygotes with smarca5 mutations and transgenic zebrafish with fluorescence, and screening from the offspring for fluorescent thrombosis zebrafish with smarca5 mutant phenotypes and fluorescence; abnormal aggregation of red blood cells occurs in the fluorescent thrombosis zebrafish model; Among them, the zebrafish heterozygote with smarca5 mutation is smarca5 zko1049a heterozygous mutant.

2. The method according to claim 1, characterized in that, the zebrafish heterozygotes with smarca5 mutations are obtained by editing the chromosomes of zebrafish by the CRISPR / Cas9 method.

3. The method according to claim 1, characterized in that, the transgenic zebrafish with fluorescence are transgenic zebrafish with different fluorescent labels.

4. The method according to claim 3, characterized in that, using different fluorescent labels to label red blood cells and vascular endothelial cells respectively.

5. The method according to any one of claims 1 to 4, characterized in that, obtaining zebrafish heterozygotes with smarca5 mutations by gene editing methods and determining zebrafish heterozygotes F0 with smarca5 mutations; the steps of mating the zebrafish heterozygotes with smarca5 mutations and transgenic zebrafish with fluorescence and screening from the offspring for fluorescent thrombosis zebrafish with smarca5 mutant phenotypes and fluorescence include: mating smarca5 mutant heterozygotes F0 with transgenic zebrafish with fluorescence to obtain smarca5 mutant F1 generation with fluorescence; selecting heterozygotes with smarca5 mutations from the F1 generation mutants for male-female mating to obtain F2 generation mutant embryos; genetically identifying the homozygotes with smarca5 mutations and fluorescence in the F2 generation mutants as the fluorescent thrombosis zebrafish model.

6. The method according to claim 1, wherein, the fluorescent thrombosis zebrafish model is a fluorescent thrombosis zebrafish embryo.

7. Use of the fluorescent thrombosis zebrafish model constructed by the method according to any one of claims 1 to 6 in screening for drugs for promoting thrombolysis or reducing thrombosis formation in a subject.

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